res Pe oe — ee ‘ei — ae or ; es = VIRUSES - _ Selected! by _ GUNTHER S. STENT T 2557200 TOE0 O MOL A 1OHM/18IN ae 3) on, ; ea? i) ists ; COMP § [ps dn AVAL | >. ; at is) : a : Ye Thu) Oat PAPERS ON BACTERIAL VIRUSES Ea Boe, way aes” « ft (? PAPERS ON BACTERIAL VIRUSES Selected by GUNTHER S. STENT University of California, Berkeley Boston Toronto LITTLE, BROWN AND COMPANY COPYRIGHT © 1960, LITTLE, BROWN AND COMPANY, INC. ALL RIGHTS RESERVED. NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. LIBRARY OF CONGRESS CATALOG CARD NO. 60-53146 Published simultaneously in Canada by Little, Brown & Company (Canada) Limited PRINTED IN THE UNITED STATES OF AMERICA FOREWORD In 1951, the collection Papers in Microbial Genetics; Bacteria and Bacterial Viruses appeared under the editorship of Joshua Lederberg. That collection has been an indispensable aid to us and many others in the teaching of micro- bial genetics. It has enabled every student to read a cross-section of the original literature and thus become directly acquainted with the basic experi- mental data that often outlive the conclusions based upon them. The Lederberg collection, however, appeared just at the eve of great dis- coveries which, since that time, have enormously advanced our understanding of the structure and function of the hereditary substance of bacteria and their viruses. The transduction of bacterial genes by bacteriophages, the polarity of bacterial conjugation, the role of DNA as the germinal substance of bacterial viruses, the structure of DNA itself — all of these were discovered within the two-year period following the publication of Lederberg’s collection. New techniques have also greatly changed the course and nature of research in microbial genetics; in particular, the use of isotopic tracers in bacterial virus research and the development of recombination tests of high resolving power for the analysis of genetic fine structure have brought microbial genetics to the molecular level. Hence it seemed urgent that a new, more up to date, collection should now be made available; we have decided to assume this task, and hope that the present book and its companion volume, Papers on Bacterial Genetics, will answer the needs of at least the immediate future. The extent of the changes that have taken place is indicated by the fact that of the more than fifty papers we have selected for the present two volumes, only five have been retained from the twenty papers of Lederberg’s collection. In addition to the reprinted papers, each volume contains an introductory text with its own bibliography, in order to compensate for the necessarily in- complete and arbitrary nature of the selections. The selections themselves were made for the sole purpose of providing students with a cross-section of the literature, at a reasonable cost; in the words of Professor Lederberg “no apologies need be offered for a selection which must be largely arbitrary.” In conclusion, we would like to express our deep appreciation to the copy- right owners and the authors who permitted us to reprint these papers, as well as to those who have supplied us with rare reprints from which some of the copy has been made. GUNTHER S. STENT Epwarp A. ADELBERG Berkeley, California, 1960 a CONTENTS Introduction, by GUNTHER S. STENT Discussion on the Bacteriophage (Bacteriolysin). I. The Nature of Bacteriophage, by I. D’HéreEuuE; I. The Bacteriophage: The Breaking Down of Bacteria by Associated Filter-passing Lysins, by F. W. Tworrt; III. Concerning the Theories of the So-called “Bacteriophage,” by J. Borpet; IV. ANDRE GRATIA Adsorption of Bacteriophages to Homologous Bacteria. II. Quantitative Investigations of Adsorption Velocity and Saturation. Estimation of the Particle Size of the Bacteriophage, by M. ScHLesinGER The Growth of the Bacteriophage, by Emory L. Exuis and Max De.priick The Growth of Bacteriophage and Lysis of the Host, by M. De.tBriicx The Intracellular Growth of Bacteriophages. I. Liberation of Intracellular Bacteriophage T4 by Premature Lysis with Another Phage or with Cyanide, by A. H. DozERMANN Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage, by A. D. HmrsHry and Marrua CHASE Nucleic Acid Transfer from Parental to Progeny Bacteriophage, by J. D. Watson and O. MAAL@E The Synthesis of Bacterial Viruses; Origin of Phosphorus Found in Deoxyribonucleic Acids of T2 and T4 Bacteriophages, by SEYMOUR 8. COHEN Enzymatic Synthesis of Deoxyribonucleic Acid. VI. Influence of Bacteriophage T2 on the Synthetic Pathway in Host Cells, by ARTHUR KORNBERG, STEVEN B. ZIMMERMAN, 5S. R. KoRNBERG, and JOHN JOSSE ; The Frequency Distribution of Spontaneous Bacteriophage Mutants as Evidence for the Exponential Rate of Phage Reproduction, by S. E. Luria Genetic Recombination between Host-Range and Plaque-Type Mutants of Bacteriophage in Single Bacterial Cells, by A. D. HersHry and RaQUEL ROTMAN Genetic Recombination and Heterozygosis in Bacteriophage, by A. D. HersHEY and MartHa CHASE Vil 1X 105 116 139 151 179 Vill Contents The Structure of DNA, by J. D. Watson and F. H. C. Crick Fine Structure of a Genetic Region in Bacteriophage, by Seymour BENZER Induction of Specific Mutations with 5-Bromouracil, by Seymour BENZER and Ernst FREESE Experiments on Photoreactivation of Bacteriophages Inactivated with Ultraviolet Radiation, by R. DuLBEcco Genetic Recombinations Leading to Production of Active Bacteriophage from Ultraviolet Inactivated Bacteriophage Particles, by 8. E. Luria and R. DuLBEcco Inactivation of Bacteriophages by Decay of Incorporated Radioactive Phosphorus, by GUNTHER S. Stent and CLARENCE R. FUERST Resistance to Ultraviolet Light as an Index to the Reproduction of Bacteriophage, by S. BENZER Investigations on a Lysogenic Bacillus Megaterium, by ANDRE LWoFF and ANTOINETTE GUTMANN Induction of Bacteriophage Lysis of an Entire Population of Lysogenic Bacteria, by ANDRE Lworr, Louis StminovitcH and NIELS KJELDGAARD Lysogeny and Genetic Recombination in Escherichia coli K12, by Evie L. WouLtMAN and FRANGOIS JACOB Spontaneous Induction of the Development of Bacteriophage ) in Genetic Recombination of Escherichia coli K12, by FRANGoIs JACOB and E11e WoLLMAN Transduction of Lysogeny in Escherichia coli, by FRANGoIS JACOB Recombination between Related Temperate Bacteriophages and the Genetic Control of Immunity and Prophage Localization, by A. D. Katsrer and F. JAcosB Pagination of the papers in this collection is indicated by the boldface number centered at the bottom of the page. Other page numbers appearing on certain papers refer to original publication. 193 209 220 228 247 280 298 312 332 334 336 339 303 INTRODUCTION BACTERIAL VIRUSES were discovered in 1915, when F. W. Twort (149) isolated a filtrable virus which produces a “glassy transformation” of micrococcal colonies during their growth on an agar surface. Twort’s paper remained relatively unnoticed, until two years later F. d Hérelle (64) published his own observations on a filtrable agent, the “bacteriophage,” capable of serially trans- missible lysis of growing cultures of enteric bacilli. D’Hérelle’s announcement caused an immediate sensation among medical bacteriologists, since the bactericidal properties of the bacteriophage offered promise of a generalized prophylaxis and therapy of bacterial diseases. Within two or three years of the publication of his first paper, dHeérelle had carried out many incisive experiments which allowed him to recognize the essential aspects of these bacterial viruses. Twort and dHérelle did not remain the only bacteriophage workers for very long, and the study of bacterial viruses rapidly became so popular that most of the leading bacteriologists of the decade following the first World War tried their hand at it. This soon led to a number of violent controversies concerning the nature and mode of action of bacteriophages. Some of these controversies were aired at a discussion on the bacteriophage organized by the British Medical Association at its Glasgow meeting of 1922. The presenta- tions which d’Hérelle, Twort, Bordet, and Gratia prepared for this meeting form the first paper of this collection. In his discussion, dHérelle demonstrates the self-reproducing, or viral, character of the bacteriophage, a view for which, as is evident from the remarks of Bordet and of Gratia, he had been under attack. Gratia’s discussion, however, rectifies two errors of d’Hérelle: the claim that the phenomena discovered by Twort and by d’Hérelle are fundamentally different and the assertion that all bacteriophages represent the same antigen. In 1926, dHérelle (65) thus summarized his earlier findings on the multi- plication of bacterial viruses: “The first act of bacteriophagy consists in the approach of the bacteriophage corpuscle toward the bacteria, then in the fixation of the corpuscle to the latter ... The bacteriophage corpuscle pene- trates into the interior of the bacterial cell. When, as a result of its faculty of multiplication, the bacteriophage corpuscle which has penetrated into the bacterium forms a colony of a number of elements, the bacterium ruptures suddenly, liberating into the medium young corpuscles which are then ready to continue the action.” The first workers to study in some detail the initial step in bacterial virus x Introduction growth, i.e. the fixation, or adsorption, of the bacteriophage to its bacterial host cell, were Krueger (91) and Schlesinger (129, 130). In his paper (130), the second of this collection, Schlesinger demonstrates that phage adsorption usually is an irreversible process which follows the kinetics expected from a two-body collision model involving freely diffusing virus particles and bacterial cells. Schlesinger’s work, which probably represents the first rigorous applica- tion of physicochemical principles to the study of bacterial viruses, was extended by Delbriick (39), who showed that the physiological state of the bacteria affects the rate of adsorption, and by Garen and Puck (59), who demonstrated clearly that, under certain conditions, a reversible union between phage and bacterium can take place. Contrary to the predictions of the simple two-body collision model of Schlesinger, however, it turned out that the rate of phage adsorption reaches a maximum at high bacterial concentrations and is strongly temperature-dependent. Hence, it appears that the irreversible fixa- tion of the bacteriophage is a two-step process, involving at least one further, temperature-sensitive, step in addition to the collision of virus and host cell (146). Subsequent electron-optical observations by T. F, Anderson revealed that the organ of adsorption is the phage tail (5), in particular, that it is the thin tail fibers (155) which are the structures undergoing the stereospecific fixation reaction with the phage receptors on the bacterial surface. (Reviews: 58, 148, 73, 125, 154.) Convincing support for d’Hérelle’s conception that the infecting phage particle multiplies within the bacterium and that its progeny are liberated upon lysis of the host cell was adduced in 1929 by Burnet (31), who showed that 20 to 100 viruses suddenly appear some 20 minutes after a bacterial suspension is infected with a single phage particle. The final demonstration, however, that a burst of progeny of the parent virus is liberated by each infected bacterial cell after a latent period was only provided in 1939 in the one step growth experiment of Ellis and Delbriick (52), whose paper is presented in this collection. In their publication, Ellis and Delbriick also describe for the first time the single burst experiment, which made possible the study of phage growth in individual infected bacteria, rather than in mass culture. The appearance of this paper marks the beginning of modern phage research. The interpretation of the phage-induced lysis of infected bacterial cultures was one of the violent controversies during the first 20 years of phage research. While d’Hérelle (65) correctly thought that intracellular phage growth leads to lysis of the host cell and liberation of the virus progeny, Bordet and Ciuca (20, 22) maintained that the phage-induced dissolution of bacterial cultures is merely the consequence of a stimulation of lytic enzymes endogenous to the bacteria. Other workers, such as Bronfenbrenner (30), or Krueger and Nor- throp (92), thought that lysis of the bacteria is only a secondary phenomenon, which may or may not follow the growth of phage, and imagined that the bacteriophage can pass freely in and out of bacterial cells. Delbriick (40) Introduction XI finally showed, in his second paper of this collection, that these arguments were bedevilled by the circumstance that there exist not one but two com- pletely different processes by which bacteriophages can lyse susceptible bac- terial cells. One of these, lysis-from-without, represents an immediate dissolu- tion of bacteria, often encountered when the multiplicity of infection is much greater than one phage per bacterium (9, 123, 92, 124). Loss of the input phages, rather than their multiplication, ensues from this form of lysis. Only the second of the lytic processes, lysis-from-within, is really the form of lysis properly connected with intracellular phage multiplication, and its onset signals the end of the latent period. The one-step growth experiment demonstrated clearly the nature and kinetics of the process by which bacterial viruses multiply within cultures of susceptible bacteria. It thus brought into focus the question of fundamental biologic interest: what is taking place inside the infected cell during the latent period while the parental phage particle replicates itself several hundredfold? In order to study the kinetics of intracellular phage multiplication, Doermann (47) broke open phage-infected bacteria at various times during the latent period and assayed the infectivity of the material released by premature lysis. The result of this experiment, published in the paper included in this collec- tion, was that the infectivity associated with the original parental virus is lost at the outset of the reproductive process, since no infective particles whatso- ever can be found in any of the bacteria lysed within ten minutes after their infection. After more than ten minutes have elapsed, however, ever-increasing numbers of infective progeny viruses make their intracellular appearance, until the final crop of progeny has been attained which would have been released by spontaneous lysis-from-within at the end of the normal latent period. The stage of intracellular bacterial virus growth during which the in- fected host cell contains no material capable of infecting another bacterium is the eclipse (107). Subsequent studies showed that the actual multiplication of the infecting virus takes place during the eclipse, i.e. that the phage multiplies in a non-infectious form, the vegetative phage (48). Schlesinger was also the first to purify a bacterial virus, a feat which he accomplished by high-speed centrifugation of phage lysates (131). Chemical analysis of the purified virus revealed that it consists of approximately equal proportions of protein and deoxyribonucleic acid (DNA) (132). Later studies by Anderson (3, 4) and by Herriott (66) showed that the viral DNA resides within a proteinaceous head membrane, from which it can be released by osmotic shock. Hershey and Chase (75) then demonstrated that the two viral moieties, protein and DNA, have independent functions in the infection process. For, as can be seen in their first paper included in this collection, Hershey and Chase found that practically all of the viral protein remains at the surface of the infected cell, and that it is mainly the viral DNA which enters the bacterium at the outset of intracellular phage growth. The bulk of the phage protein appears to be relieved of any further function in the intra- Xil Introduction cellular reproductive process after the proteinaceous tail has attached the virus particle to the bacterial surface and the DNA has safely entered the interior of the host cell. This historic discovery showed that it must be the viral DNA that is the carrier of the hereditary continuity, i.e. the germinal substance of the extracellular, resting phage. The release of the DNA from its protein envelope at the very moment of infection also accounts for the existence of the eclipse period at the early stages of intracellular virus development. For having been divested of its attachment and injection organs, the DNA of the infecting phage naturally is unable to gain entrance into any further bacterial cells to which it may be presented in the infectivity test. What happens to the viral DNA after its injection into the host cell? In 1950 Putnam and Kozloff (126) devised an experiment directed toward the question of whether any of the atoms of the parental DNA ultimately reappear among the progeny viruses. In this “transfer experiment,” bacteria are infected with phage particles whose DNA is isotopically labeled, and the phage yield issuing from such infected cells assayed for its content of parental isotope. The outcome of Putnam and Kozloff’s transfer experiment was that about half of the atoms of the parental DNA were found to be transferred to the progeny. This work was confirmed and extended with improved experimental techniques by Watson and Maalge (120, 152), one of whose publications is included in this collection. In view of the inference that it is the DNA of the virus which carries the genetic continuity into the host cell, it seemed likely that an under- standing of the mechanism of transfer of DNA atoms from parent to offspring might afford valuable insight into the nature of the reproductive process. Further investigations have revealed that the parental DNA complement of a single parental virus is not transferred intact to a single progeny virus, but that the molecular patrimony is dispersed over several offspring phages (76, 143, LOL, 145). Some theories of the nature of phage multiplication envisaged that there are present, within the normal host bacterium, bacteriophage precursors whose metamorphosis into mature bacteriophages is merely triggered by the infecting phage particle (93). This view was finally dispelled in 1948 by an experiment of Cohen (37), designed to determine the origin of the substance of the progeny phages. This work, reported in a paper of this collection, represents the first use of radioisotopes in the study of bacterial viruses. By exposing bacterial cultures to P**, either only prior to or only subsequent to their infec- tion with phage, and analyzing the virus progeny for their relative content of radioisotope, Cohen could show that most of the phage DNA is synthesized from materials still in the growth medium at the moment of infection; hence, the phage particles cannot have been derived from pre-existing bacterial pre- cursors. The complete kinetics of assimilation of phage DNA phosphorus were studied subsequently by modifications of Cohen’s original method, by either adding to or withdrawing from the growth medium of bacterial cultures P*” at various times before or after their infection. The results of this work led to Introduction Xill the idea that, prior to its incorporation into intact, infective progeny particles, the phage DNA exists in an intrabacterial phage precursor pool (144, 71). Further insight into the process of phage multiplication was gained by the discovery of a variety of “incomplete” phage structures which possess one or another of the properties of the virus without being endowed with the power of self-reproduction, the most complex of all its attributes. Thus, premature lysis of infected bacteria at late stages of the eclipse period liberates newly synthesized proteinaceous material already possessing some of the antigenic properties of the intact bacteriophage (119). The total amount of phage antigen finally liberated upon spontaneous lysis of the cells, furthermore, generally exceeds that incorporated into infective progeny (119, 32, 46). Electron-optical observations of such lysates, furthermore, reveal the presence of structures whose morphology bears some resemblance to the characteristic shape of mature bacterial virus particles (161, 63). Prominent among these structures are the “doughnuts” which Levinthal and Fisher (103) found to appear during the eclipse and then to increase in number at about the same rate as the complete phage particles. Later studies have shown that the dough- nuts are, in fact, empty phage heads, and that the “maturation” of infective progeny at the end of the eclipse seems to represent the stable union of phage precursor DNA with phage precursor protein into structurally intact virus particles (88, 89). Phage precursor protein and phage precursor DNA are not the only materials whose synthesis within the host cell is induced, or presided over, by the DNA of the infecting parental virus. For, at the outset of intracellular phage growth, the formation of some non-precursor proteins must proceed before replication of the viral DNA can begin. One of these “early” proteins was identified by Flaks and Cohen (53, 54) as the enzyme deoxycytidylate hydroxymethylase, essential for the synthesis of the specific components of the viral DNA, 5-hydroxymethylcytosine (160). Studies by Kornberg, Zimmer- man, Kornberg, and Josse (90), presented in a paper of this collection, revealed the phage-induced formation of four further enzymes, all of which are demonstrably involved in the synthesis and replication of the viral DNA. It is important to realize, therefore, that the phenotypic expression of the genetic substance of the phage is not confined solely to the construction of materials that find incorporation into the mature, infective progeny virus. In common with other organisms, bacterial viruses sport occasional hereditary variants, or mutants, in the course of their growth (34, 68, 133). These mutants can differ from their parents in a variety of characteristics, such as the type of plaque formed on agar seeded with sensitive indicator bacteria (67), the strains of bacteria which the phage can infect (105), or the physical or chemical properties of the virus particle (2, 43, 26). The mutation of the vegetative phage during its intracellular growth was used by Luria (108) to probe the nature of the self-duplication of the hereditary material of the infecting particle, as shown in a paper of this collection. In his experiment, XIV Introduction Luria examined the individual phage yield of many thousands of phage- infected bacterial cells and scored the single bursts for the presence of progeny viruses possessing a certain plaque-type mutant character. On the basis of the observed clonal frequency distribution of these mutants, Luria was able to infer that the replication of the hereditary material of the phage is geo- metric, i.e. that it proceeds by a number of successive cycles of self-duplica- tion, since other conceivable reproductive models, e.g. successive replications of the initial parental element or chain replication of the last element produced, would have led to mutant distributions quite different from that actually found. In 1946, a most important discovery was made independently by Delbriick and Bailey (44) and by Hershey (67), who examined the genetic character of the phage yield issuing from bacterial cells infected with two related parent viruses differing from each other in two mutant factors. It was found that among the progeny of such mixed infection there appear virus offspring carry- ing one of the mutant factors of one and one of the mutant factors of the other of the two parents, demonstrating that bacterial viruses can undergo genetic recombination. The first detailed study of genetic recombination in phage was undertaken by Hershey and Rotman (77), their paper being included in our collection. This work showed that, on the basis of the frequency with which recombinant progeny for various mutated characters appear in such “crosses, it is possible to construct a genetic map of the phage on which the mutant loci can be arranged in a linear order. Hershey and Rotman also examined the frequency of complementary recombinant types in the yields of individual mixed infected bacteria and found that the formation of comple- mentary types does not seem to occur in a single event [Bresch (28) was able to establish this conclusion even more convincingly in a later study]. This fact led Hershey and Rotman to entertain the notion that recombination in phage might not be the consequence of a reciprocal exchange of preformed genetic structures, such as chromosomal recombination in higher forms, but that it might be an act incidental to the replication of the genetic material itself. This hypothesis, which came to be called “partial replicas” (69), or “copy choice” (97), now forms one of the basic concepts in the understanding of the molecular basis of self-duplication and genetic recombination. As more data concerning the process of genetic exchange in phage accumulated, it became evident that the theoretical analysis of a phage “cross” is a problem in population genetics. It was seen that within each mixedly infected bacterial cell, growth and recombination of the numerous vegetative phage replicas proceed concurrently. In 1953, Visconti and Delbriick (150) developed, there- fore, a theory which succeeded in explaining quantitatively the recombinant frequency observed in different phage crosses under various conditions. This theory assumes that replication and recombination of vegetative phages pro- ceeds in an intrabacterial pool, in which phages repeatedly mate pairwise and at random until lysis of the host cell, and from which pool the vegetative phages are withdrawn irreversibly for maturation into infective progeny Introduction XV phages. A concise statement of this theory can be found in Adams’ book (1), and a more generalized formulation is presented in two later analyses of this problem (137, 29). An important clue to the nature of the elementary recombinational event in phage was uncovered by Hershey and Chase (74), described in their second paper of this collection. They noted that among the progeny of mixed infec- tions about 2% of the particles are heterozygous, in that these individuals carry homologous loci, or alleles, from both parents of the cross. The heterozy- gosity is only partial, however, in that in any one heterozygote virus only a very limited segment of the genome is actually of biparental provenance, most of its loci being homozygous, or derived from only one or the other of the parents, The structure and behavior of these heterozygotes suggested to Hershey and Chase that the formation of heterozygotes and the formation of recombinants might be related processes. The nature of heterozygotes was considered further by Levinthal (100), who demonstrated that such viruses are recombinant for genetic loci on opposite sides of the limited region of heterozygosity. Levinthal then inferred that recombinant phages, in fact, arise through the formation of heterozygotes in the course of phage reproduction by the partial replica, or copy-choice, recombination mechanism. This inference found further support from Levinthal’s calculation that the observed frequency of heterozygotes is great enough to explain the observed frequency of recom- binational events. Once the viral DNA had been identified as the germinal substance, it became possible to consider in actual chemical terms how the hereditary information is stored in the resting phage and how it is replicated in the vegetative phage. After deoxyribonucleic acid was discovered by Miescher in 1871, some 60 years of chemical study of this substance revealed that its building block is the nucleotide, composed of one molecule each of phosphoric acid, deoxyribose, and either adenine, guanine, thymine, or cytosine. More recently, it was established that DNA molecules are, in fact, polymers of very high molecular weight, each molecule containing more than 10* nucleotide units joined through phosphate diester bonds linking successive deoxyribose molecules (cf. 36). The actual molecular architecture of DNA was worked out finally by Watson and Crick (151), whose paper is presented here. Wat- son and Crick showed that the DNA molecule consists of two helically inter- twined polynucleotide chains laterally held together by a pair of hydrogen bonds between a complementary pair of purine and pyrimidine residues on opposite chains. The nature of the DNA molecule suggests that the only specific aspect which could distinguish one DNA macromolecule from another is the precise sequence of the four possible purine-pyrimidine base pairs along the complementary nucleotide chains, i.e. that the hereditary information is a message written into the DNA macromolecule in an alphabet containing four letters. This structure also suggested to Watson and Crick a mechanism by which the DNA molecule could replicate itself; for if the two complementary XVI Introduction polynucleotide chains separate and each parental chain acts as the template for the de novo synthesis of a complementary daughter chain, a pair of DNA molecules would be generated, each half-old, half-new, whose specific purine- pyrimidine base pair sequence is identical to that of the parent molecule. A genetic mutation, from this point of view, would then be a rare copy error in the replication process by which a nucleotide carrying an incorrect base is introduced into the replica nucleotide chain, thus producing a change in the genetic information. Even though a number of modifications of this replication scheme of Watson and Crick were subsequently proposed (45), later experi- ments have shown that in the replication of bacterial DNA the distribution of the atoms of the parental molecules appears to proceed by the semi-conserva- tive route (122), a central feature inherent in the Watson-Crick scheme. One of the first successful attempts to bridge the gap between chemistry and genetics was made by Benzer (12) in his first paper of this collection. Benzer discovered a method for scoring very rare recombinant viruses appear- ing in phage crosses between parents bearing extremely closely linked mutant loci. This allowed him to construct a fine structure map of a large collection of mutants situated in a very restricted region of the phage genome. In consequence of this work, the concept of the gene, traditionally regarded as the unit of recombination, mutation and function, became clarified. For Benzer showed that these three aspects of the genetic material are opera- tionally separable and hence cannot share a common unit. Translated into molecular terms, the unit of recombination appears to represent one, or a few, nucleotide pairs along the DNA molecule, whereas the unit of mutation can be of variable length, ranging from the alteration of a single nucleotide pair, in case of a point mutation, to long-span alterations of the phage genome, covering hundreds or thousands of nucleotide pairs. Finally, the unit of function, or cistron, assumed to determine the specific chemical structure of an enzyme protein, or more precisely, of a polypeptide chain, is of the order of 1000 nucleotides in length (13). After Benzer had arranged his set of closely linked spontaneous phage mutants into a linear linkage map, it became obvious that there exists a great variability in mutability of different genetic sites within a single functional group, or cistron, since at some loci, or “hot spots,” spontaneous mutations recur with much greater frequency than at other, nearby loci. This differential mutability of individual genetic sites very probably reflects the chemical structure of the hereditary molecule corresponding to each locus; e.g. the chance of making a spontaneous copy error at a given site in the course of viral DNA replication might depend on which particular sequence of purine and pyrimidine residues obtains there and which particular base substitution will produce the mutant genotype in question (13). Benzer and Freese (14), therefore, examined also distribution, or mutational spectrum, of mutants induced by the action of chemical mutagens, in particular by replacement of thymine by its analog 5-bromouracil in the viral DNA, which replacement, as Introduction XVil Litman and Pardee had found (104), is highly mutagenic in bacterial viruses. Benzer and Freese’s investigation, presented in this collection, showed that the set of mutants induced by the action of 5-bromouracil is completely different from the set of spontaneous mutants in the same general region of the viral genome, demonstrating that “the mutagen does not merely enhance the over-all mutation rate, but acts at specific locations in the hereditary struc- ture.” Mutational spectra of other chemical mutagens were subsequently established, and it turned out that each of these substances raises the proba- bility of mutation at a restricted number and individually characteristic set of sites (27, 57). Further insight into the chemical nature of the induced muta- tions was provided by studies that determined the connection between the induction of a mutation at a specific site by a given mutagen and the ability of the same, or of another mutagen, to revert this mutation to the original state. On the basis of these results, Freese proposed that there exist two basic types of point mutation in the viral genetic material: transversions, corresponding to the substitution of a purine by a pyrimidine residue, or vice versa, and transi- tions, corresponding to the replacement of one type of pyrimidine by the other or of one type of purine by the other (56). Not long after the discovery of the bacteriophage it was found that ultra- violet light (UV) kills the virus particle (65), and since then, UV has been the inactivation agent whose effects have been most extensively studied (136, 95, 110, 139). This work has shown that in addition to simply destroying the reproductive power, UV also produces a number of important physiological and genetic effects. The inactivated phages, furthermore, are by no means inert, being still capable of adsorbing to and killing bacteria, and of interfer- ing with the growth of other, unirradiated phages in the same host cell (111). Some of the lethal effects of UV, finally, are reversible under appropriate con- ditions. An important example of such reversibility is the existence of photo- reactivation, discovered by Dulbecco (49) in the work presented herein. Dul- becco found that viability is restored to UV-inactivated phages if bacteria infected with such “dead” particles are illuminated with visible light. Dul- becco’s quantitative analysis of photoreactivation showed that a fraction of the UV lesions, the photoreactivable sector, is restored by a light-activated enzyme system of the bacterial host cell. Later work by Bowen (23, 24) revealed that photoreactivation consists of two steps: the first step is a dark reaction requiring no light, which generates the substances adsorbing and “activated” by the quanta of visible light for the second, actually reactivating step. Experiments by Lennox, Luria, and Benzer (99) suggested that photo- reactivation constitutes a direct reversal rather than a bypass mechanism of the primary ultraviolet damage, a conclusion that now seems certain since Good- gal, Rupert, and Herriot demonstrated the in vitro photoreactivation of UV- inactivated transforming DNA by illuminated bacterial extracts (61). Viability can also be restored to UV-inactivated phages if two or more “dead” particles, each unable to reproduce itself in solo, happen to infect XVIi1 Introduction the same bacterial cell. This is the phenomenon of multiplicity reactivation, discovered by Luria in 1947 (106) and investigated in some detail by Luria and Dulbecco (112), whose paper is included in this collection. The quan- titative results presented here seemed to bear out Luria’s proposal that each inactivating UV lesion represents a lethal mutation in one of a certain number of genetic subunits of the phage and that multiplicity reactivation ensues from the genetic exchange of still undamaged units between the two irradiated parent viruses. In order to explain the very high frequency of reactivation, furthermore, it was assumed that phage growth occurs by the independent reproduction of each subunit, followed by reassembly of the units into com- plete phages. When Dulbecco (50) subsequently continued his multiplicity reactivation studies, he found that the results observed at very high UV doses are no longer compatible with the notion of independently multiplying sub- units. In any case, studies on genetic recombination in phage had in the mean- time indicated that the genetic material of the phage does not multiply in the form of independent subunits (150). More recently, however, modifications of the original hypothesis of multiplicity reactivation have been proposel by Baricelli (10) and by Harm (62), which still retain that most essential element of Luria’s hypothesis that reactivation proceeds by a mechanism of genetic exchange of undamaged parts and which lead to quantitative formulations in satisfactory agreement with the observed data. Another radiobiological method of inactivation bacterial viruses was dis- covered by Hershey, Kamen, Kennedy, and Gest (76), who showed that highly P*?-labeled bacteriophages lose their infectivity upon decay of radio- phosphorus atoms. From the kinetics and efficiency of this inactivation process, it could be inferred that the cause of death is the transmutation of phosphorus into sulfur atoms in the polynucleotide chains of the viral DNA, or the highly energetic nuclear recoil associated with this event. These studies were extended by Stent and Fuerst (141), whose paper appears in this collection. They found that, although the efficiency of killing of one lethal hit per ten P*? disintegrations first observed by Hershey and his co-workers also obtains in a variety of different bacteriophage strains, the fraction of disintegrations that are lethal depends on the temperature at which decay is allowed to proceed. A mechanism for the decay inactivation process of the virus was suggested on the basis of these findings. It was proposed that the high pro- portion of nonlethal decays reflects the possibility that the physiological func- tion of the double-stranded DNA molecule is preserved even after radioactive decay has interrupted only one of its polynucleotide strands. The lethal decays, in contrast, are thought to be those that result by chance in a complete cut of both strands of the DNA double helix. The decay of incorporated P*? atoms has proven a very useful tool for the study of the structure, physiology, and genetics of bacterial viruses and bacteria. (Review: 142.) In the hope of measuring the extent to which the infecting parental virus has multiplied within the host cell during the eclipse period before the appear- Introduction XIX ance of any mature progeny, Luria and Latarjet (113) irradiated phage- infected bacteria with UV at various stages of intracellular phage growth. They reasoned that if the UV sensitivity of the vegetative phage is equal to that of the free, extracellular virus, then the result of this irradiation experi- ment ought to be a family of multiple-hit survival curves from which the instan- taneous number of vegetative phages present at the time of irradiation could be inferred. The outcome of Luria and Latarjet’s experiment was contrary to their expectation, however; instead of the anticipated multiple-hit survival curves, a family of straight lines of ever decreasing slope was observed, indica- tive of the fact that the intrinsic UV sensitivity of the vegetative phage is much less than that of the free virus. This is also evident from Benzer’s (11) im- proved experimental design of the Luria-Latarjet technique, presented here as Benzer’s third paper. The meaning of the great reduction in UV sensitivity of the vegetative phage has not yet found an entirely satisfactory explanation. On the one hand, as is evident from Benzer’s report, some phages do not mani- fest this effect, so that UV irradiation of bacteria infected with such phages actually gives rise to the family of multiple-hit curves anticipated by Luria and Latarjet. On the other hand, the vegetative phage is also much more resistant to inactivation by decay of incorporated P** atoms than the extra- cellular P*2-labeled virus (138). It seems likely, however, that the reduction in radiosensitivity of the vegetative phage reflects some important aspect of the function and replication of the viral DNA, and some of the possible interpre- tations have been discussed in several reviews (94, 136, 139, 142). In any case, the method of Luria and Latarjet has found a number of valuable applications in the study of intracellular virus growth, not only with bacterio- phages but also with plant and animal viruses (134, 135, 51, 128). Within a few years of the discovery of the bacteriophage, lysogenic bacterial strains were found which appear to “carry” bacteriophages, in the sense that phage particles are always present in the culture fluid of such strains (21, 60). It was soon realized that this association of phage and bacteria cannot be of a casual nature, since it is impossible to permanently free lysogenic strains from the phage they carry by methods which ought to kill or remove the virus particles, such as heating, anti-phage serum neutraliza- tion, or single-colony purification (8, 18, 121). The nature and significance of lysogeny then remained a subject of intense controversy for about 30 years, some workers denying the existence of “true” lysogeny and others claiming that lysogeny disproves the whole notion that bacteriophagy involves an infec- tion of bacteria by virus particles. Nevertheless, a few bacteriologists, such as Burnet and McKie (35), and the elder Wollman (156), already envisaged that lysogeny represents an innate capacity of bacterial cells for phage produc- tion. In order to establish firmly some of the basic but controversial facts of lysogeny, Lwoff began a study of this phenomenon after the Second World War and in 1950 published the paper (116) presented here. In this work Lwoff and Gutmann demonstrate unequivocally that each bacterium of a lyso- XX Introduction genic strain harbors and maintains a noninfective structure, the probacterio- phage or prophage, which endows the cell with the ability to give rise to infec- tive phage without further intervention of exogenous virus particles. The actual synthesis of infective phage, however, proceeds in only a small fraction of the cells of a growing culture of lysogenic bacteria, whose intracellular content of virus particles is liberated by lysis of the phage-producing indi- vidual. Lwoff also inferred from these experiments that the induction of phage development in a lysogenic cell is under control of external factors, and his subsequent investigations in collaboration with Siminovitch and Kjelgaard (118) showed that treatment with various agents, in particular irradiation with UV light, will indeed induce phage production and ultimately lysis of almost every cell of a culture of lysogenic bacteria. The first pre- liminary report of this finding (117) is included in this collection. After the publication of Lwoffs papers and reviews (114), the study of lysogeny not only flowered into a distinct branch of bacterial virus research but also became the bridge leading from the genetics of virus to that of host cell. In fact, the recognition of the existence and nature of the provirus state engendered entirely new ideas concerning the origin, evolution, and biological function of viruses (79). (Reviews: 114, 17, 85. ) What is the relationship of the prophage to the remainder of the lysogenic cell? The great stability of the lysogenic character implies that the prophage is transmitted to daughter cells at each bacterial division. This could happen in one of two ways: either the prophage represents numerous autonomous structures, replicating in the bacterial cytoplasm in synchrony with the rest of the bacterium and being partitioned at random at each division over the daughter cytoplasms, or the prophage is integrated into the nuclear apparatus of the host cell and participates in the specific replication and segregation process which assures, nolens volens, that each daughter cell obtains one complete set of parental hereditary factors. The first of these alternatives soon appeared unlikely, when indirect estimates of the number of prophages revealed that each cell seems to carry only one or two prophages per bacterial nucleus (16, 81), and that a given type of prophage appears to saturate a limited number of sites on some bacterial structure (15). Positive indications that the prophage is integrated into the bacterial nucleus became available from experiments in bacterial conjugation, in which non-lysogenic bacteria were crossed with lysogenic bacteria, and a linkage of the lysogeny character with other known genetic factors of the cell inferred from the segregation pattern of the recombinants (96, 158, 7, 55). After the discovery of high- frequency-recombination (Hfr) bacterial strains and of the oriented transfer of the bacterial chromosome from donor to recipient cell, Wollman and Jacob (159) could show very clearly in their first paper of this collection that at least one particular prophage has its specific location on the bacterial chromosome: their later work, furthermore, revealed that different prophages have different specific chromosomal sites (83, 84). At the same time, Jacob and Wollman Introduction XXI (82) also discovered the existence of zygotic induction, described here in their second paper. For if a chromosome fragment of a donor bacterium bearing a prophage enters a non-lysogenic recipient cell, then the prophage becomes induced, enters the vegetative state, leading to the production of infective progeny and lysis and loss of the bacterial zygote. This phenomenon accounted for the discrepancies that had been observed in earlier attempts to determine the chromosomal location of the prophage by bacterial conjugation experi- ments, from which different linkage relations could be inferred, depending upon which of the two parents of the cross carried the prophage (160). A completely independent confirmation of the specific location of the pro- phage on the bacterial chromosome was provided through transduction experiments by Jacob (78), whose paper is presented here. Transducing virus particles carry a small genetic segment of closely linked loci of a donor bacterium, i.e. the last host cell, into a recipient bacterium, i.e. the next host cell. In this way, recombinant bacteria can arise which have derived a very limited part of their genome from the donor cell (164, 163, 147, 98). Jacob thus showed that a transducing virus can carry the prophage of an entirely unrelated virus strain from a lysogenic donor into a non-lysogenic recipient bacterium, usually in association with the contiguous region of the donor chromosome. The chromosomal region, moreover, turned out to be the same in which the prophage had been already placed by bacterial conjugation studies. The presence of the prophage not only endows the bacterium with the capacity to produce phage but also confers upon the cell an immunity to infection by a homologous phage (8, 18, 121, 157). Such virus particles are usually adsorbed to immune lysogenic bacteria, but the particles neither multiply to give rise to infective progeny nor affect growth and division of the immune cells in any way (16, 15, 81). The immune character is highly specific, in that a bacterium lysogenic for, and hence immune to, infection by one type of phage is not immune to infection by other viruses whose prophage the cell does not happen to carry. It became possible to study the genetic basis of immunity when Wollman and Jacob (84) discovered a number of related phage strains that undergo genetic recombination with one another but that differ in their immune specificity as well as in the locations of their prophages on the host linkage map. The final paper of our collection presents the work of Kaiser and Jacob (87) which established by means of crosses of these related phage strains that there is a definite segment of the viral genome, the C region, which determines the immune specificity of the phage. This same segment also controls the ability of the virus to establish itself as pro- phage and the locus at which the prophage is situated on the bacterial chromo- some. Later experiments by Jacob and Campbell (80) have shown that im- munity derives from the presence of a repressor substance in the cytoplasm of the lysogenic cell. The specificity of both formation and action of this repressor appears to be determined by the C region of the phage genome. XXil Introduction Before closing this introduction, we must once more call attention to the necessarily very limited and quite arbitrarily restricted scope of this collection. Not only do the twenty-five papers presented make up a very small fraction of the important contributions to the study of bacterial viruses, but even the references listed on the following pages are by no means an adequate, or even representative, coverage of the relevant literature. Some impression of the extent of that literature can be gained from the astounding bacteriophage bibliography prepared by Raettig (127), which lists no fewer than 5655 papers published between 1917 and 1956! The reader who wishes to acquire a more profound knowledge of this subject without working his way through these 5655 papers may do so by studying some of the books and reviews on bacterial viruses which have appeared over the years. This study might begin with Adams’ book Bacteriophages (1), the most authoritative and compre- hensive treatment available at present, and with the more specialized chapters in Burnet and Stanley’s treatise The Viruses (115, 58, 140, 102, 85, 136). More easily digestible works are Weidel’s little book Virus (153), a sprightly intro- duction to bacterial viruses, and Afinsen’s The Molecular Basis of Evolution (6) which contains an account of the role of bacterial viruses in avant-garde molecular biology. A first-hand acquaintance with the evolution of bacterial virus research can be obtained by perusal of the reviews of Bronfenbrenner of 1928 (30), Bordet of 1931 (19), Burnet of 1934 (33), Delbriick of 1942 and 1946 (41, 42), Cohen of 1949 (38), Luria of 1950 (107) and Hershey of 1952, 1953, 1956, and 1957 (69, 70, 72, 73). Finally, some other important reviews that treat special aspects of bacterial viruses are those of Luria (109), Brenner (25), and Jacob and Wollman (86, 79). BIBLIOGRAPHY 1. Adams, M. H. ‘Bacteriophages,’ Interscience, New York, 1959. 2. Adams, M. H., and Lark, G. Mutation to heat resistance in coliphage T5. J. Immunol., 64, 335 (1950). 3. Anderson, T. F. The reactions of bacterial viruses with their host cells. Botan. Rev., 15, 464 (1949). 4, Anderson, T. F. Destruction of bacterial viruses by osmotic shock. J. Appl. Phys., 21, 70 (1950). 5. Anderson, T. F. The morphology and osmotic properties of bacteriophage systems. Cold Spring Harbor Symp. Quant. Biol., 18, 197 (1953). 6. Anfinsen, C. B. ‘The Molecular Basis of Evolution.” John Wiley and Sons, New York, 1959. 7. Appleyard, R. Segregation of lambda lysogenicity during bacterial recombination in Escherichia coli K12. Genetics, 39, 429 (1954). 8. Bail, O. Der Colistamm 88 von Gildemeister und Herzberg. Wed. Klin. (Munich), OF 2h 925): Bibliography XXili 9. Bail, O., and Matusmoto, T. Die Anhéufungsmoglichkeit von Bakteriophagen und 10. 1A 12. 13. 14. 15. 16. ice 18. 8) 20. 21. 22. 23. 24. 25. 26. 27. 28. Bakterien, Med. Klin. (Munich), 19, 1579 (1923). Baricelli, N. A. A “chromosomic’”’ recombination theory for multiplicity reactivation in phages. Acta biotheoretica (Leiden), 11, 107 (1956). Benzer, 8S. Resistance to ultraviolet light as an index to the reproduction of bacteri- ophage. J. Bacteriol., 63, 59 (1952). Benzer, S. Fine structure of a-genetic region in bacteriophage. Proc. Natl. Acad. Sci. U.S., 41, 344 (1955). Benzer, 8. 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On the mechanism of DNA replication, in “The Chemical Basis of Heredity,’ W. D. McElroy and B. Glass, eds., p. 699. The Johns Hopkins Press, Baltimore, (1957). DeMars, R. I. The production of phage-related material when bacteriophage development is interrupted by proflavine. Virology, 1, 83 (1955). Doermann, A. H. The intracellular growth of bacteriophages. I. Liberation of intracellular bacteriophage T4 by premature lysis with another phage or with cyanide. J. Gen. Physiol., 35, 645 (1952). Doermann, A. H. The vegetative state in the life cycle of bacteriophage: Evidence for its occurrence and its genetic characterization. Cold Spring Harbor Symp. Quant. Biol., 18, 3 (1953). Dulbecco, R. Experiments on photoreactivation of bacteriophages inactivated with ultraviolet radiation. J. Bacteriol., 59, 329 (1950). Dulbecco, R. A critical test for the recombination theory of multiplicity reacti- vation. J. Bacteriol. 63, 199 (1952). Dulbecco, R., and Vogt, M. Biological properties of poliomyelitis virus as studied by the plaque technique. Annals New York Acad. Sct., 61, 790 (1955). Ellis, E. L., and Delbriick, M. The growth of bacteriophage. J. Gen. Physiol., 22, 365 (1939). Flaks, J. G., and Cohen, 8. §., Virus-induced acquisition of metabolic function. I. Enzymatic formation of 5-hydroxymethyldeoxycytidylate. J. Biol. Chem., 234, 1501 (1959). 54. 55. 56. 57. 58. Bibliography XXV Flaks, J. G., Lichtenstein, J., and Cohen, 8. S., Virus-induced acquisition of meta- bolic function. II. Studies on the origin of the deoxycytidylate hydroxymethylase of bacteriophage-infected EF. coli. J. Biol. Chem., 234, 1507 (1959). Fredericq, P. Localisation du prophage sur le chromosome. Son intervention dans le taux de ségrégation apparent des recombinants d’ Escherichia coli K12. Compt. rend. Soc. Biol., 148, 1501 (1954). Freese, E. 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There exist in the intestinal contents of all living beings principles which have the property of dissolving certain bacteria. These principles pass porcelain filters; hence it is possible to separate them from intestinal bacteria. Each filtering principle, isolated from intestinal contents of normal indi- viduals, dissolves a certain number of bacterial species, belonging generally to the coli-typhoid-dysentery group. The action, therefore, of these principles is not strictly specific; but belongs, for a given filtrate, to a certain number of microbial species. In convalescents from an infectious disease a principle endowed with an energetic dissolving action on the bacterial species which causes the disease is always met with. It is not only towards the intestinal bacteria that these lytic principles exist: for instance, one can isolate from the excreta of convalescents from bubonic plague a principle which dissolves B. pestis; the same phenomenon exists in various animal septicaemias. I cannot enter here into an examination of all the facts relating to bacterio- phage, facts most of which have been already confirmed by the investigations of other workers, But if the facts themselves have not been contested, it is not so in regard to the hypothesis which I have put forward to explain the nature of the principle which brings about the dissolution of bacteria. Reprinted by permission of the British Medical Association from the British MepIcAL JOURNAL, 2 (3216), 289-297, August 19, 1922. 3 Ave. 19, 1922 | SECTION OF MICROBIOLOGY. [er ee A preliminary remark presents itself. Many authors who have formulated hypotheses as to the nature of the lytic principle have simply taken a particular fact supporting their own point of view and have neglected the whole assembly of experimental facts which renders such a thesis untenable, thereby forgetting that experiment is the final criterion of the truth of a theory. Let us try to resolve the question objectively. The choice between the different hypotheses a priori possible to explain the origin of the lytic principle can only be made in accordance with a funda- mental rule of logic — namely, in order that a hypothesis becomes admissible it is necessary that it should explain all the experimental facts and that it is not contradicted by any of them; further, in order that this admissible hypothesis may be considered as being in conformity with the nature of things, it must be proved that all the experimental facts cannot be explained if one abandons or modifies this hypothesis. The bacteriolysis produced under the influence of the principle which we have named “bacteriophage” consists in a total dissolution of the microbial body; at the end of this action there remains no visible residue. A total dissolu- tion of a microbial substance can only be due to a transformation or decompo- sition of the proteins of the microbe by proteolytic enzymes. Whence, it may be asked, come these proteolytic enzymes? Four hypotheses may be con- sidered. 1. The enzymes may be derived from the animal organism which is attacked by the given bacteria. The enzymes would then be the result of a defensive reaction on the part of the organism. This is the hypothesis of Kabeshima, Bordet and Ciuca, and Ann Kuttner. Kabeshima?’ does not specify the particular tissue of the animal from which the enzymes originate; Bordet and Ciuca* indicate the leucocytes; Ann Kuttner* incriminates any tissue. 2. The enzymes may come from intestinal bacteria as the result of a micro- bial antagonism. For instance, one knows the bacteriolytic action of filtrates of old cultures of B. pyocyaneus. This is the hypothesis of Lisbonne et Carrére,® for whom the lytic enzymes are secreted by intestinal bacilli such as B. coli, B. proteus, etc. 3. The enzymes may be secreted by the bacterium itself which undergoes the lysis. These enzymes would therefore be autolysins. This is the hypothesis of Weinberg and Aznar.° Under the action of a cause x the bacteria would acquire the property of secreting autolytic enzymes. Bail’ had already indicated a similar origin, but his hypothesis was more complicated, and that in order to harmonize his conception with certain experimental facts to which we shall refer later. The bacteria undergoing lysis would become fragmented into filtrable corpuscles (the “splitters” hypothesis of Bail). These corpuscles would secrete enzymes capable of dissolving the bacteria from which they spring. With the same view, Otto and Winkler* have put forward a hypothesis Aue. 19, 1922 | THE BACTERIOPHAGE. [eee Mepicar JourNAL closely related to that of Bail, but for them the question resolved itself into one of bacterial albuminoid micelles, which do not reproduce themselves, but which secrete the lytic enzymes. 4. The enzymes may be secreted by an ultramicroscopic virus, which is a parasite of bacteria. This is the hypothesis by which I have held since my first publication.° Those four hypotheses cover all the possibilities. Let us now see which hypothesis conforms best with the experimental facts, at the same time eliminating those which fail to do so. First Facr: The dissolution of bacteria under the influence of the bacterio- phagic principle takes place in series. That is to say, a filtrate containing the bacteriophagic principle, when added to a culture in liquid medium of a bacterium towards which the principle manifests its lytic action, provokes the entire dissolution of the bacteria. A trace of the latter, inoculated into a second bacterial culture, leads to the latter in its turn undergoing total lysis. A trace of this second dissolved culture, inoculated into a third bacterial culture, produces the same phenom- enon, and so on ad infinitum. After more than a thousand passages, the thousandth bacteriolysed culture contains a bacteriophagic principle as active as, and generally much more active than, that of the primitive filtrate. The phenomenon goes on in series in the same way, whether the culture is inoculated with the previously dissolved non-filtered culture, or with the dissolved culture filtered through a porcelain filter: the filtrate contains the bacteriophagic principle as active as the lysed non-filtered culture. A simple enzyme action would cease to show itself from the first tubes of the series, because of the greater and greater dilution of the enzyme solution in the course of the successive passages. One calculates easily that at the thousandth passage (each passage being carried out on 10 c.cm. of bacterial emulsion inoculated with 1/1000 c.cm. of the preceding dissolved culture) the titre of the dilution in the thousandth tube of the series is given in cubic kilo- metres by the the number 10*°*?. To appreciate this incommensurable number it is interesting to note that, at the twenty-second passage only, the drop of primitive filtrate introduced into the first tube of the series finds itself diluted in 107° cubic kilometres of liquid; such a cube of liquid is so great that a light ray would take one billion years to cross one edge of it. It is impossible to conceive of an enzyme, contained in a single drop of primitive filtrate, still existing, without any diminution of its activity, after being diluted to this extent. That the action may become manifest, each dilution must be allowed sufficient time (four to six hours) to permit the development of the bacterio- phagic principle. On the contrary, when the dilution is utilized immediately, the bacteriophagic action ceases about the fourth or fifth dilution. These experiments establish clearly that a regeneration of the bacterio- phagic principle occurs at each passage. Consequently the lytic enzymes are Ave. 19, 1922 | SECTION OF MICROBIOLOGY. ase oe Mepicat JourNAL produced by a living being, which regenerates itself in the course of the successive passages. It will be obvious, then, that this transmissibility in series eliminates hypotheses 1 and 2. The authors who have put forward these hypotheses must admit that the principle derived from the organism which is attacked by para- sites (hypothesis 1), or from the foreign bacteria (hypothesis 2), plays solely the role of a transformer—that is to say, it gives rise in the bacteria which undergo the transmissible lysis to a special state of “autolysability.” We come now to the third hypothesis. Only Bordet and Ciuca have attempted to give an explanation of this. For them the leucocytic principle would provoke in the bacteria a “hereditary vitiation of nutritional nature.” I must say that I do not understand how a filtered liquid can transmit a hereditary property. Besides, the whole theory of Bordet and Ciuca is based on an experience entitled “leucocytic exudates,” which I have elsewhere stated it is impossible to repeat—a statement which has not been challenged by Bordet.'? The result obtained by him in one experiment would appear to be purely accidental."! But it is to no purpose to discuss here, point by point, the different theories; it is sufficient to show that they are contrary to the facts. Hypotheses 1 and 2 being eliminated, there only remain to consider hypotheses 3 and 4. SeconD Fact: The lytic enzymes emanate from material corpuscles which traverse filters; these corpuscles multiply in the course of the bacteriolysis. EXPERIMENT A. In a well-developed bacterial culture, showing a marked turbidity, let us add a small quantity (say 1/10,000,000 c.cm.) of a filtrate containing the bacteriophagic principle, shake, withdraw 1/20 c.cm. and spread it over the surface of an agar slope. Thus we spread over the agar a great number of bacteria and a very small quantity of bacteriophagic principle. After incubation (eighteen to twenty-four hours at 37°) we note that the surface of the agar shows a bacterial growth with a certain number of circular bare spaces here and there, where the agar presents no trace of growth. These vacant spaces once formed are unchangeable; they never spread, and they are never invaded by the surrounding culture. It is the presence of these immutable bare spaces, which are perfectly circular, that characterizes what we have named “bacteriophage.” Before concluding that any bacteriolytic phenomenon is bacteriophagic, it becomes necessary to verify if it gives rise to such bare spaces on agar culture. If not, the bacteriolysis is not of bacteriophagic nature. The number of spaces depends simply on the quantity of filtrate added to the bacterial culture. If into various bacterial emulsions we introduce variable quantities of the filtrate, the number of bare spaces is strictly proportional to the quantity of filtrate added. On the other hand, the number of the bare spaces is independent of the number of bacteria contained in the medium; whether one introduces a given quantity of filtrate into a bacterial emulsion containing one hundred millions or ten billions of bacteria per cubic centi- metre, the number of bare spaces on the agar is practically always the same. The phenomenon of these vacant spaces is only comprehensible on the Auc. 19, 1922 | THE BACTERIOPHAGE. Tue Barrisn Mepicar JourNAL supposition that the bacteriophagic principle is of corpuscular nature. The following experiments give the proof thereof. EXPERIMENT B. Given a filtrate containing the bacteriophagic principle, let us determine its force in the production of bare spaces. It is well known that to determine the number of living bacteria contained in an emulsion agar plates are inoculated with a dilution of this emulsion; after incuba- tion, the number of colonies which develop multiplied by the titre of the dilution gives the number of bacteria per cubic centimetre of the primitive emulsion. The enumeration of the bacteriophagic corpuscles contained in a filtrate is estimated in exactly the same way. But as the bacteriophage only grows at the expense of living bacteria, we must make the dilutions of the filtrate in a bacterial emulsion. For this we pipette over agar the bacterial emulsion containing a given quantity of filtrate: after incubation we obtain a bacterial layer strewn with circular bare spaces, each of these spaces being a colony of bacteriophage issued from one corpuscle. The number of bare spaces multiplied by the titre of the dilution gives the number of ultramicroscopic bacteriophagic corpuscles contained in 1 c.cm. of the primitive filtrate. This experiment shows that the behaviour of the bacteriophage is exactly the same as that of any ordinary microbe. But this last develops at the expense of the nutritive substances contained in the medium; the bacteriophage develops at the expense of the bacterial bodies which constitute its nutritive medium. The bare spaces represent places cleared up by the growth of the ultramicroscopic bacteriophagic corpuscles. EXPERIMENT C. Now dilute a filtrate so as to obtain a dilution such that 1 c.cm. contains one bacteriophagic corpuscle. Dilute this 1 c.cm. with 9 c.cm. of sterile water, and inoculate ten tubes of bacterial emulsion each with 1 c.cm. It will be obvious that only one of the ten tubes will contain the generator of a bare space; the nine others will not contain any. Place the ten tubes in the incubator at 37° for twenty-four to forty-eight hours; it will be seen that only one of the ten microbial emulsions shows bacteriolysis; the nine others will remain unchanged, the bacteria remain liv- ing, normal, and subculturable. The lytic action, therefore, is complete when only one generator of a bare space is introduced into a bacterial emulsion; the action is nil in the contrary case. This experiment can only be explained on the supposition that the bactericphagic principle, the source of the lytic enzymes, is a corpuscle; and that each corpuscle deposited on the agar in the midst of the bacteria gives rise to a coiony of these ultramicroscopic corpuscles, such a colony being repre- sented by a bare space. EXPERIMENT D. Inoculate a bacterial emulsion with a bacteriophagic filtrate of known corpuscular strength (that is, of known bare spaces forming force); make agar slopes every hour in the same way as in Experiment A. In this way, after six hours, we will have six agar slopes inoculated, and, on incubation, it will be seen that the number of bacteriophagic spaces (each bare space corresponding to a corpuscle) increases in proportion as the lytic action progresses in the emulsion. The maximum number of bare spaces is given when the lysis of the original emulsion is complete; at this Aue. 19, 1922 | SECTION OF MICROBIOLOGY. [este Basase Mepicar JourNAL moment there are no longer any living bacteria in the emulsion—it is a pure culture of the ultramicroscopic bacteriophagic corpuscles. The bacteriophagic power of such a lysed culture remains at its maximum level during many weeks—it is practically “fi > xed. The foregoing experiments show that the lytic enzymes are originated by ultramicroscopic corpuscles which multiply and reproduce themselves. Hence those hypotheses explaining the phenomenon as being due to a soluble auto- lytic enzyme become eliminated. Of all the hypotheses put forward, only that of Bail, and perhaps that of Otto and Winkler, could not be incompatible with these experiments, since they suppose that the element which secretes the lytic enzymes can be furnished by ultra-microscopic corpuscles, but derived from the bacteria. It is difficult to reconcile these hypotheses with the fact that the bacteriophagic action is not specific, and that the same strain of bacteriophage can dissolve bacteria of different species. Indeed, if one can understand how a principle originated by a species of bacteria can provoke the formation of a similar principle in a culture of the same bacterial species, it is hard to admit how this principle could provoke the formation of the bacteriophagic principle in a culture of a different bacterial species. Besides, these two hypotheses are rendered untenable by the following fact. Tuirp Facr: All bacteriophagic ultramicroscopic corpuscles, grown at the expense of any bacterial species, constitute one and the same antigen. I have up to the present isolated various strains of bacteriophage active towards different bacteria: B.typhosus, B. paratyphosus A and B, B. dysen- teriae (Shiga, Flexner, Hiss), B. coli, B. pestis, B. proteus, B. gallinarum, bac- teria of haemorrhagic septicaemias, staphylococcus, etc. We have seen above what constitutes a culture of the ultramicroscopic bacteriophagic corpuscles. Inoculate an emulsion of B. dysenteriae with a filtrate containing an active bacteriophage towards this bacterium; after a few hours the medium becomes limpid, the bacilli are dissolved, and the bacterio- phage corpuscles have multiplied; the culture of the bacteria has become a culture of antidysenteric bacteriophage. Similarly, a culture of B. pestis dissolved under the action of the bacteriophagic principle (isolated from the intestinal contents of a convalescent from bubonic plague) becomes a culture of antipestic bacteriophage. And so on for any other microbe. One knows that the serum of a rabbit prepared by injections of B. dysen- teriae contains an amboceptor towards B. dysenteriae, but does not contain any amboceptor towards any other bacteria—for example, B. pestis. Now prepare a rabbit with a culture of B. dysenteriae dissolved by bac- teriophagic action; one verifies that its serum does not contain any amboceptor towards normal B. pestis, but contains an amboceptor for B. pestis dissolved by the action of the bacteriophage. And this is true for no matter what bac- terial species; the serum of an animal prepared by a culture of any bacterial species dissolved by bacteriophage contains an amboceptor which fixes itself on any other bacteriophaged culture. Hence the amboceptor is specifically Auc. 19, 1922 | THE BACTERIOPHAGE. ee Mepicat JourNAL antibacteriophagic, and not antibacterial. Therefore the bacteriophage is an autonomous antigen, and consequently must be considered, in the present state of knowledge, as a definite ultramicroscopic virus constituted by cor- puscles, such a virus being necessarily an ultramicrobe. Only the hypothesis of a filtrable micro-organism which is an obligatory parasite of bacteria is compatible with the foregoing facts. It now remains to show that this only admissible hypothesis is not contradicted by any experi- mental fact. FourtH Fact: The ultramicroscopic corpuscles possess a variable virulence. We have seen that the above ultramicroscopic organism, which we have designated by the name of “bacteriophage,” is constituted by living corpuscles which multiply and which it is possible to count. Now, experience shows that the lytic activity of these corpuscles varies from one strain to another. For instance, certain strains recently isolated from the organism are unable to provoke the dissolution of a bacterial emulsion, and no matter what number of corpuscles are inoculated into this bacterial emulsion, one can only perceive the presence of these bacteriophagic corpuscles by the production of pin-point bare spaces on agar. On the contrary, with very active strains giving on agar large bare spaces (4 to 5 mm. in diameter), the inoculation of one corpuscle is sufficient to provoke a total dissolution of all the bacteria contained in the emulsion. It is easy to prove that this difference in action is due to a difference in the multiplication of the corpuscles inoculated. In the case of feeble strains reproduction is slow; in the case of strong strains it is rapid. The active particle, therefore, is endowed with a variable virulence, the term “virulence” being taken in its real bacteriological sense of “vegetative power in vivo.” To explain this fact one must consider the active corpuscle as a microbe, because only a microbe possesses a variable virulence from strain to strain. Moreover, an enzyme only acts by its quantity; a microbe by its virulence and its toxicity combined. Firtu Facr: By successive passages, it is possible to increase the virulence of a feeble strain of bacteriophage. For instance, inoculate a feeble strain of bacteriophage into a culture of B. typhosus; filter the mixture after incubation; introduce a drop of this filtrate into a fresh culture of B. typhosus, which is filtered in turn, and so on for a few passages. The virulence is found to increase little by little, and after a certain number of passages the virulence becomes such that a trace of the filtrate is sufficient to provoke the total dissolution of a fresh emulsion of this bacillus. The technique of the exaltation of virulence is really the same as for any microbe: we exalt the virulence of a bacterium for a given animal by serial passages in this animal species; we exalt the virulence of the bacteriophage for a given bacterial species by serial passages in this bacterium. This fact is only compatible with the hypothesis that bacteriophage is a microbial parasite, since adaptation is the prerogative of living beings. Ave. 19, 1922 | SECTION OF MICROBIOLOGY. lee Beer Mepicat JourNAL SixtH Facr: Experience shows that bacteria attacked by bacteriophage do not remain passive; they defend themselves and are even capable, under certain conditions, of acquiring an immunity towards the parasite. This defence of the bacteria manifests itself by a double mechanism: the bacteria surround themselves with a capsule, and secrete “aggressines” capable of paralysing the lytic enzymes of the bacteriophage. Only the hypothesis of bacteriophage being a parasite of bacteria conforms with these facts. SEVENTH Facr: The behaviour of bacteriophage towards physical and chemical reagents is that of a living being, and does not agree with that of an enzyme. The resistance of bacteriophage to the action of physical and chemical reagents is intermediate between that of the vegetative and the spore forms of ordinary bacteria. The resistance is less than that of certain ultramicrobes, notably that of the tobacco mosaic. It is destroyed at 74°-75°C., but ceases to develop at 43°. Between 43° and 74° it possesses no lytic action. As to the action of the antiseptics, the bacteriophage is killed after twenty-four hours’ contact with a 1 per cent. solution of a neutral salt of quinine, after forty-eight hours’ contact with 95 per cent. alcohol, and by eight days’ contact with glycerin. The last-named liquid is precisely the medium employed for preserving indefinitely an enzyme in soluble form. Ercutu Fact: It is possible to extract the lytic enzymes free from the living bacteriophage mirco-organism. If to one part of a culture of bacteriophage is added nine parts of absolute alcohol and the mixture left forty-eight hours in contact, a precipitate is obtained which contains bacteriolytic enzymes, while the bacteriophagic germs are killed, which is proved by the failure of transmissibility in series of the phenomenon, One can only understand this fact on the supposition of an ultramicroscopic microbial parasite: the bacteria are dissolved by enzymes secreted by the living ultramicroscopic germs, hence the action in series. In the above experiment the ultramicroscopic germs are killed, while the enzymes already secreted are preserved intact; hence only a lytic action is obtained which is not transmissible in series. Nintu Fact: Bacteriophage is capable of adaptation. The bacteriophage is very sensitive to the action of acids—much more sensitive than ordinary microbes. It can be accustomed progressively to live in media containing quantities of an acid which would have been destructive beforehand. The same fact is observed with glycerin. Adaptation is a preroga- tive of living things. Tentu Facr: The properties of bacteriophage are essentially variable. It is impossible to isolate two strains of bacteriophage which are absolutely identical as to their range of action on different bacterial species, and as to their intensity of action towards each of these bacterial species. For a given 10 Aue. 19, 1922 | THE BACTERIOPHAGE. ~ Tue Britisu Mepicar JouRNAL strain of bacteriophage one can vary experimentally this intensity. Variability is an essential characteristic of life. We have seen, first, that of all the possible hypotheses concerning the nature of the bacteriophage, the hypothesis of an ultramicroscopic parasite of bacteria is alone admissible. Moreover, we have just seen that this hypothesis is not contradicted by any of the facts of bacteriophagic phenomena; and, furthermore, all of the facts cannot be explained if this hypothesis is aban- doned or modified. It follows logically, therefore, that such a hypothesis be- comes a certitude. Further, I would remark that I do not specify in any way the species to which the ultramicroscopic organism, which I have given the name of Bac- teriophagum intestinale, belongs; the name simply recalls its characteristic property and the place where it was at first found. Is it a protozoon, a fungus, a bacterium? Does it belong to a kingdom which is neither the vegetable nor the animal—a still simpler form of life than any which we at present know? These are questions which cannot at present be answered. All that we know of it is that it is an ultramicroscopic organism, a filtrable being, parasite of bacteria, endowed with functions of assimilation and reproduction—functions which characterize the living nature of the being which possesses these proper- ties. That is all that experiment actually shows us.* The foregoing discussion can only apply to the phenomenon of bacteriolysis in series presenting the special characters that I have indicated, This being so, a final question arises—that of the plurality of the serial phenomena. In 1915 Twort'? described a bacterial transformation taking place in series, under the influence of a principle which passes through porcelain filters. His researches have been made chiefly with a micrococcus isolated from vaccine. In addition he observed, an identical phenomenon with two other bacteria—a large indeterminate bacillus, and a bacillus of the coli-typhoid group. Twort isolated the active principle in the following manner. “Some interesting results, however, were obtained with cultivations from glycerinated calf vaccinia. Inoculated agar tubes, after twenty-four hours at 37°, often showed watery-looking areas, and in cultures that grow micrococci it was found that some of these colonies could not be subcultured, but if kept became glassy and transparent. On examination of these glassy areas nothing but minute granules, staining reddish with Giemsa, could be seen.” The transforming principle is contained in this transparent material. “The transparent material when diluted (one in a million) with water or saline was found to pass the finest porcelain filters with ease, and one drop of the filtrate, pipetted over an agar tube, was sufficient to make that tube unsuitable for the growth of the micrococcus. That is, if the micrococcus was inoculated down the tube as a streak, this would start to grow, but would soon become dotted with transparent points which would rapidly extend over the whole growth. If in an infected tube * T will mention, in passing, the hypothesis of Salimbeni: the enzymes would be secreted by a myxobacteria, microscopically and even macroscopically visible, given filtrable spores. All the workers who have studied the question have failed to observe this would-be myxobacteria, which besides, according to the description of Salimbeni, presents a mycelium (2). That can only mean an impurity, as I have elsewhere pointed out. 11 Aue. 19, 1922 | SECTION OF MICROBIOLOGY. Exc eee Mepicat Journat small areas of micrococci are left—and this usually happens when the microcoecus has grown well before being infected—these areas will start to grow again and extend over the transparent portion. .. .” According to this description of Twort, it is not a question of a real bac- terial dissolution, but a transformation of a normal culture on agar into a glassy and transparent one. This phenomenon is totally different from that produced by Bacteriophagum intestinale. Indeed, under the influence of this latter—no matter what bacterial species is dealt with, no matter what virulence, feeble or strong, of the strain of the bacteriophage, operating exactly in the same experimental conditions as Twort—in no circumstances does one observe the formation of transparent material or anything presenting such an appear- ance. Pipette over an agar tube a drop of a diluted filtrate containing a bacterio- phage active towards staphylococcus; then inoculate this tube as a streak with a pure culture of staphylococcus; after incubation we obtain a surface growth of the organism, which macroscopically, microscopically, and biologically is normal. This normal bacterial culture is strewn here and there with circular clear spaces; in the interior of these spaces the agar is bare, without any visible trace of any material. These bare spaces never spread over the sur- rounding culture, even after months in the incubator. Furthermore, they never become invaded by the surrounding bacterial culture: once formed, the bare spaces remain immutable. Confusion, therefore, between the phenomenon observed by Twort and the phenomenon provoked by the bacteriophage is in no way possible. What is the nature of the principle which acts in the phenomenon of bac- terial transformation observed by Twort? From a consideration of the observa- tions made on this phenomenon, and in view of the fact that the lytic agent and the bacteria are destroyed at the same temperature, it is probable that it is derived from the bacterium itself, which is capable of splitting up into fragments. Twort himself favours this view, but further experiments will be necessary to settle this question. This conclusion, however, does not detract in any way from the interest attaching to the important researches of Twort. One can already see that the phenomenon observed by this author may play an important role in the etiology of the so-called filtrable virus diseases, as he indeed seems to have foreseen. On the other hand, the bacteriophage undoubtedly plays a part in the defence of the organism in the course of infectious diseases, as I have shown elsewhere. From a survey of the results already arrived at in the domain of scientific investigation, it is easy to foresee that there exists a whole series of phenomena, quite unsuspected a few years ago, whose study should play a large part in the advance of scientific medicine. REFERENCES. 1For details see: Le Bactériophage, son role dans limmunité, in the collection, Mono- graphs of the Pasteur Institute (Masson, Paris), of which an English edition is in prepara- tion (Williams and Wilkins, Baltimore, publishers). ?Sur un ferment d’immunité bactériol- 12 Aue. 19, 1922 | THE BACTERIOPHAGE. Tue Brirtisu Mepicat JourNAL ysant, C.R. Soc. Biologie, \xxxiii, February 28th, 1920, p. 219. ‘Le bactériophage de dHerelle, sa reproduction, son interprétation, C.R. Soc. Biologie, |xxxiii, October 9th, 1920, p. 1296. 4On the influence of the tissue enzymes on the bacteriophage principle, Proceed. Exp. Biol. and Med., xviii, April 20th, 1 21, p. 222. 5Antagonisme microbien et lyse trans- missible, C.R. Soc. Biologie, Ixxxvi, March 18th, 1922, p. 569 ®Autobactériolysne et le phénoméne de dHerelle, C.R. Soc.Biologie, \xxxvi, April 29th, 1922, p. 833. 7Bakerio- phagen Wirkungen gegen Flexner- und Koli-Bakterien, Wien. klin. Woch., xxxiv, No. 37, September 15th, 1921. SUber die Natur des d’Herelle’schen Bakteriophagen, Deut. med. Woch., No. 21, 1922. Sur un microbe invisible, antagoniste des bacilles dysentériques, C.R. Acad. des Sciences, clxv, September 10th, 1917, p. 373. 1°Exsudats leucocytaires et autolyse microbienne transmissible, C.R. Soc. Biologie, |xxxiii, October 9th, 1920, p. 1293. N]T/ultramicrobe bactériophage, C.R. Soc. Biologie, October 29th, 1921, p. 721. 12An investigation on the nature of ultramicroscopic viruses, Lancet, December 4th, 1915. Il—_THE BACTERIOPHAGE: THE BREAKING DOWN OF BACTERIA BY ASSOCIATED FILTER- PASSING LYSINS. By F. W. Twort, M.R.C:S., L.R.C.P. (From the Laboratories of the Brown Institution, London. ) Tuts phenomenon of bacteriolysis, which in France has been called the “bac- teriophage,” consists, as you know, of a breaking down and dissolving up of bacteria by a filter-passing material which in certain circumstances may be associated with pure cultures. My first experiments were carried out at the Brown Institution during 1914 and 1915, for the Local Government Board, and the results were published in the Lancet (December 4th, 1915) under the title of “An investigation on the nature of ultramicroscopic viruses.” The con- dition, in my opinion, is distinct from the various degenerative changes which have been so often described in bacterial cultures, and it will be necessary for me to consider in detail experiments on degenerative changes. It is also impossible in the time at my disposal to discuss every aspect of the subject; moreover, other speakers will no doubt deal with the many interesting experi- ments which they have carried out. In this paper I propose to give you as shortly as possible my original experiments as they were published in 1915, and I shall pass on to consider certain aspects of the subject which have since become points of controversy between different workers, and shall then deal shortly with certain more recent experiments which I have been doing on the biology of bacteria, as these may throw some light on the use of the lytic material. When starting the research my object was to discover, if possible, the na- ture and life-history of the ultramicroscopic group of viruses. The experiments were carried out with these views in my mind: In the first place we do not know for certain the nature of an ultramicroscopic virus. It may be a minute bacterium that will grow only on living material, or it may be a tiny amoeba which, like ordinary amoebae, thrives on living bacteria or animal tissue. On the other hand, it must be remembered that if the living organic world has 13 Aue. 19, 1922 | SECTION OF MICROBIOLOGY. [ ae eae Mepicat JouRNAL been built up slowly in accordance with the theories of evolution, then an amoeba and a bacterium must be recognized as highly developed organisms in comparison with much more primitive forms which once existed, and probably still exist, in nature. It is quite possible that an ultramicroscopic virus belongs somewhere in this vast field of life where the organization is lower than that of the bacterium or amoeba. It may be living protoplasm that forms no definite individuals, or an enzyme with power of growth. In the first instance attempts were made to demonstrate the presence of non-pathogenic filter-passing viruses. As is well known, in the case of ordinary bacteria, for every pathogenic micro-organism discovered many non-patho- genic varieties of the same type have been found in nature, and it seems highly probable that the same rule will be found to hold good in the case of ultra- microscopic viruses. It is difficult, however, to obtain proof of their existence, as pathogenicity is the only definite evidence we have at the present time of the presence of an ultramicroscopic virus. On the other hand, it seems probable that if non-pathogenic varieties exist in nature these should be more easily cultivated than the pathogenic varieties. The first experiments were carried out with such materials as soil, dung, grass, hay, straw, and with water from ponds. Cultivations were made on a large number of special media and under special conditions. Experiments were also carried out with pathological material obtained from distemper in dogs, from vaccinia, and from various other sources. The first results of interest were obtained with vaccinia. Inoculations were made on to ordinary agar tubes, and on to tubes of special egg media such as I used in my experi- ments for the cultivation of Johnes’s bacillus and the lepra bacilli of man and of rats. It will be impossible to describe all these in detail, but the essential part of one series consists in the incorporation in the media of the dead bodies of certain acid-fast bacilli such as Bacillus phlei, which proved to be so success- ful in the cultivation of Johnes’s bacillus. The egg media usually grew a number of colonies of micrococci and diphtheroids, while the agar tubes grew only a few colonies of micrococci. In the case of the egg media it was noted that in a few days certain parts of the micrococcus growth sometimes became dull, and in appearance rather resembled bacterial growth in which amoebae were also growing. On the agar media the colonies of micrococci ocassionally showed a translucent or transparent change, which started as more or less clear spots at the margins of the colonies. It was also found that some of these colonies could not be subcultured, but if kept the transparent change extended over most of the colony. On examination of these glassy areas nothing but minute granules, staining reddish with Giemsa, could be seen. Further experiments showed that if a colony of the white micrococcus that had started to become transparent was plated out instead of being subcultured as a streak, then the micrococcus grew and a pure streak culture from certain of these colonies could be obtained. On the other hand, if the plate cultures (made by inoculating the condensation 14 Tue Britisu Aue. 19, 1922 | THE BACTERIOPHAGE. eases JourNAL water of a series of tubes and floating this over the surface of the medium) were left, the colonies, especially in the first dilution, soon started to turn transparent, and the micrococci were replaced by fine granules. This action, unlike an ordinary degenerative process, started from the edge of the colonies; and further experiments showed that when a pure culture of the white or the yellow micrococcus isolated from vaccinia is touched with a small portion of one of the glassy colonies the growth at the point touched soon starts to become transparent or glassy, sometimes killing out all the micrococci and replacing these by fine granules. Experiments showed that the action is more rapid and complete with vigorous growing young cultures than with old ones, and there is very little action on dead cultures or on young cultures that have been killed by heating to 60°C. Anaerobia does not favour the action, although it appears best in tubes that are capped with gutta-percha tissue. The transparent growth when diluted (one in a million) with water or saline was found to pass the finest porcelain filters (Pasteur-Chamberland F. and B. and Doulton White) with ease, and one drop of the filtrate pipetted over an agar tube was sufficient to make that tube unsuitable for the growth of the micrococcus—that is, if the micrococcus was inoculated down the tube as a streak, this would start to grow, but would soon become dotted with transparent points which would rapidly extend over most of the growth. The number of points from which this starts depends upon the dilution of the transparent material, and in some cases it is so active that the growth is stopped and turned transparent almost directly it starts. This condition or disease of the micrococcus when transmitted to pure cultures of the micrococcus can be conveyed to fresh cultures for an indefinite number of generations; but the transparent material will not grow by itself on any medium. If in an infected tube small areas of micrococci are left—and this usually happens when the micrococcus has grown well before becoming infected— these areas will start to grow again and extend over the transparent portions, which shows that the action of the transparent material is stopped or hindered in an overgrown tube; but it is not dead, for if a minute portion be transferred to another young culture of the micrococcus it soon starts to dissolve up the micrococci again. Although the transparent material shows no evidence of growth when placed on a fresh agar tube without micrococci, it will retain its power of activity for over six months. It also retains its activity when made into an emulsion and heated to 52°C., but when heated to 60°C. for an hour it appears to be destroyed. It has some action, but very much less on Staphylococcus aureus and albus isolated from boils of man, and it appears to have no action on members of the coli group or on streptococci, tuberc’e bacilli, yeasts, etc. The transparent material was inoculated into various animals, and was rubbed into the scratched skin of guinea-pigs, rabbits, a calf, a monkey, and a man, but all the results were negative. When continuing my investigation of infantile diarrhoea and vomiting, for 15 Tue Britisu Aue. 19, 1922 | SECTION OF MICROBIOLOGY. ixcastedeaT eee the Local Government Board in 1915, similar experiments were carried out with material obtained from the intestinal tract. After certain difficulties had been overcome it was found that in the upper third of the intestine, which contained numerous bacilli of the colon group, some larger forms were also present. In some cases these grew in far greater number than the ordinary types of bac- teria, but this was only so when precautions were taken to eliminate the action of a dissolving substance which infected the colonies so rapidly that they were dissolved before attaining a size visible to the eye. Here, then, was a similar condition to that found in vaccinia, and the greatest difficulty was experienced in obtaining the bacilli free from the transparent dissolving material, so rapidly was the infection increased and carried from one colony to another. At first this bacillus was not believed to be a member of the dysentery-typhoid-coli group, but I have now no doubt that it was a special large form of a member of this group. Unfortunately I lost my cultures during the war before all the characters of the bacillus were completely worked out. Similar though not such definite results were also obtained with a micro- coccus and a member of the coli-typhoid group of bacilli which were obtained from the intestinal mucous membrane of a dog suffering from acute distemper, and I obtained some evidence that the difficulty often experienced in isolating certain known pathogenic micro-organisms might be due to the same cause. In my paper I also pointed out that results similar to those obtained with vaccinia and with bacilli from the intestinal tract would probably be obtained in cases of true dysentery; but I was unable at the time to investigate dysen- tery, as my scheme for research on this disease which was submitted to the War Office in 1914 was not accepted. Shortly after the publication of my work, however, I went to Salonica in charge of the base laboratory. While there in 1916 the subject was again discussed on several occasions with the English, French, and Canadian bacteriologists; but at the time the phenom- enon was not accepted as one of much importance, and my fresh proposals for continuing the work on dysentery failed to meet with approval. As, how- ever, I had predicted, a similar condition was later found to occur in cultures of true dysentery bacilli, particularly by Dr. d’Herelle, who carried out a series of important experiments on these bacilli. Dr. d’Herelle’s published researches appear to me to confirm in the main my own results with micrococci and members of the coliform group of bacilli. I will now pass on to consider the conclusions I drew from my experiments. In the case of vaccinia it is clear that the transparent material contains an enzyme, and this is destroyed on heating to 60°C. for one hour. It also increases in quantity when placed on an agar tube containing the micrococcus, and this can be carried on indefinitely from generation to generation. If it is part of the micrococcus it may be either a stage in its life-history which will not grow on ordinary media but stimulates fresh cultures of the micrococcus to pass into the same stage, or an enzyme secreted by the micrococcus which leads to its own destruction and the production of more enzyme. The fact that 16 Ave. 19, 1922 | THE BACTERIOPHAGE. Seteeecoracoe the transparent portion cannot be grown except on the micrococcus makes it impossible to obtain any definite evidence on these points. There is this, however, against the idea of a separate form of life: if the white micrococcus is repeatedly plated out a colony from the last plates may give a good white growth for months when subcultured at intervals on fresh tubes; eventually, however, most pure strains show a transparent spot, and from this the trans- parent material can be obtained once again. Of course, it may be that the micrococcus was never quite free from the transparent material, or this may have passed through the cotton-wool plug and contaminated the micrococcus, but it seems much more probable that the material was produced by the micro- coccus. Incidentally, this apparent spontaneous production of a self-destroying material which, when started, increases in quantity, may be of interest in connexion with cancers. In any case, whatever explanation is accepted, I do not think my experiments definitely disproved the possibility of its being an ultramicroscopic virus, because we do not know for certain the nature of such a virus. If the transparent portion were a separate virus, it might be vaccinia, or it might be some contaminating non-pathogenic ultramicroscopic virus, for it is conceivable that whereas a non-pathogenic variety might grow on micro- cocci or bacilli, a pathogenic variety might grow only in the animal it infects. As the animal experiments were negative, there is no evidence that it is vaccinia, although such a virus might lose its virulence when grown outside the body. On the other hand, no evidence was obtained that it was a non- pathogenic variety. On the whole it seems probable, though by no means certain, that the active lytic material is produced by the micrococcus, and since it leads to its own destruction and can be transmitted to fresh healthy cultures, it might almost be considered as an acute infectious disease of micro- cocci. I have now described my original experiments and conclusions, and I will pass on to consider the views of other workers in this field. Where differences of opinion exist the controversy has centred chiefly round the experiments that have been carried out to determine the source and nature of the lytic material. I have already pointed out that when this material is diluted and filtered and pipetted on to a tube of medium just before inoculating the tube with the micro-organism to be lysed, then the action starts at definite points throughout the growth. Dr. d’Herelle obtained a similar result in his experi- ments with dysentery bacilli, and he considers this a strong point in favour of the view that the lytic material is a separate living micro-organism. I can- not, however, for several reasons, agree that such experiments prove this. First, it is well known, notably in the case of certain starches, that the diastatic enzymes do not in every case dissolve up the grains evenly, but start at certain points, causing pitting and erosion of the grains; at the same time some grains are more susceptible to the enzyme than others. In this case no one has sug- gested that the unevenness of the action proves the diastatic enzyme to contain a living ultramicroscopic virus. I have noted also with bacteria that not only 17 Aue. 19, 1922 | SECTION OF MICROBIOLOGY. [ieee oes Mepicar JouRNAL does the lytic agent attack some members of a culture before it attacks others, but individual members may be pitted and eroded, and particularly is this so with certain large forms of dysentery bacilli which I shall consider later. Again, it is well known in the case of all bacteria that certain members of a culture are more resistant to chemicals and to specific lysins produced in animals, and it may well be that the lytic action on the bacteria starts from a number of distinct points because these points happen to contain specially susceptible micro-organisms, and it is only when the action is started and the lytic sub- stance increases in quantity and in concentration that the more resistant members become lysed. Then there are the very interesting experiments of Professor Bordet and Dr. Ciuca. These workers found that when a lysin was produced by an animal against a coliform bacillus, this lysin not only dissolved up fresh cultures of the coliform bacillus, but the lytic effect could be transmitted from culture to cul- ture. These results certainly do not favour the view that the lytic agent is a definite living micro-organism. Moreover, there are my own original experi- ments where, after obtaining normal growths of micrococci for a number of generations, eventually some fresh subcultures started to become lysed, apparently spontaneously. But these experiments appear to me to be evidence not only against the view that the lytic material is a definite living organism, but also against the view of Bordet and Ciuca that the lytic agent arises from an association of the bacterium with cells from the animal body. dHerelle has also obtained the lytic agent by associating bacteria with filtrates of soil, etc., and I agree with him that these experiments do not support Bordet’s view, although at the same time I do not think they support the view that the lytic agent is a definite living micro-organism. The apparent spontaneous pro- duction of the lytic agent in some of my pure cultures is, I think, evidence against both the views mentioned. It may, of course, be argued, as I men- tioned at the time, that my cultures were never really quite free from the lytic agent, but if this view is accepted regarding my repeatedly plated cultures, then it is reasonable to suggest the same regarding the cultures used by dHerelle and by Bordet. Recent experiments, in fact, have in no way changed my views, and I repeat my original opinion regarding the lytic agent of the micrococcus—namely, that “the possibility of its being an ultramicroscopic virus has not been definitely disproved,” and that “it seems probable, though by no means certain, that the active transparent material is produced by the micrococcus”; and I hold the same view regarding the lytic agent which various workers and myself have found associated with the dysentery-typhoid-coli group of bacilli. However, as I have already pointed out, it is just possible that an ultra- microscopic virus may be of the nature of an enzyme, and if so, the original source of such a virus might be the cell it infects: in remote ages possibly a normal enzyme which has gradually developed to take on a pathological action as it has passed through an infinite number of generations of cells, either 18 [ Tue Britisy Mepicat JourNAL Aue. 19, 1922 | THE BACTERIOPHAGE. bacterial, vegetable, or animal, according to its source, and which possesses the power either of directly increasing in quantity or of stimulating the cell to produce more pathological enzyme. On the other hand, it is conceivable that if this lytic agent be a pathological enzyme, it might bear the same relation to the normal enzyme that the cancer cell does to the normal cell. These possibilities may sound very improbable, but at the same time some such explanation would account for the extremely specific nature of many of the ultramicroscopic viruses, and it would certainly explain the absence of visibility and of growth on artificial media. After all, excluding such organisms as the lepto-spirillum of yellow fever, which probably belongs to an entirely different group, there are fundamental differences between the smallest micro- organisms known and the various ultramicroscopic viruses. Cultivations of the minutest micro-organisms known show all the charac- teristics of ordinary bacteria. If these are obtained from the soil many of them grow well. On the other hand, if ultramicroscopic viruses are simply more minute members of the group of bacteria, there must be thousands of wild varieties in the soil, and yet no one has succeeded in obtaining a definite growth of a single variety on any solid medium. Minuteness of size might account for their being invisible, but it will not explain the absence of visible growth on artificial solid media. It is true that certain workers at one time claimed to have obtained growths of pathogenic varieties in Noguchi’s medium, but in the absence of definite confirmation these experiments need not be considered here. Professor Bordet and Dr. Ciuca have carried out some most important experiments with the dysentery-typhoid-coli group of bacilli on the specificity of the lytic material, and have obtained results which indicate not only that the lytic agent can be made to break down allied bacilli, but also that certain resisting and otherwise changed strains can be obtained. As I have already mentioned, my micrococcus lysin had little effect on Staphylococcus aureus; but I did not carry out many experiments of this nature after obtaining the apparent spontaneous production of the lytic agent in my pure cultures of micrococci, as this result appeared to me to make it difficult to draw definite conclusions from such experiments. Many other workers, but particularly Dr. dHerelle and Dr. André Gratia, have also carried out important experiments in this branch of the subject, but I must leave other speakers to deal with it, as it is impossible in the space at my disposal to do justice to their work. There is, however, another aspect of the subject which I should like to mention. As is well known, in pure cultures of such bacilli as dysentery, typhoid, and coli, one sometimes meets with forms which are considerably larger and longer than the average bacillus, and these may be found in patho- logical material containing these bacilli, being not uncommon in urine in cases of cystitis. Hort and others have described these large forms in considerable detail. In certain experiments dealing with the lytic material I observed these long 19 Tue Britisu Ave. 19, 1922 | SECTION OF MICROBIOLOGY. [ser ieee forms in greater number than usual, and it was thought that they might in some way be connected with the lytic agent associated with this group of bacilli. There was also the possibility that they might be distinct but symbiotic bacilli growing only with the dysentery or other bacilli forming the culture; they might be mutations, or, again, they might be special forms with special work to do, like bees in a swarm, and, alone or in association with the lytic agent, might prove to be of importance in connexion with the pathogenicity of cultures and the production of immunity in the host. The work was interrupted by the war, but eventually the results were published in the British Journal of Experimental Medicine (October, 1920), and I shall do no more now than consider their possible relation to the lytic agent. In the first place, it must be noted that these large forms occur much, more frequently in pathological material than they do in cultures on artificial media. In cases of infantile diarrhoea and vomiting I have found them in large number associated with a lytic agent in the upper part of the intestinal tract, and it is only after the action of the lytic agent is eliminated, that they can be easily cultivated. The nature of the bacilli from such conditions is now being investigated. Most of my experiments were carried out with the Shiga type of dysentery bacillus. The first point of importance to be noted is that the large or “special forms” occur in all pure cultures of bacilli belonging to the dysentery-typhoid- coli group, besides occurring in cultures of the influenza bacillus and other micro-organisms. Further, they are much more numerous in very young cul- tures than in older ones, and practically disappear in cultures that are twenty- four hours old. I observed also, in cultures that had been growing for six to twelve hours, that these “special forms” were often, partially dissolved. From these early experiments I concluded that the bacilli normally produce “special forms,” and that these, when presumably of no use to the bacterial community of a pure culture, are dissolved by a lytic agent which is also present, and that this lysin prevents the special forms from multiplying, and interferes with their isolation as growths free from the normal small bacilli. I found, however, two methods by which these “special forms” could be isolated. They may be obtained either by repeatedly plating out on litmus-maltose agar tubes, or by growing in an emulsion of dead coliform, typhoid, or some similar bacillus, and then plating out on the maltose agar medium. Three fairly distinct and stable types were isolated, but it will be unnecessary to consider these in detail. The chief points which I wish to bring to your notice are these: The large bacilli proved to be “special forms” of the bacterium from which they were obtained, and were easily agglutinated by the specific serums. I also obtained some evidence that they were more pathogenic when produced by the normal bacilli than after repeated multiplication by division. When isolated and grown they became more resistant to the lytic agent. In most of the cultures numerous free granules were present, while many of the bacilli contained similar looking granules in the fusiform and round swelling which they presented. 20 Aue. 19, 1922 | THE BACTERIOPHAGE. eee Mepicat JourNAL These researches seem to indicate another possible explanation of the lytic action of the substance under review. If this substance is, after all, not patho- logical to the bacterium, but is a normal product of its activity, then it must be produced for some special purpose which is advantageous to the life of that variety of bacterium. If the “special forms” of bacilli which are found in normal cultures and are so soon dissolved up are produced for the benefit of the bacterial community of a pure culture, then it may be that the lytic agent is formed for the purpose of carrying out this lytic action, and possibly setting free toxins contained in the special forms. Or it may be that it sets free “anti- toxins,” or substances that will neutralize the toxic substances of the host it infects, or such substances as are produced by other varieties of bacteria with which it struggles for existence when outside the animal body. Probably all bacteria produce substances of a toxic nature, but the possibility of their pro- ducing antibodies which will neutralize the toxins of other varieties is one that has interested me for some time. My experiments in this direction lead me to believe that this may be the case, but a research of this nature is rather difficult to carry out technically, and I shall do many more experiments before com- mitting myself to this view. If, however, additional experiments should con- firm my opinion, and if the special forms play some part in this process, one can quite understand that the special forms would be useless among the mem- bers of a pure culture growing on artificial media. In fact, they might be directly detrimental to the bacterial community, in that they would use up the food supply and overgrow the other bacilli which are necessary for a con- tinuation of the life of that species or variety. In this case the lytic agent would perform a good purpose in getting rid of the useless special forms. It may be argued that this will not explain the lysis of the normal forms of bacilli, but then the conditions of cultivation on artificial media are not those of nature. In fact, in the process of evolution the bacterium did not develop its characters under the influence of cultivation in incubators, and an action that might be restrained to the limits of useful purposes in nature might very well extend beyond those limits under such an abnormal environment as that presented by an uncontaminated tube of agar. Some of my views and the possibilities I have suggested are no doubt open to criticism, but I claim that the discovery of the filter-passing lytic agent in association with bacteria offers a large field for research, and I suggest that this field has been further extended by the isolation of “special forms” of bac- teria and by the possibility of demonstrating the production of bacterial anti- toxins for the neutralization of toxins produced by other varieties of bacteria. Moreover, it must be remembered that all the vital processes of a bacterium have some relation to each other, and it is therefore necessary to study these problems together, or the true significance of any one may not be fully appre- ciated. 21 Ave. 19, 1922 | SECTION OF MICROBIOLOGY. ie ae Mepicav JourNAL IIl.—_CONCERNING THE THEORIES OF THE SO-CALLED “BACTERIOPHAGE.” By J. Borvet, M.D., Director of the Pasteur Institute, Brussels. [Tue following explanatory statement was read by Dr. Gratia for Professor Bordet. | Through Dr. Gratia I obtained access to the paper Dr. d Herelle intends to present on the lytic phenomenon due to the so-called “bacteriophage.” I was not a little surprised to find that Dr. d'Herelle in this paper attributes to my co-worker, Dr. Ciuca, and to myself, as regards the intimate nature of this phenomenon, an opinion which is wholly different from what we felt entitled to uphold from the very beginning of our studies on the subject.’ Dr. d’'Herelle quotes our names next to Kabeshima’s, and enlists us among the authors who assume that the lytic principle is a leucocytic secretion. In fact, this view seems to us altogether untenable, and is almost the reverse of the opinion we have constantly emphasized. I think we were first to advocate the view that the lytic principle is pro- duced by the microbe itself which shows the lysis—in other words, that the transmissible lysis is in reality an autolysis betraying a nutritive vitiation primarily started by external influences, an example of which may be the con- tact with a leucocytic exudate. No doubt it would be quite unnecessary to translate literally the many passages of our papers where this assumption is advocated. Some lines, however, may be quoted: “External influences such as that of a leucocytic exudate modify the bacterium, inducing the latter to elaborate a lytic substance capable of diffusing itself and bring- ing about the same autolytic phenomenon through successive cultures. When the autolytic process occurs a large number of the microbes present may perish, but some of them, being more resistant, are, during a certain length of time, still capable of reproduction in spite of their producing the active principle, thus imparting to new cultures of the same microbe the same autolytic tendency.” In another paper we add: “According to d’Herelle, the lysis is due to a living being, to a filtering virus. We, on the contrary, believe that the lytic principle originates from the bacteria them- selves, which, when touched by this active substance, are capable of regenerating it, the factor responsible for the phenomenon being thus unceasingly reproduced—on the condition, however, that the bacteria be still living and provided with the alimentary substances necessary to their growth.” I wonder how Dr. d’Herelle could possibly give such an erroneous account of our work as in his paper. The many authors who have written on the subject did not, like Dr. d’Herelle, misinterpret a theory which we have so often and so distinctly outlined and explained. I shall allow myself to quote, for instance, the paper recently published by Dr. Bruynoghe,” who writes: “According to Bordet and Ciuca, the microbes undergo—throuch the agency of a leucocytic exudate—a modification by which they are henceforth capable of elaborating an autolytic principle, this property being further transmitted to the 22 Ave. 19, 1922 | THE BACTERIOPHAGE. Vine ioe following generations by the germs which were sufficiently resistant, and thus could multiply. This interesting view permits the understanding of the fact that the lytic principle is only regenerated when the bacteria are living, since the theory asserts that this principle is produced by the bacteria themselves.” I think there is no need to dwell longer on the subject. But one must agree that I could not refrain from correcting d’Herelle as regards our views, nor from presenting them again as they are expressed in all of our papers. The mere titles of these are clear enough; we always designate the phenomenon under the name of “the microbian transmissible autolysis.” REFERENCES. 1C.R. Soc. Biologie, October, 1920. °Le Scalpel, March, 1922. IV._ANDRE GRATIA, M.D., Pasteur Institute, Brussels. 1. The Twort phenomenon and the d’Herelle phenomenon are identical. They are two different aspects of one and the same phenomenon: the trans- missible lysis of bacteria. When the “dissolving material” of Twort found in diseased agar cultures of micrococci obtained from vaccinia lymph is transplanted into a young broth culture of staphylococci a dissolution of the latter occurs, and the filtrate of the dissolved culture exhibits all the characteristics of a typical staphylococcus bacteriophage according to the definition of d’Herelle. On the other hand, typical staphylococcus bacteriophage could be obtained also by other means—namely, by the leucocytic exudate technique of Bordet and Ciuca, or by the puncture of a subcutaneous abscess. When small amounts of this staphylococcus lytic agent are introduced in melted agar which is afterwards slanted and seeded with sensitive staphylococci a culture results, apparently normal at the beginning, but which, a little later, turns into the typical glassy transparent material of Twort. In other words, the Twort phenomenon leads to the dHerelle phenomenon, and, inversely, the d’'Herelle phenomenon leads to the Twort phenomenon. 2. There are no unquestionable proofs that the bacteriophage is a living organism. The assumption of the bacteriophage being a filtrable virus for bacteria was suggested by two main facts: (a) The power of reproduction possessed by the lytic agent, and (b) the localization of the lysis to certain round spots of clarification when a very diluted lytic agent is poured over the surface of an agar culture of sensitive bacteria. Although easily explained by the virus theory, yet both facts are not unquestionable proofs of the living nature of the bacteriophage, because they are by no means exclusive features of living beings. Fire is not living, and yet fire is endowed with power of reproduction. When once lighted, thanks to an initial impulsion such as an electric spark or 23 Aue. 19, 1922 | SECTION OF MICROBIOLOGY. Prerersa ee the mere striking of a match, it can be indefinitely reproduced if fuel is pro- vided. A still more striking, because more biological, example is found in blood coagulation. Suppose a series of tests tubes containing a stable plasma—bird’s plasma, for instance—which will remain indefinitely fluid. To the first tube we add just a few cubic centimetres of distilled water. As a result of that initial thromboplastic action, which does not need to be repeated to the future, thrombin suddenly appears in the first tube and the plasma clots. If a few drops of the exudate serum in the first tube are pipetted off and poured in the next tube, this second tube clots, in its turn, with a new regeneration of throm- bin, which, transferred in the third tube, brings about the coagulation of that tube with again a new production of thrombin, and so on indefinitely. In this way we realize the transmissible coagulation of blood in series, with the con- tinuous regeneration of thrombin, and thrombin is not a living being. The localization of the lytic action of diluted bacteriophage can be explained by tha hypothesis of a chemical substance as well. It must be kept in mind that a culture is not a homogeneous whole, but made up of organisms showing all kinds of qualitative and quantitative individual differences—that is, as far as their susceptibility to the lytic agent is concerned. When a very concentrated lytic agent is poured over the surface of an agar culture an almost complete dissolution occurs, with the exception of just a few organisms resistant enough to overwhelm the strong action of the concentrated lytic agent. On the other hand, when a diluted lytic agent is used only the few extremely sensitive bacteria will be influenced, and each of them becomes a centre of regeneration of the lytic agent, which, diffusing evenly in every direction, produces perfectly round spots of clarification very often surrounded by a kind of halo of diffusion. Between these two extreme conditions all kinds of intermediate degrees exist. Further, any substance, living or not, is com- posed of particles, molecules, atoms, or ions. When we pour out a glass of soda water, there appear on the wall of the glass small round bubbles of gas, the size of which, increases exactly as the so-called colonies of bacteriophage, and yet gas is not a virus. 3. The idea of the bacteriophage being a product of bacterial activity is suggested by the close parallelism existing between the regeneration of the lytic agent on the one hand, and the activity of growth of the bacteria on the other hand. No regeneration ever occurs in dead cultures, nor in living cultures when put in such conditions that they cannot grow—in saline emulsions of bacteria, for instance, or at low temperature. A slight lysis, with but a small regeneration of lytic agent, is induced in the slow-growing culture of B. coli in a syn- thetic medium. On the contrary, an abundant regeneration occurs in a fast- growing culture in broth. A recently seeded broth culture to which is added just a trace of lytic agent will not be inhibited; but a few hours later, at the very moment the culture reaches its acme of growth, a rapid dissolution occurs with an abundant regeneration of lytic agent. 24 [ Tue Brirtisu Mepicat JouRNAL Auc. 19, 1922 | THE BACTERIOPHAGE. 4. The conception of the bacteriophage being a chemical substance is favoured by the chemical-like affinity existing between a given lytic agent and the corresponding susceptible strain. I first observed that small amounts of lytic agents lose a certain part of their activity when put together with too thick emulsions of sensitive bacteria. Bordet, with a different technique, could even obtain the complete disappear- ance of traces of lytic agent in the same condition. Still more convincing are the results of Yaumain and of Da Costa, who observed the absorption of rela- tively important amount of lytic agent by dead emulsions of the corresponding sensitive bacteria. This specific affinity which is the necessary condition for a lytic agent for inducing the dissolution of a given bacterium is not favourable to the virus theory, because we question how a virus could be definitely fixed by dead bacilli, which, however, it is unable to attack. 5. The bacteriophage is not one and the same antigen. Several lytic agents showing antigenic specificity must be considered. The coli lytic agent can be completely neutralized by proper amounts of corresponding coli antilytic serum, but is not at all affected by staphylococcus antilytic serum, which, on the other hand, is only able to neutralize staphylo- coccus lytic agent and not coli lytic agent. This neutralization reaction is thus specific, and demonstrates the plurality of the bacteriophage. The non-specific results obtained with the alexin fixation reaction and advocated by d’Herelle in favour of the unicity of the bacteriophage, are of no value, because they are vitiated, as can be easily demonstrated, by the presence in the bacteriophage of bacterial dissolution products which have lost their specificity and play therefore the rdle of common antigen between different lytic agents. 25 Adsorption of Bacteriophages to Homologous Bacteria II. Quantitative Investigations of Adsorption Velocity and Saturation. Estimation of the Particle Size of the Bacteriophage By M. ScCHLESINGER Institute for Colloid Research, Frankfurt A.M. It was shown in the preceding communication that bacteriophages of a lysate do not represent a uniform population; instead they are composed of several classes of widely varying adsorbabilities. The following equation expressing the kinetics of adsorption of phages to homologous bacteria was derived: 2.3 log a = kbt , (1) where n, represents the initial concentration of free phage, n; the concentration after a reaction time ¢ and 6 the concentration of bacteria per ml. However, this equation is applicable only to experimental conditions under which the observed change in the concentration of free bacteriophages is mainly due to the adsorption of a single class of particles of uniform adsorbability. Lysates of the coli 88 bac- teriophage employed in our experiments were found to consist almost entirely of a uniform class of rapidly and irreversibly adsorbable phages when conditions were such that the concentration of sensitive bacteria and the reaction time was varied only within limits that allowed the decrease in concentration of free phage not to exceed 90 to 95%. It shall be shown in this communication that under these conditions equation (1) is so well satisfied that wide variations of the different experimental param- eters produce no change in the value of the velocity constant k calculated from the experimental results. After establishing the value of k for adsorption to dead as well as to living bacteria, experiments will be presented that are designed to determine the bacterial “saturation capacity”’, z.e. the number of phage particles that a single bacterial cell can adsorb. Finally, an attempt will be made to calculate the particle size of the phage from the adsorption velocity constant. Further insight shall be gained into the adsorption mechanism by a comparison of the values so calculated with the results of direct determinations of the particle size. Translated from the German and reprinted by permission of the Springer Verlag from ZEITSCHRIFT FUR HYGENIE UND IMMUNITAETSFORSCHUNG, 114, 149-160 (1932). 26 Adsorption of Bacteriophages to Homologous Bacteria Measurement of the Adsorption Velocity. In order to test equation (1), we shall determine, as a function of time, the in- fectivity of mixtures of known concentration of heat-killed bacteria and input bacteriophages, so that the value of the expression whose constancy is demanded by equation (1), can be calculated. These experiments are carried out in the following way. A heat-killed bac- terial suspension of known concentration is diluted into broth, so that after ad- dition of the phage the concentration of bacteria in the reaction mixture is b. After this dilution has been warmed to 37°, a measured amount of a lysate di- lution of exactly known phage titer is added and the time noted. In order to keep the temperature constant, the reaction mixture is placed in an incubator, in the case of long experiments, or, otherwise, in a water bath. After various times have elapsed, a measured aliquot of the reaction mixture is quickly diluted 10 or 100- fold, so that for all practical purposes, the reaction is stopped'. In this manner, reaction times down to 2 minutes can be investigated, after some practice. For reaction times of more than | hour, in cases where the anticipated titer permits this procedure, the sample is plated directly on agar plates. Under the present experimental conditions it is unnecessary to separate the dead bacteria from the reaction mixture before plating, since only irreversible adsorption is involved. The detailed results of one experiment, and the calculations based thereon, shall be presented here as an illustration. 0.25 ml of the heat-killed broth culture of coli bacteria, containing 4 X 10® bacteria ml, was added to 4.65 ml broth. After warming the bacterial suspension to 37°, 0.1 ml of a thousand-fold dilution of a lysate of titer 8.5 X 108 was added toit. The mixture was placed in the incu- bator; after 1, 2, and 3 hours, 0.1 ml aliquots were removed from the mixture and spread at once on agar plates. The initial titer of the mixture, n,, was 1.7 X 104; the titers, after 1, 2, and 3 hours were found to be 5.8 X 10%, 2.4 & 10%, and 7.2 xX 102. With the known value of 6 = 2.0 X 107 and the time measured in seconds, k can be calculated. After 1 hour ee 2S ystems 2.3 Wes as bt °F nn, 2X 107 X 3600 5.8 xX 10: = 3.2 X 10-!! log 2.9 = 1.5 X 107}! After 2 hours k= log 7:1 = v4 107% SO < ll de Zz, After 3 hours Ore xg lOn k= 3 log 23.6 = 1.5 X 10-1! 1A 100-fold dilution reduces the reaction velocity by a factor of 100. 27 M. Schlesinger The values of & (multiplied by 10!'), calculated on the basis of our most exten- sive experiments, are summarized in Table I. For these experiments, a phage lysate of titer 6.5 X 10% and a heat-killed bac- terial suspension (70° for 1 hour) were used. Prior to heating, the bacterial suspension was grown from a slant agar culture to a density of 5.8 X 108, as de- termined by colony count. However, the value of 6.5 X 10® used in the calcula- tions was determined by microscopic cell count after heat-killing. TABLE [| b = 1.6 X 108 |b = 6.5 X 107 |b = 3.3 X 107 Time in Seconds 6.5 X 105 6.5 X 10° Grob 104 6.5 x 104 6.5 X 108 6.5 X 103 6.5 X 108 b = 3.3 X 108 No Oo No 120 240 360 720 ; 12)) kes 1080 ; Tey lial 1440 1800 EO. |) one 3600 He Ma [oe 7200 : 1.2 1.3 10800 : 0.9 1.5 14400 1.3 len 28800 ; ; 1.3 43200 | 1.5 —» »|b = 6.5 X 108 NS? aot So tb bv It is apparent that, neglecting variations due to experimental error, the value of k remains virtually constant, in spite of the fact that in the different experiments the initial titer of phage has been varied by a factor of 100, the concentration of bacteria by a factor of 200 and the reaction time by a factor 360. The average value of the velocity constant in these experiments is 1.2 K 10711. In five additional, less extensive, experiments, carried out with cultures of coli 88 grown either in broth or on agar, the following values of k were found: 1.0 X 10-11, 1.4 < 107-14, 1.6 10° 4 10K 105*4,and 1.4 X 10° *" (average valitesrat 3, 7, 9, 4, and 7 determinations). These fluctuations are no greater than the uncertainty in the determination of the bacterial concentrations’. The average value of the velocity constant k, computed on the basis of 58 single determi- nations for the binding to dead bacteria, was 1.3 K 1071). 2As has already been mentioned, the heat-killing was carried out by heating to 70° for 1 hour. This temperature treatment has no important effect on the binding capacity of the bacteria. In a series of experiments, aliquots from a single broth culture of coli were heat-killed at 70° for 3, 1, 2 and 3 hours; the adsorption velocity constants determined in experiments utilizing these suspensions were 1.7 X 10711, 1.6 x 10-#, 1.3 X 1071! and 1.4 X 10711. 28 Adsorption of Bacteriophages to Homologous Bacteria The velocity of adsorption of phage to living bacteria was measured either di- rectly, or by adding bacteriophages to a mixture of dead and living bacteria and then determining their distribution over the two components. A 6-hour-old agar slant of coli 88 is suspended in broth and freed of larger clumps by short centrifugation; half of this suspension is placed on ice, and the other half is heated for 30 minutes in a water bath at 70°. The cell concentration in each of the cultures is then determined by microscopic counts. 0.9 ml aliquots of each culture, as well as equal volumes of a two-fold dilution, are placed in the water bath at 37° and infected with 0.1 ml of a 1000-fold dilution of the phage lysate. After 3 and 6 minutes, or after 6 and 12 minutes, aliquots of each mixture are diluted 10-fold and centrifuged at once at 8000 r.p.m. and the titer of the supernatant liquid determined. The values of k, calculated from the experi- mental results, are presented in Table II. (The uncentrifuged mixtures are also titrated; in the experiment with living bacteria, the infective titer remains equal to that of the original lysate; whereas, in the experiment with dead bacteria, the surviving infectivity is always equal to that of the infectivity of the supernatant liquid.) The suspensions of living and dead bacteria are then mixed with one another in the following proportions: 5+ 5, 3+7, 2+ 8, and1+9. To each of 0.9 ml of these mixtures, as well as to a suspension of only living bacteria, 0.1 ml of the lysate dilution is added and an aliquot of 0.1 ml of each mixture spread on agar plates after 15 minutes. During this time no multiplication of the bacterio- phage takes place, whereas most of the phages are already adsorbed. Since any phage adsorbed to a living bacterium manifests itself by plaque formation, one may infer that any observed decrease in phage titer must represent phages ad- sorbed to dead bacteria. This decrease thus permits a calculation of the ratio of the adsorption velocity constants k (living) k (dead) which apparently indicates the distribution of phages over two competing components. The result of this experiment is presented in Table ITI. Microscopic counts of the suspension of dead bacteria indicated a concen- tration of 4.7 X 108, of the suspension of living bacteria a count of 5.8 & 108/ml. These figures are reduced by 10% in the reaction mixture through dilution upon addition of the phage lysate. The results of direct measurement of the adsorption velocity are presented in Table IT. TaB_Le II Time in Heat-killed bacteria Living bacteria seconds b = 4.8 X 108 bi 24 >< 1108 b = 4.8 X 108 b= 274 108 180 FOR AOe! : 2.4 X 1071! : 360 1.0 X 107}! 1.0 < 10712 2.8 X 107-11 2.7 X 10711 720 : 0.8 X 107}! : 2.3 X 10-1! Thus, according to this experiment, with the ratio of the two velocity constants being 2.6, the average value of kis 1.0 X 1071! for dead bacteria and 2.6 X 1071! 29 M. Schlesinger for living bacteria. The distribution of bacteriophages in a mixture of living and dead bacteria is presented in Table ITI. TABLE III In 10 parts of the mixture Plaques per 0.1 c.c. k (living) / k (dead) Living Heat-killed bacteria bacteria 10 ‘ 1000 5 5 720 2.6 3 7 420 lez ) 8 360 2.2 1 9 280 3.5 Average 2.5 The results of this method are thus in agreement with those obtained from the first method. Hence adsorption of phages to young living bacteria proceeds 2 to 3 times more rapidly than adsorption to dead bacteria. If we multiply the value of k of 1.8 & 107-11, determined in numerous experiments with dead bacteria, by the proportionality constant 2.6, we obtain the velocity constant of adsorption to living bacteria: k = 3.4 X 107?1. Determination of the Saturation Capacity of Bacteria. The following method was selected for determining the maximum number of bacteriophages, referred to as the saturation capacity of the bacterium, that the average heat-killed bacterium can adsorb: samples are prepared which contain exactly the same number of dead bacteria but to which increasing initial concen- trations of bacteriophages are added. After a sufficient length of time has elapsed, all of the free bacteriophages capable of irreversible adsorption will have disappeared from each sample. The remaining phage titer will thus be independ- ent of the absolute value of the initial phage concentration, or, at any rate, will represent only a small fraction of it. In fact, the approximate constancy of the relative decrease in free phage should persist even when the allowed reaction time is insufficient for the attainment of the final state; for both parameters im- portant for the velocity of adsorption, the concentration of the bacteria and the relative proportion of phages of differing adsorbability, are constant for all of the samples. This picture will change, however, and change drastically, whenever the initial concentration of bacteriophages attains that multiple of the bacterial concentration which corresponds to the saturation capacity. For instance, when the initial concentration is only half of the saturation capacity, then the adsorp- tion velocity could be reduced, but only near the end of the adsorption process; when, however, the initial concentration of phage is twice that of the total bacterial capacity, then irrespective of the length of time of the experiment, only half of the initial phage population can be adsorbed. The saturation range thus manifests itself by a rapid increase in the ratio of final to initial phage titer in such an adsorption experiment. This ratio will vary from a value of about 0.01 prior to a saturating phage concentration to a value of about 1.0, once saturation has 30 Adsorption of Bacteriophages to Homologous Bacteria been attained. If no saturation is observable in such a series of experiments, even with the highest initial concentrations of phages obtainable, then the concentra- tion of bacteria must be decreased in a second series of experiments. In that event, of course, the reaction time must be correspondingly prolonged. Tas.e IV. Bacterial Concentration 5.0 X 107. Reaction Time: 1 Day. Dennen eee eee Initial Titer | Final Titer | Ratio Phage/Bacteria | Relative Decrease 5.0 X 108 2.1 X 106 10:1 1:240 2 108 1.0 X 106 5:1 1:250 1.0 X 108 3.3 < 10° 2:1 1:300 5.0 X 107 Ie xasLO2 1:1] 1:450 2x 101 1.8 X 10° 0.5:1 1:140 OX, 107 3.1 X 104 0.2:1 1:320 TaBLE V. Bacterial Concentration 1.0 X 107. Reaction Time: 4 Days 9.0 x 108 4.6 X 106 90:1 1:200 4.5 X 108 2.0 X 106 45:1 1:220 1.8 X 108 5.0 X 10° 18:1 1:360 9.0 X 107 2.6 X 10° Ort 1:340 TaB_e VI. Bacterial Concentration 1.0 106. Reaction Time: 8 Days (Two samples became unsterile) ee x<< 1102 1 S102 1100:1 neil pep >< 108 : 550:1 : DOESN 8.4 x 107 220:1 1:2°6 1.1 X 108 110:1 Tasie VII. Bacterial Concentration 1.8 X 106. Reaction Time: 8 Days 7.6 xX 108 Call << 103 420:1 Leila 3.8 X 108 1.4 x 108 210:1 LSPA a LEDS 108 8.2 xX 106 84:1 1:18 ox 107 4.5 X 105 42:1 1:170 3.8 X 107 6.6 < 104 Ziel 1:570 15S 104 2 x M04 8.4:1 1:550 The experimental results are summarized in Tables IV to VII. The experi- ments were carried out with different dilutions of a three-hour broth culture of coli 88, heat-killed at 70°, containing 1.0 X 108 bacteria/ml (Table IV-VI) and with a dilution of a similar culture containing 1.8 X 10% bacteria ml (Table VII). As controls, samples or dilutions of the lysates were always placed in the incu- bator and their titers similarly determined at the conclusion of the experiments; these titers always agreed with the earlier assays of the lysates. It follows from the data of Table IV that the saturation capacity must be at least 10, while the data of Table V show that this capacity must exceed at least 90. In the last two tables, the region of saturation has been attained, and their third and second lines, respectively, permit a more exact estimation. According to Table VI, 1.0 X 10° bacteria are saturated by (2.2 — 0.8) X 10° bacterio- phages. Hence, one bacterium is saturated by 140 phage particles. Similarly, it follows from the data of Table VII that the saturation capacity has the value of 130. 31 M. Schlesinger Therefore, a coli bacterium killed by heating to 70° is capable of irreversibly adsorbing an average maximum of 130 to 140 bacteriophages. Calculation of the Particle Size of the Bacteriophage from the Adsorp- tion Velocity and from the Saturation Capacity. Conclusions: a) Calculation of Particle Size from the Adsorption Velocity. The assumption was made in the derivation of equation (1), a formal description of the adsorption kinetics, that contact between one phage and one bacterium occurs entirely by chance and without benefit of any orienting or attractive forces whatsoever. The collisions were thought to be mainly due to the Brownian movement of the particles*, to which, obviously the movement of the much smaller bacteriophages made the principal contribution. Since there exist well-known relations between Brownian movement, the diffusion coefficient, and the particle size, it is thus possible theoretically to correlate the latter with the adsorption velocity of the phage. It is, however, first necessary to make an assumption concerning the relation which might exist between the Brownian movement collision frequency of phages and bacteria and the number of adsorptive events which actually ensue as a consequence of these collisions. The simplest assumption in this connection is that every collision leads to an irreversible fixation. If this assumption is valid, then the relation of the adsorp- tion velocity to the diffusion constant, D, of the phage follows at once from the formula on which M. v. SMoLucHowsky has based his theory of the kinetics of coagulation’. This formula states that the quantity Jdt of a solute which diffuses in a time dt onto a sphere of radius R capable of fixing solute particles which touch it is Jdt = 4rDRcadt where c is the concentration of the substance®. If we equate R to the radius of a supposedly spherical bacterial cell, substitute for c the number of free bacterio- 3It is also possible that convection or mechanical agitation of the fluid could play a certain role. The error arising from neglecting the Brownian movement of the bacteria depends on the ratio of the bacterial diameter to that of the phage, and probably causes the estimate of the phage diameter to be too small by 10% in the following calculation. 4Z. Physik. Chem., 92, 140 (1917). An attempt — though insufficient — to apply the formula of the v. Smoluchowsky coagulation theory to bacteriophage adsorption has already been made by v. Angerer [Arch. f. Hyg., 92, 312 (1924)]. The complete formula is R Jdts= 4D Re ea | dt VrDt where ¢ indicates the time which has elapsed since the start of the experiment. As in the coagulation experiments of Zsigmondy analyzed by v. Smoluchowsky, so also, under our R experimental conditions is the quantity ar small compared to unity, and can be neglected Vr without significant error. Incidentally, Jdt represents in v. Smoluchowsky’s application, as well as in ours, not the number of particles which actually diffuse onto the sphere in the time dt (after all, in our experiments there is only one bacteriophage for every hundredth or thousandth bacterial cell), but only the probability of an encounter of a particle with the adsorbing sphere. 32 Adsorption of Bacteriophages to Homologous Bacteria phage particles n, replace Jdt by the decrease —dn of the number of free phages which occurs in the time dt, and bear in mind that for the adsorption process there is available not one but simultaneously b adsorbing bacteria (referring our entire consideration to the unit of volume), then we can write —dn = 4rDRbn dt. If we compare this expression with the differential equation on which equation (1) was based —dn = kbndt then it follows that k rae the desired relation between adsorption velocity and diffusion constant of the bacteriophage. Now it will be recalled that the values of the velocity constant for adsorption to living and dead bacteria are not the same, since k for living bacteria is 2.6 times greater than k for dead bacteria. The reduced adsorption velocity for heat-killed bacteria evidently implies that a smaller fraction of the contacts leads to adsorption, since the collision frequency is the same whether the bacteria are living or dead. The assumption that every collision leads to fixation can therefore be excluded a priori for dead bacteria, while it could still be valid for living bacteria. In the latter case, the number of collisions is probably greater than that estimated since the motion of the coli cell was neglected in the calcu- lations. Nevertheless, the relatively slow motion of the bacteria is insufficient to explain the entire difference between the adsorption velocity to dead and to living bacteria, since the movement of the cells could hardly triple the collision fre- quency. Therefore, our comparison of the D values for living and dead bacteria will be based on our calculations of their respective k values. For the purpose of our calculations we shall equate R to the radius of that sphere whose surface is equal to the surface of a cylinder of 1.2 u length and 0.5 u width, in which case R is equal to 4.3 X 10°°. The diffusion coefficient of the phage at the experimental temperature of 37° thus is i ee oe Eg Cees ee if the time is reckoned in seconds, as in the calculation of k. If this value is reduced to the unit of time generally used in diffusion experiments, i.e., the day, it follows that D is equal to 0.0055. On the basis of this value of the diffusion coefficient, one estimates from Einstein’s formula® a diameter of the bacterio- i = 47D or Dp) = 6This formula is me Cae e 6xNnD where p is the particle radius, C the gas constant (8.32 107 erg/degree), 7 the absolute temperature, N Avogadro’s number (60.7 X 1022), and n the viscosity. It thus follows that fe 8.32 < 107 (273 + 37) 6x X 60.7 X 1022 X 0.007 XK 6.3 X 1078 or that the diameter is 102 mu. p = 5.1 X 106 cm 33 M. Schlesinger phage of 100 my. On the other hand, utilizing k for killed bacteria (1.3 X 1071), one calculates a particle diameter of 260 mu. This latter value is undoubtedly too high; thus, in the case of heat-killed bacteria, it seems certain that not every collision can lead to fixation. It follows from this calculation that the adsorption velocity of the bacteriophage is not too great to be fully explained by Brownian movement alone. If one reverses this calculation and estimates the adsorption velocity constant from the known particle size of the bacteriophage (80 to 90 mu; see below), one obtains a value of approximately 4 x 10~!!. It seems probable that this agreement in order of magnitude of different, independent estimates of the particle diameter can be taken as confirmation of the essential validity of the concepts on which these estimates were based. b) Calculation of the Particle Size from the Saturation Capacity. The particle size of the bacteriophage can be calculated from the saturation capacity, if it is assumed that the bacterial cell absorbs that maximum number of phages which can completely cover its surface with a monolayer of particles. The premise that the adsorption takes place in a single layer, i.e., that it results only from direct contact between phage and bacteria, is justified on the basis of the irreversible and specific nature of the adsorption process’. On the other hand, the assumption® that saturation occurs only after the entire surface of the bacterium has been covered by phages is inherent in the previous hypothesis that every contact between phage and bacterium leads to fixation. For every contact can lead to fixation only if the entire bacterial surface is capable of phage fixation®. In case of close-packing, 140 spheres of diameter 6 cover a surface of 140 62. If this value is equal to the surface of the bacterial cell, that is to say 2.3 v2, then it follows that 6 = 127 mu. If one considers, however, that random collisions of phage particles will hardly cover the bacterial surface in the closest packing assumed here, and that the number 140 refers to the adsorption capacity of In the case of heat-killed bacteria, there can be no question of any penetration of the bacteriophage into the interior of the cell; at least there is no indication of such a possibility. 8Obviously, this hypothesis refers to conditions under which there can be no question of any interference with adsorption by partial saturation; that is to say, under conditions of great excess of bacteria. The assumption that this hypothesis is not fully applicable to heat-killed bacteria will be considered below. 9However, even if the entire surface of the bacterium were capable of phage fixation, it would not necessarily follow that every collision would lead to fixation; other prerequisites of fixation might be appropriate direction and adequate intensity of the collision. In the event that only a number of isolated points, instead of the entire surface, is capable of fixation, then only those collisions which bring the phages into contact with these ‘‘valence points’ could lead to fixation. The saturation capacity (S) then represents the number of such points. This assumption also makes possible an estimate of the phage diameter 6, on the basis of the equation br k = 4crDR IR™ This equation states that the adsorption velocity is proportional to the fraction of the bacterial surface capable of adsorbing the phage. The calculation, for which & for heat-killed bacteria must be employed since S refers to the adsorption capacity of heat-killed bacteria, leads to a value of 5 = 19 mu. If we assume the correctness of our direct determination of particle size of the phage (80-90 mz) mentioned below, then this low estimate of the phage diameter indicates that almost the entire bacterial surface must be covered by “‘valence points.” 34 Adsorption of Bacteriophages to Homologous Bacteria heat-killed bacteria, whose 2.6-fold reduction in adsorption velocity possibly reflects in inactivation of the phage receptors of a corresponding fraction of the cell surface, then one might revise this estimate of the phage diameter down to values of 61 to 120 mu. Thus, two completely independent sets of data, the adsorption velocity and the saturation capacity, yield roughly the same value for the diameter of the bacterio- phage: 100 my and 60-120 my respectively. These figures appear much too high when compared with most of the values for the particle diameter given in the literature. In recent months, however, we have succeeded in measuring the sedimentation velocity in a centrifuge run at 7000 to 8000 r.p.m., of phage particles from lysates of the same coli phage selected for the present adsorption experiments. These measurements, which no doubt exceed all others in reliabil- ity, allowed us to calculate a particle size of 80 to 90 mu. By means of a successive series of repetitive centrifugations, we were finally able to sediment into a pellet up to 99.99% of the active particles without encountering any decrease in the sedimentation velocity, 1.e., without detecting any particles of smaller diameter. ! The agreement of the values of the phage diameter calculated indirectly from the adsorption velocity and from the saturation capacity, and the similarity of these values to the phage diameter determined directly, substantiates the validity of our assumptions. In any case, the formal kinetics of the process as well as the actual numerical values of the relevant parameters can be integrated, without contradiction, into the following simple picture. In each suspension of a mixture of bacteria and bacteriophages collisions occur between the individual particles of the two components. These collisions are entirely random, and their frequency is mainly determined by the Brownian movement of the bacteriophages. In the case of bacteriophage particles of maximum adsorption affinity (and in our col lysate most of the particles are of this nature), the first such random collision between phage and bacterial cell leads to an irreversible fixation. More precisely, in the case of young, living bacteria almost every collision leads to fixation and in the case of heat-killed bacteria approximately every third collision does!!. In the case of phage particles of reduced affinity, only a fraction of the collisions leads to fixation; and here also the fixation is less stable and partially reversible. The bacteria conserve their ability to find additional bacteriophage particles as long as their surfaces are not completely covered by a monolayer of such particles. Investigations on the influence of the medium on the absorption process shall be communicated in a future paper. Summary 1. The equation describing the adsorption kinetics derived in a previous paper 2.3 log = = kkbt t The results of this investigation will be published in this journal. This statement refers only to the experimental conditions communicated here, that is to say, to a broth medium and to the temperature of the incubator. 35 M. Schlesinger is satisfied for very wide variations in the parameters n,, b, and t (initial econcen- tration of phage, concentration of bacteria, and reaction time), provided that these variations remain in such limits that the adsorption velocity is still deter- mined by the same group of bacteriophages of maximal adsorptive affinity (i.e., n/N must not fall below 1/20). 2. The adsorption velocity constant k has the value of 1.3 & 1071! for adsorp- tion of phage to heat-killed bacteria; k has an approximately 2.5 times greater value for phage adsorption to living bacteria. 3. A heat-killed coli bacterium is capable of adsorbing, on the average, a maximum of 130 to 140 bacteriophage particles; this number is referred to as the saturation capacity. 4. On the basis of the simplest assumptions, two values for the diameter of the bacteriophage have been calculated; one from the adsorption velocity constant and the other from the saturation capacity; the former yields an approximate particle diameter of 100 mu, the latter a value of 60 to 120 my. A direct size determination of the same bacteriophage from its sedimentation velocity leads to a particle diameter of 80 to 90 mu. 36 THE GROWTH: OF BACTERIOPHAGE By EMORY L. ELLIS anp MAX DELBRUCK* (From the William G. Kerckhoff Laboratories of the Biological Sciences, California Institute of Technology, Pasadena) (Accepted for publication, September 7, 1938) INTRODUCTION Certain large protein molecules (viruses) possess the property of multiplying within living organisms. ‘This process, which is at once so foreign to chemistry and so fundamental to biology, is exemplified in the multiplication of bacteriophage in the presence of susceptible bacteria. Bacteriophage offers a number of advantages for the study of the multiplication process not available with viruses which multiply at the expense of more complex hosts. It can be stored indefinitely in the absence of a host without deterioration. Its concentration can be determined with fair accuracy by several methods, and even the individual particles can be counted by d’Herelle’s method. It can be concentrated, purified, and generally handled like nucleoprotein, to which class of substances it apparently belongs (Schlesinger (1) and Northrop (2)). The host organism is easy to culture and in some cases can be grown in purely synthetic media, thus the conditions of growth of the host and of the phage can be controlled and varied in a quanti- tative and chemically well defined way. Before the main problem, which is elucidation of the multiplication process, can be studied, certain information regarding the behavior of phage is needed. Above all, the “natural history” of bacteriophage, z.e. its growth under a well defined set of cultural conditions, is as yet insufficiently known, the only extensive quantitative work being that of Krueger and Northrop (3) on an anti-staphylococcus phage. The present work is a study of this problem, the growth of another phage (anti-Escherichia coli phage) under a standardized set of culture conditions. * Fellow of The Rockefeller Foundation. Reprinted by permission of the authors and The Rockefeller Institute from THE JouRNAL OF GENERAL PuHysioLoey, 22 (3), 365-384, January 20, 1939. 37 366 GROWTH OF BACTERIOPHAGE EXPERIMENTAL Bacteria Culture.—Our host organism was a strain of Escherichia coli, which was kindly provided by Dr. C. C. Lindegren. Difco nutrient broth (pH 6.6- 6.8) and nutrient agar were selected as culture media. These media were selected for the present work because of the complications which arise when synthetic media are used. We thus avoided the difficulties arising from the need for accessory growth factors. Isolation, Culture, and Storage of Phage.—A bacteriophage active against this strain of coli was isolated in the usual way from fresh sewage filtrates. Its homo- geneity was assured by five successive single plaque isolations. The properties of this phage remained constant throughout the work. The average plaque size on 1.5 per cent agar medium was 0.5 to 1.0 mm. Phage was prepared by adding to 25 cc. of broth, 0.1 cc. of a 20 hour culture of bacteria, and 0.1 cc. of a previous phage preparation. After 34 hours at 37° the culture had become clear, and contained about 10° phage particles. Such lysates even though stored in the ice box, decreased in phage concentra- tion to about 20 per cent of their initial value in 1 day, and to about 2 per cent in a week, after which they remained constant. Part of this lost phage activity was found to be present in a small quantity of a precipitate which had sedimented during this storage period. Therefore, lysates were always filtered through Jena sintered glass filters (5 on 3 grade) immediately after preparation. The phage concentration of these fil- trates also decreased on storage, though more slowly, falling to 20 per cent in a week. However, 1:100 dilutions in distilled water of the fresh filtered lysates retained a constant assay value for several months, and these diluted preparations were used in the work reported here, except where otherwise specified. This inactivation of our undiluted filtered phage suspensions on standing is probably a result of a combination of phage and specific phage inhibiting sub- stances from the bacteria, as suggested by Burnett (4,5). To test this hypothesis we prepared a polysaccharide fraction from agar cultures of these bacteria, ac- cording to a method reported by Heidelberger ef al. (6). Aqueous solutions of this material, when mixed with phage suspensions, rapidly inactivated the phage. Method of Assay.—We have used a modification of the plaque counting method of d’Herelle (7) throughout this work for the determination of phage concentra- tions. Although the plaque counting method has been reported unsatisfactory by various investigators, under our conditions it has proven to be entirely satis- factory. Phage preparations suitably diluted in 18 hour broth cultures of bacteria to give a readily countable number of plaques (100 to 1000) were spread with a bent glass capillary over the surface of nutrient agar plates which had been dried by inverting on sterile filter paper overnight. The plates were then incubated 6 to 24 hours at 37°C. at which time the plaques were readily distinguishable. The 0.1 cc. used for spreading was completely soaked into the agar thus prepared in 38 EMORY L. ELLIS AND MAX DELBRUCK 367 2 to 3 minutes, thus giving no opportunity for the multiplication of phage in the liquid phase. Each step of each dilution was done with fresh sterile glassware. Tests of the amount of phage adhering to the glass spreaders showed that this quantity is negligible. The time of contact between phage and bacteria in the final dilution before plating has no measurable influence on the plaque count, up to 5 minutes at 25°C. Even if phage alone is spread on the plate and allowed to soak in for 10 minutes, before seeding the plate with bacteria, only a small decrease in plaque count is apparent (about 20 per cent). This decrease we attribute to failure of some phage particles to come into contact with bacteria. Under parallel conditions, the reproducibility of an assay is limited by the sampling error, which in this case is equal to the square root of the number of plaques (10 per cent for counts of 100; 3.2 per cent for counts of 1000). To test the effect of phage concentration on the number of plaques obtained, successive dilutions of a phage preparation were all plated, and the number of plaques enumerated. Over a 100-fold range of dilution, the plaque count was in linear proportion to the phage concentration. (See Fig. 1.) Dreyer and Campbell-Renton (8) using a different anti-coli phage and an anti- staphylococcus phage, and a different technique found a complicated dependence of plaque count on dilution. Such a finding is incompatible with the concept that phage particles behave as single particles, 7.e. without interaction, with respect to plaque formation. Our experiments showed no evidence of such a complicated behavior, and we ascribe it therefore to some secondary cause inherent in their procedure. Bronfenbrenner and Korb (9) using a phage active against B. dysenteriae Shiga, and a different plating technique found that when the agar concentration was changed from 1 per cent to 2.5 per cent, the number of plaques was reduced to 1 per cent of its former value. They ascribed this to a change in the water supplied to the bacteria. With the technique which we have employed, variation of the agar concentration from 0.75 per cent to 3.0 per cent, had little influence on the number of plaques produced, though the size decreased noticeably with increasing agar concentration. (See Table I.) Changes in the concentration of bacteria spread with the phage on the agar plates had no important influence on the number of plaques obtained. (See Table I.) The temperature at which plates were incubated had no significant effect on the number of plaques produced. (See Table I.) In appraising the accuracy of this method, several points must be borne in mind. With our phage, our experiments confirm in the main the picture proposed by d’Herelle, according to which a phage particle grows in the following way: it becomes attached to a susceptible bacterium, multiplies upon or within it up to a critical time, when the newly formed phage particles are dispersed into the solution. In the plaque counting method a single phage particle and an infected bac- terium containing any number of phage particles will each give only one plaque. 39 368 GROWTH OF BACTERIOPHAGE This method therefore, does not give the number of phage particles but the number of loci within the solution at which one or more phage particles exist. These loci will hereafter be called ‘infective centers.’”? The linear relationship between phage concentration and plaque count (Fig. 1) does not prove that the number of plaques is equal to the number of infective centers, but only that it is proportional to this number. We shall call the fraction of infective centers which Plaque count Relative concentration of phage Fic. 1. Proportionality of the phage concentration to the plaque count. Successive twofold dilutions of a phage preparation were plated in duplicate on nutrient agar; 0.1 cc. on each plate. The plaque counts from two such series of dilutions are plotted against the relative phage concentration, both on a loga- rithmic scale. produces plaques the “‘efficiency of plating.”” With the concentrations of phage and bacteria which we have used this coefficient is essentially the fraction of infected bacteria in the suspension spread on the plate, which goes through to lysis under our cultural conditions on the agar medium. After plating, the phage particles released by this lysis infect the surrounding bacteria, increasing only the size, and not the number of plaques. 40 EMORY L. ELLIS AND MAX DELBRUCK 369 TABLE I Independence of Plaque Count on Plating Method Agar concentration Plates were prepared in which the agar strength varied, and all spread with 0.1 cc. of the same dilution of a phage preparation. There is no significant difference in the numbers of plaques obtained. Agar concentration, per cent................ 0.75 1,5 3.0 In aquexcountsiare aes seer on Soe ee 394 373 424 408 430 427 376 443 455 411 465 416 373 404 469 ESV EIARE wee trcentarteieln ase ieee oe ere Oe ae ees 392 423 438 IP aquelsize, Mb tes oe ack ate a atoeeon 2 0.5 0.2 Concentration of plating colt A broth suspension of bacteria (10° bacteria/cc.) was prepared from a 24 hour agar slant and used at various dilutions, as the plating suspension for a single phage dilution. There are no significant differences in the plaque counts except at the highest dilution of the bacterial suspension, where the count is about 15 per cent lower. Concentration Plaque count 1 920 1/5 961 1/25 854 1/125 773 Temperature of plate incubation Twelve plates were spread with 0.1 cc. of the same suspension of phage and bacteria, divided into three groups, and incubated at different temperatures. There were no sig- nificant differences in the plaque counts obtained. MemperatureysG. ts. .c. cca ssee ese cele es 37 24 10 PIA GWEN ie 6 aa 9 «eo bdbsvvigh ares oe cele 352 384 405 343 405 377 386 403 400 422 479 406 AV CTA 6s, peepee tard ks es tienes mare ii 376 418 397 41 370 GROWTH OF BACTERIOPHAGE The experimental determination of the efficiency of plating is described in a later section (see p. 379). The coefficient varies from 0.3 to 0.5. This means that three to five out of every ten infected bacteria produce plaques. The fact that the efficiency of plating is relatively insensitive to variations in the tempera- ture of plate incubation, density of plating coli, concentration of agar, etc. indi- cates that a definite fraction of the infected bacteria in the broth cultures do not readily go through to lysis when transferred to agar plates. For most experiments only the relative assay is significant; we have therefore, given the values derived directly from the plaque counts without taking into account the efficiency of plating, unless the contrary is stated. Growth Measurenients The main features of the growth of this phage in broth cultures of the host are shown in Fig. 2. After a small initial increase (discussed below) the number of infective centers (individual phage particles, plus infected bacteria) in the suspension remains constant for a time, then rises sharply to a new value, after which it again remains constant. Later, a second sharp rise, not as clear-cut as the first, and finally a third rise occur. At this time visible lysis of the bacterial suspension takes place. A number of features of the growth process may be deduced from this and similar experiments, and this is the main con- cern of the present paper. The Initial Rise When a concentration of phage suitable for plating was added to a suspension of bacteria, and plated at once, a reproducible plaque count was obtained. If the suspension with added phage was allowed to stand 5 minutes at 37°C. (or 20 minutes at 25°C.) the number of plaques obtained on plating the suspension was found to be 1.6 times higher. ‘This initial rise is not to be confused with the first ‘‘burst”’ which occurs later and increases the plaque count 70-fold. After the initial rise, the new value is readily duplicated and remains constant until the start of the first burst in the growth curve (30 minutes at 37° and 60 minutes at 25°). This initial rise we attribute not to an increase in the number of infective centers, but to an increase in the probability of plaque forma- tion (z.e. an increase in the efficiency of plating) by infected bacteria in a progressed state; that is, bacteria in which the phage particle has commenced to multiply. That this rise results from a change in the 42 EMORY L. ELLIS AND MAX DELBRUCK Sil efficiency of plating and not from a quick increase in the number of infective centers is evident from the following experiment. Bacteria were grown for 24 hours at 25°C. on agar slants, then suspended in broth. Phage was added to this suspension and to a suspension of bacteria grown in the usual way, and the concentration of infective Time Fic. 2. Growth of phage in the presence of growing bacteria at 37°C. A diluted phage preparation was mixed with a suspension of bacteria con- taining 2 < 10° organisms per cc., and diluted after 3 minutes 1 to 50 in broth. At this time about 70 per cent of the phage had become attached to bacteria. The total number of infective centers was determined at intervals on samples of this growth mixture. Three such experiments, done on different days, are plotted in this figure. The same curve was easily reproducible with all phage prepara- tions stored under proper conditions. centers was determined on both. The initial value was 1.6 times higher in the agar grown bacteria than in the control experiment, and remained constant until actual growth occurred. The initial rise was therefore absent in this case, clearly a result of an increase in the efficiency of plating. A sufficient number of experiments were per- 43 372 GROWTH OF BACTERIOPHAGE formed with bacteria grown on agar to indicate that in other respects their behavior is similar to that of the bacteria grown in broth. The bacteria grown in this way on agar slants are in some way more sus- ceptible to lysis than the broth cultured bacteria. Adsorption The first step in the growth of bacteriophage is its attachment to susceptible bacteria. ‘The rate of this attachment can be readily measured by centrifuging the bacteria out of a suspension containing phage, at various times, and determining the amount of phage which remains unattached in the supernatant (cf. Krueger (10)).! According to the picture of phage growth outlined above, phage cannot multiply except when attached to bacteria; therefore, the rate of attachment may, under certain conditions, limit the rate of growth. We wished to determine the rate of this adsorption so that it could be taken into account in the interpretation of growth experiments, or eliminated if possible, as a factor influencing the growth rate. Our growth curves show that there is no increase in the number of infective centers up to a critical time; we could therefore, make measurements of the adsorption on living bacteria suspended in broth, so long as the time allowed for attachment was less than the time to the start of the first burst in the growth curve. ‘The adsorption proved to be so rapid that this time interval was ample to obtain adsorption of all but a few per cent of the free phage if the bacteria concentration was above 3 X 107. The number of bacteria remained constant; the lag phase in their growth was longer than the experimental period. The rate of attachment was found to be first order with respect to the concentration of free phage (Ps) and first order with respect to the concentration of bacteria (B) over a wide range of concentrations, in agreement with the results reported by Krueger (10). That is, the concentration of free phage followed the equation n ve = kal P;)(B) 1A very careful study of the adsorption of a coli-phage has also been made by Schlesinger (Schlesinger, M., Z. Hyg. u. Infektionskrankh., 1932, 114, 136, 149). Our results, which are less accurate and complete, agree qualitatively and quantitatively with the results of his detailed studies. 44 EMORY L. ELLIS AND MAX DELBRUCK Ss) in which k, was found to be 1.2 * 10-®cm.3/min. at 15° and 1.9 x 10-9 cm.*/min. at 25°C. These rate constants are about five times greater than those reported by Krueger (10). With our ordinary 18 hour bacteria cultures (containing 2 < 108 B. coli/cc.) we thus obtain 70 per cent attachment of phage in 3 minutes and 98 per cent in 10 minutes. The adsorption follows the equation accurately until more than 90 per cent attachment has been accomplished, and then slows down somewhat, indicating either that not all the phage particles have the same affinity for the bacteria, or that equilibrium is being approached. Other experiments not recorded here suggest that, if an equilibrium exists, it lies too far in favor of adsorption to be readily detected. This equation expresses the rate of adsorption even when a tenfold excess of phage over bacteria is present, indicating that a single bacterium can accommodate a large number of phage particles on its surface, as found by several previous workers (5, 10). Krueger (10) found a true equilibrium between free and adsorbed phage. The absence of a detectable desorption in our case may result from the fixation of adsorbed phage by growth processes, since our conditions permitted growth, whereas Krueger’s experiments were conducted at a temperature at which the phage could not grow. Growth of Phage Following adsorption of the phage particle on a susceptible bac- terium, multiplication occurs, though this is not apparent as an in- crease in the number of plaques until the bacterium releases the resulting colony of phage particles into the solution. Because the adsorption under proper conditions is so rapid and complete (as shown above) experiments could be devised in which only the influence of the processes following adsorption could be observed. The details of these experiments were as follows: 0.1 cc. of a phage suspension of appropriate concentration was added to 0.9 cc. of an 18 hour broth bacterial culture, containing about 2 X 108 B. coli / cc. After standing for a few minutes, 70 to 90 per cent of the phage was attached to the bacteria. At this time, the mixture was diluted 50- fold in broth (previously adjusted to the required temperature) and incubated. Samples were removed at regular intervals, and the concentration of infective centers determined. 45 374 GROWTH OF BACTERIOPHAGE The results of three experiments at 37°C. are plotted in Fig. 2, and confirm the suggestion of d’Herelle that phage multiplies under a spatial constraint, z.e. within or upon the bacterium, and is suddenly liberated in a burst. It is seen that after the initial rise (discussed above) the count of infective centers remains constant up to 30 minutes, and then rises about 70-fold above the initial value. The rise corresponds to the liberation of the phage particles which have multiplied in the initial constant period. This interpretation was verified by measurements of the free phage by centrifuging out the infected bacteria, and determining the number of phage particles in the supernatant liquid. The free phage concentration after adsorp- tion was, of course, small compared to the total and remained constant up to the time of the first rise. It then rose steeply and became sub- stantially equal to the total phage. The number of bacteria lysed in this first burst is too small a fraction of the total bacteria used in these experiments to be measured as a change in turbidity; the ratio of uninfected bacteria to the total possible number of infected bacteria before the first burst is 400 to 1, the largest number of bacteria which can disappear in the first burst is therefore only 0.25 per cent of the total. The phage particles liberated in the first burst are free to infect more bacteria. ‘These phage particles then multiply within or on the newly infected bacteria; nevertheless, as before, the concentration of infective centers remains constant until these bacteria are lysed and release the phage which they contain into the medium. This gives the second burst which begins at about 70 minutes from the start of the experiment. Since the uninfected bacteria have been growing during this time, the bacteria lysed in the second burst amount to less than 5 per cent of the total bacteria present at this time. There is again therefore, no visible lysis. This process is repeated, leading to a third rise of smaller magnitude starting at 120 minutes. At this time, inspection of the culture, which has until now been growing more turbid with the growth of the unin- fected bacteria, shows a rapid lysis. The number of phage particles available at the end of the second rise was sufficient to infect the re- mainder of the bacteria. These results are typical of a large number of such experiments, at 46 EMORY L. ELLIS AND MAX DELBRUCK 315 37°, all of which gave the 70-fold burst size, z.e. an average of 70 phage particles per infected bacterium, occurring quite accurately at the time shown, 30 minutes. Indeed, one of the most striking features of these experiments was the constancy of the time interval from adsorption to the start of the first burst. The magnitude of the rise (70-fold) was likewise readily reproducible by all phage preparations which had been stored under proper conditions to prevent deterioration (see above). Multiple Infection The adsorption measurements showed that a single bacterium can adsorb many phage particles. The subsequent growth of phage in these ‘‘multiple infected” bacteria might conceivably lead to (a) an increase in burst size; (6) a burst at an earlier time, or (c) the same burst size at the same time, as if only one of the adsorbed particles had been effective, and the others inactivated. In the presence of very great excesses of phage, Krueger and Northrop (3) and Northrop (2) report that visible lysis of the bacteria occurs in a very short time. It was possible therefore, that in our case, the latent period could be shortened by multiple infection. To determine this point, we have made several experiments of which the following is an example. 0.8 cc. of a freshly prepared phage suspension containing 4 X 10° particles per cc. (assay corrected for efficiency of plating) was added to 0.2 cc. of bacterial suspension containing 4 < 10° bacteria per cc. The ratio of phage to bacteria in this mixture was 4 to 1. 5 minutes were allowed for adsorption, and then the mixture was diluted 1 to 12,500 in broth, incubated at 25°, and the growth of the phage followed by plating at 20 minute intervals, with a control growth curve in which the phage to bacteria ratio was 1 to 10. No significant difference was found either in the latent period or in the size of the burst. The bacteria which had adsorbed several phage particles behaved as if only one of these particles was effective. Effect of Temperature on Latent Period and Burst Size A change in temperature might change either the latent period, 7.e. the time of the burst, or change the size of the burst, or both. In order to obtain more accurate estimates of the burst size it is desirable 47 376 GROWTH OF BACTERIOPHAGE to minimize reinfection during the period of observation. This is obtained by diluting the phage-bacteria mixture (after initial contact to secure adsorption) to such an extent that the rate of adsorption then becomes extremely small. In this way, a single ‘‘cycle” of growth, (infection, growth, burst) was obtained as the following example Relative phage concentration Time Fic. 3. One-step growth curves. A suitable dilution of phage was mixed with a suspension of bacteria con- taining 2 < 10° organisms per cc. and allowed to stand at the indicated tempera- ture for 10 minutes to obtain more than 90 per cent adsorption of the phage. This mixture was then diluted 1:10* in broth, and incubated. It was again diluted 1:10 at the start of the first rise to further decrease the rate of adsorption of the phage set free in the first step. The time scales are in the ratio 1:2:6 for the temperatures 37, 25, and 16.6°C. Log P/Po is plotted, Po being the initial con- centration of infective centers and P the concentration at time ¢. The broken line indicates the growth curve of the bacteria under the corresponding conditions. 48 EMORY L. ELLIS AND MAX DELBRUCK SUE shows. 0.1 cc. of phage of appropriate and known concentration was added to 0.9 cc. of an 18 hour culture and allowed to stand in this con- centrated bacterial suspension for 10 minutes at the temperature of the experiment. This mixture was then diluted 1:104 in broth and incubated at the temperature chosen. Samples of this diluted mix- ture were withdrawn at regular intervals and assayed. ‘The results of three such experiments are plotted in Fig. 3. The rise corresponds to the average number of phage produced per burst, and its value can be appraised better in these experiments than in the complete growth curve previously given (Fig. 2) where there is probably some over- lapping of the steps. In these experiments the rise is seen to be practically identical at the three temperatures, and equals about sixty particles per infected bacterium, but the time at which the rise oc- curred was 30 minutes at 37°, 60 minutes at 25°, and 180 minutes at 16.6°. This shows that the effect of temperature is solely on the latent period. We have also made separate measurements of the rate of bacterial growth under the conditions of these experiments. They show that the average division period of the bacteria in their logarithmic growth phase varies in the same way with temperature, as the length of the latent period of phage growth. The figures are: Temperature Division period of B Latent period of P growth ¢ min. min. 16.6 About 120 180 25 42 60 37 21 30 There is a constant ratio (3/2) between the latent period of phage growth and the division period of the bacteria. This coincidence suggests a connection between the time required for division of a bacterium under optimum growth conditions, and the time from its infection by phage to its lysis. Individual Phage Particle The growth curves described above give averages only of large numbers of bursts. They can, however, also be studied individually, as was first done by Burnett (11). 49 378 GROWTH OF BACTERIOPHAGE If from a mixture containing many particles very small samples are withdrawn, containing each on the average only about one or less particles, then the fraction p; of samples containing r particles is given by Poissons’ (12) formula, ne” br = (1) r! where m is the average number of particles in a sample and e is the Napierian logarithm base. If the average number is unknown, it can be evaluated from an experimental determination of any single TABLE II Distribution of Individual Particles among Small Samples A suitably diluted phage preparation was added to 5 cc. of 18 hour bacteria culture and 0.1 cc. samples of this mixture were plated. The distribution of particles among the samples is that predicted by formula (1). pr (experimental) br (calculated) 0 plaques on 13 plates 0.394 0.441 1 plaque “ 14 “ 0.424 0.363 2 plaquesii, 159) 0.151 0.148 Suet so) Aaplate 0.033 0.040 pO ke “0 plates 0.000 0.008 Dili Cane Sy a 1.002 1.000 one of the ,, for instance from a determination of po, the fraction of samples containing no particles: n = —In po (2) Let us now consider the following experiment. A small number of phage particles is added to a suspension containing bacteria in high concentration. Within a few minutes each phage particle has at- tached itself toa bacterium. The mixture is then diluted with a large volume of broth, in order to have the bacteria in low concentration so that after the first burst a long time elapses before reinfection, as in the one step growth curves. Samples (0.05 cc.) are removed from this mixture to separate small vials and incubated at the desired temperature. If these samples are plated separately (after adding a drop of bacterial suspension to each vial) before the occurrence of bursts, the fraction of the plates containing 0, 1, 2, etc. plaques is found 50 EMORY L. ELLIS AND MAX DELBRUCK 379 to conform to formula (1) (see Table II). In this experiment we could also have inferred the average number of particles per sample, using formula (2), from the fraction of the plates showing no plaques (giving 0.93 per sample) instead of from the total number of plaques (27/33 = 0.82 per sample). Experimental Measure of Efficiency of Plating If the samples are incubated until the bursts have occurred, and then plated, the samples which had no particles will still show no plaques, those with one or more particles will show a large number, depending on the size of the burst, and on the efficiency of plating. In any case, if we wait until all bursts have occurred, only those samples which really contained no particle will show no plaques, quite inde- pendent of any inefficiency of plating. From this fraction of plates showing no plaques we can therefore evaluate the true number of particles originally present in the solution, and by comparison with the regular assay evaluate the efficiency of plating. In this way we have determined our efficiency of plating to be about 0.4. For instance, one such experiment gave no plaques on 23 out of 40 plates, and many plaques on each of the remaining plates. This gives 23 po = 40° 0.57 from which m = 0.56 particles per sample. A parallel assay of the stock phage used indicated 0.22 particles per sample; = 0.39. This plating effi- a 0.2 the plating efficiency was therefore 0.56 ciency remains fairly constant under our standard conditions for assay. The increase in probability of plaque formation which we suppose to take place following the infection of a bacterium by the phage particle, z.e. the initial rise, brings the plating efficiency up to 0.65. The Burst Size Single particle experiments such as that described above, revealed a great fluctuation in the magnitude of individual bursts, far larger than one would expect from the differences in size of the individual bacteria in a culture; indeed, they vary from a few particles to two hundred or more. Data from one such experiment are given in Table ITI. 51 380 GROWTH OF BACTERIOPHAGE We at first suspected that the fluctuation in burst size was con- nected with the time of the burst, in that early bursts were small and late bursts big, and the fluctuation was due to the experimental superposition of these. However, measurement of a large number of bursts, plated at a time when only a small fraction of the bursts had occurred, showed the same large fluctuation. We then suspected that the particles of a burst were not liberated simultaneously, but over an interval of time. In this case one might expect a greater homogeneity TABLE III Fluctuation in Individual Burst Size 97.9 per cent of phage attached to bacteria in presence of excess bacteria (10 minutes), this mixture diluted, and samples incubated 200 minutes, then entire sample plated with added bacteria. 25 plates show O plaques Bursts 1 plate shows 1 plaque 14 plates show bursts Average burst size, taking account of probable doubles = 48 130 58 26 123 83 9 31 5 53 48 72 45 190 9 i (0) 6: Learner berm ee ree tt cnr ora cise! didlo Oran ch Gl eae 882 plaques in burst size, if measurements were made at a late time when they are at their maximum value. This view also was found by experiment to be false. The cause of the great fluctuation in burst size is therefore still obscure. DISCUSSION The results presented above show that the growth of this strain of phage is not uniform, but in bursts. These bursts though of constant 52 EMORY L. ELLIS AND MAX DELBRUCK 381 average size, under our conditions, vary widely in individual size. A burst occurs after a definite latent period following the adsorption of the phage on susceptible bacteria, and visible lysis coincides only with the last step-wise rise in the growth curve when the phage parti- cles outnumber the bacteria present. It seemed reasonable to us to assume that the burst is identical with the lysis of the individual bacterium. Krueger and Northrop (3), in their careful quantitative studies of an anti-staphylococcus phage came to an interpretation of their results which differs in some important respects from the above: 1. Their growth curves were smooth and gave no indication of steps; they concluded therefore that the production of phage is a continuous process. 2. In their case, the free phage during the logarithmic phase of a growth curve was an almost constant small fraction of the total phage. This led them to the view that there is an equilibrium between intra- cellular and extracellular phage. With an improved technique, Krueger (10) found that the fraction of free phage decreased in propor- tion to the growth of the bacteria, in conformity with the assumption of an equilibrium between two phases. 3. Krueger and Northrop (3) found that visible lysis occurred when a critical ratio of total phage to bacteria had been attained, and they assumed that there was no lysis in the earlier period of phage growth. To appreciate the nature of these differences it must be born in mind that their method of assay was essentially different from ours. They used, as a measure of the “activity” of the sample of phage assayed, the time required for it to lyse a test suspension of bacteria under standard conditions. This time interval, according to the picture of the growth process given here, is the composite effect of a number of factors: the average time required for adsorption of free phage, its rate of growth in the infected bacteria, the time and size of burst, and the average time required for repetition of this process -until the number of phage particles exceeds the number of bacteria and infects substantially all of them. Then, after a time interval equal to the latent period, lysis occurs. This lysis assay method tends to measure the total number of phage particles rather than the number of infective centers as the 53 382 GROWTH OF BACTERIOPHAGE following considerations show. Let us take a sample of a growth mixture in which is suspended one infected bacterium containing fifty phage particles. If this sample is plated, it can show but a single plaque. However, if the sample is assayed by the lysis method, this single infective center soon sets free its fifty particles (or more, if multiplication is still proceeding) and the time required to attain lysis will approximate that for fifty free particles rather than that fora single particle. Since the burst does not lead to an increase in the number of phage particles, but only to their dispersion into the solution, the lysis method cannot give any steps in the concentration of the fofal phage in a growth curve. On the other hand one might have expected a step-wise in- crease in the concentration of free phage. However, the adsorption rate of the phage used by Krueger (10) is so slow that the infection of the bacteria is spread over a time longer than the presumed latent period, and therefore the bursts would be similarly spread in time, smoothing out any steps which might otherwise appear. Moreover, their measurements were made at 30 minute intervals, which even in our case would have been insufficient to reveal the steps. The ratio between intracellular and extracellular phage would be determined, according to this picture of phage growth, by the ratio of the average time of adsorption to the average latent period. The average time of adsorption would decrease as the bacteria increased, shifting the ratio of intracellular to extracellular phage in precisely the manner described by Krueger (10). As we have indicated in the description of our growth curves, lysis of bacteria should become visible only at a late time. Infection of a large fraction of the bacteria is possible only after the free phage has attained a value comparable to the number of bacteria, and visible lysis should then set in after the lapse of a latent period. At this time the total phage (by activity assay) will be already large compared to the number of bacteria, in agreement with Krueger and Northrop’s findings. It appears therefore that while Krueger and Northrop’s picture does not apply to our phage and bacteria, their results do not exclude for their phage the picture which we have adopted. It would be of fundamental importance if two phages behave in such a markedly different way. 54 EMORY L. ELLIS AND MAX DELBRUCK 383 SUMMARY 1. An anti-Escherichia coli phage has been isolated and its behavior studied. 2. A plaque counting method for this phage is described, and shown to give a number of plaques which is proportional to the phage concen- tration. The number of plaques is shown to be independent of agar concentration, temperature of plate incubation, and concentration of the suspension of plating bacteria. 3. The efficiency of plating, 7.e. the probability of plaque formation by a phage particle, depends somewhat on the culture of bacteria used for plating, and averages around 0.4. 4. Methods are described to avoid the inactivation of phage by substances in the fresh lysates. 5. The growth of phage can be divided into three periods: adsorp- tion of the phage on the bacterium, growth upon or within the bac- terium (latent period), and the release of the phage (burst). 6. The rate of adsorption of phage was found to be proportional to the concentration of phage and to the concentration of bacteria. The rate constant kg is 1.2 X 10-9 cm.3/min. at 15°C. and 1.9 x 10-9 cm.?/min. at 25°, 7. The average latent period varies with the temperature in the same way as the division period of the bacteria. 8. The latent period before a burst of individual infected bacteria varies under constant conditions between a minimal value and about twice this value. 9. The average latent period and the average burst size are neither increased nor decreased by a fourfold infection of the bacteria with phage. 10. The average burst size is independent of the temperature, and is about 60 phage particles per bacterium. 11. The individual bursts vary in size from a few particles to about 200. The same variability is found when the early bursts are mea- sured separately, and when all the bursts are measured at a late time. One of us (E. L. E.) wishes to acknowledge a grant in aid from Mrs. Seeley W. Mudd. Acknowledgment is also made of the assistance of Mr. Dean Nichols during the preliminary phases of the work. 35 384 GROWTH OF BACTERIOPHAGE Oo fe WNW ~I REFERENCES . Schlesinger, M., Biochem. Z., Berlin, 1934, 273, 306. . Northrop, J. H., J. Gen. Physiol., 1938, 21, 335. . Krueger, A. P., and Northrop, J. H., J. Gen. Physiol., 1930, 14-, 223. , Burnet, ho Ms brit Je ean Pai.. 1927. 8 121, . Burnet, F. M., Keogh, E. V., and Lush, D., Australian J. Exp. Biol. and Med. Sc., 1937, 15, suppl. to part 3, p. 227. }. Heidelberger, M., Kendall, F. E., and Scherp, H. W., /. Exp. Med., 1936, 64, 559. . d’Herelle, F., The bacteriophage and its behavior, Baltimore, The Williams & Wilkins Co., 1926. . Dreyer, C., and Campbell-Renton, M. L., J. Path. and Bact., 1933, 36, 399. . Bronfenbrenner, J. J., and Korb, C., Proc. Soc. Exp. Biol. and Med., 1923, 21, 315. . Krueger, A. P., J. Gen. Physiol., 1931, 14. 493. . Burnet, F. M., Brit, J. Exp. Path., 1929, 10, 109. . Poisson, 8. D., Recherches sur la probabilité des jugements en matiére criminelle et en matiére civile, précédées des régles générales du calcul des probabilités, Paris, 1837. 56 THE GROWTH OF BACTERIOPHAGE AND LYSIS OF THE HOST By M. DELBRUCK* (From the William G. Kerckhoff Laboratories of the Biological Sciences, California Institute of Technology, Pasadena) (Received for publication, January 29, 1940) Introduction and Statement of Main Result Bacteriophage grows in the presence of living susceptible bacteria. In many but not all cases the growth of phage leads finally to a lysis of the bacterial cells, a phenomenon which in dense cultures manifests itself to the naked eye as a clearing of the bacterial culture. The exact nature of the connection between the growth of the phage and the dissolution of the cells has been a subject of controversy since the original discoveries of d’Herelle in 1917. D’Herelle believed that lysis is the process by which the phage, which has grown within the bacterium, is liberated from the cell and dispersed in solution. Many later authors, notably Burnet, have concurred with him on this point. Last year Ellis and Delbriick (1) published detailed evi- dence that phage liberation in B. coli occurs in sudden bursts and showed that all the evidence was compatible with the assumption that in sensitive strains the bursts of phage liberation occurred only if and when a cell is lysed. Northrop and Krueger (3-5) on the other hand have developed ideas along a somewhat different line in the course of their extensive research with a strain of Staphylococcus aureus and a bacteriophage active against it. Bordet (2) had put forward the conception that phage production followed by lysis is a more or less normal physiological function of the bacteria. In lysogenic strains where visible lysis never occurs it can be put into close analogy with the production of an extra-cellular enzyme. Northrop’s and Krueger’s work served to substantiate this view also in their case where the phage growth leads finally to the dissolution of the bacteria. In their view lysis of the bacteria is a secondary and incidental activity of the phage. Krueger and Northrop (3) found first that clearing, if it occurs at all, begins when a certain threshold value in the ratio total phage/bacteria is * Fellow of The Rockefeller Foundation. Reprinted by permission of the author and The Rockefeller Institute from Tor JOURNAL OF GENERAL PuystoLoey, 23 (5), 643-660, May 20, 1940. 57 644 GROWTH OF PHAGE AND LYSIS overstepped. A considerable loss in total phage parallels the clearing of the culture. If sufficient phage is added so that the ratio phage/bacteria is greater than the threshold value lysis begins almost at once. This experi- ment was later repeated and confirmed by Northrop (4, 5) with purified concentrates of phage. Recently Northrop (5) found with a susceptible megatherium strain and homologous phage that the bulk of the phage was liberated before the cul- ture began to clear. He found further with a lysogenic strain which never showed clearing but produced phage lysing the sensitive strain, that the yield in phage from this lysogenic strain was large compared to the number of bacteria present in the culture. All these results indicate that in these strains lysis, if it occurs at all, is brought about by a mass attack of the phage on the bacteria after the phage have grown and been liberated into solution. We have now studied in more detail the relation between phage growth and lysis in a new sensitive strain of B. coli and homologous phage and have obtained results which may offer a basis for reconciling the two diverging lines of interpretation. We have found in this strain two entirely different types of lysis, which we designate as “Lysis from within” and “Lysis from without.” Lysis from without is brought about almost instantly by adsorption of phage at a threshold limit, which is equal to the adsorption capacity of that bacterium. No phage are liberated in this case, on the contrary, the ad- sorbed phage are lost. The phage attack the cell wall in such a way as to permit swelling of the cell, and its deformation into a spherical body. Lysis from within is brought about by adsorption of one (or few) phage particle(s). Under favorable conditions this one phage particle multiplies during a latent period within the bacterium up to a threshold value (which is equal to the adsorption capacity). When the threshold value is reached, and not before, the phage is liberated by a sudden destruction of the proto- plasmic membrane, which permits a rapid exudation of the cell contents without deformation of the cell wall. It would seem that the results of other observers may be explained by postulating that (a) In the case of Staphylococcus aureus the observable clearing is caused by lysis from without. Lysis from within either does not exist here and is replaced by continuous phage secretion; or it exists but leads only to a slow equalization of the refractive indices of the cell interior and the milieu. The decrease of total phage during lysis is caused by the adsorption of phage in the process of lysis from without. 58 M. DELBRUCK 645 (6) In the case of B. megatherium 36, sensitive, (Northrop (5)) the same relations hold. (c) In the case of B. megatherium 899, lysogenic, (Northrop (5)), lysis from without does not occur, although the bacteria can adsorb a few phage particles each. Both the phage production capacity and the phage ad- sorption capacity are far smaller than the corresponding value for the same phage acting on the sensitive strain. The equation Adsorption capacity = maximum yield of phage per bacterium was found to hold true both for bacteria in the phase of rapid growth, and for saturated bacterial cultures that had been aerated for 24 hours and consisted only of very small bacteria. This equality points to a material connection between the bacterial con- stituents which can adsorb the phage and the new phage formed when it grows. ‘These bacterial constituents we shall call 6. It might be assumed that 6, which the bacterium constantly produces without the help of phage (and in some cases also secretes), is part of the precursor which under favorable conditions is transformed into phage after combination of the bacterium with a phage particle from without. The complex bP might be the catalyst which im the cell transforms uncombined 0 into phage. The difference between a sensitive strain and a lysogenic strain would consist in this: in the sensitive strain the reaction b oat NLL phage (in the cell) would be faster than the production of 0 (in the cell). In the lysogenic strain 6 would be produced faster than it is converted into phage. This permits the bacterium and the phage to grow. The extremely interesting but puzzling observations of Burnet and McKie (6) on lysogenesis of different variants of one strain of B. enteriditis Gaertner, and of Burnet and Lush (7) on induction of resistance and lysogenesis by the phage in a strain of Staphylococcus albus may perhaps allow further analysis in the light of these speculations. EXPERIMENTAL The strains of B. coli and of homologous phage used in this work were obtained from the Pasadena Junior College, through the courtesy of Mr. F. Gardner. They have not been studied before and will be designated as By and Pg, in distinction to the strains B, and P; used last year by Ellis and Delbriick (1). Growth curves of Bz in Difco nutrient broth at 37° (by colony counts) are shown in Fig. 1. The maximum division rate is considerably smaller than that of Bj. 59 646 GROWTH OF PHAGE AND LYSIS TIME IN HOURS Fic. 1. Growth of bacteria in broth at 37°. The inoculating bacteria were taken from an 18 hour not aerated broth culture. Without aeration the growth reaches saturation at 108 B/cc. With aeration the growth proceeds further beyond 10° B/cc. The maximum growth rate is in both cases equal and corresponds to an average division time of 30 minutes. The cessation of growth in the unaerated culture is therefore caused by lack of air. This is further supported by the top curve, which shows how the unaerated 18 hour culture, without the addition of fresh broth, proceeds to grow, when aerated, and reaches about 2 x 10° B/cc. Fic. 2. Growth of the standard experimental culture of bacteria in broth at 37°. At time zero 0.1 cc. of a 24 hour stock culture was added to 25 cc. broth. Every 7.5 minutes platings were made for colony counts. The plotted values show complete agreement with Hershey’s (11) finding that such aerated bacteria exhibit a sudden transition from the phase of cell enlargement (lag period) to the phase of cell division. The lag period is 2 hours, if defined as the time required for increase in cell number by a factor 1.5 (compare Hershey’s discussion (11)). 60 M. DELBRUCK 647 Synthetic Medium.—The bacteria and phage were also grown with aeration in a synthetic medium, consisting of PENS PALARINE 6. Mice sa fefesi - 17 16 170 250 pAthrsaeratedsbacterlayys ca. seas eres oss « 30 22 20 20 would have been qualitatively the same but quantitatively quite different (see Fig. 5). Table III lists the respective values. The constant rate of phage liberation in the best one step growth curves 67 654 GROWTH OF PHAGE AND LYSIS Lop e (2) EN TIME {IN MINUT 5 > cos sOMEONTSO" 60 70 80 Fic. 5. One step phage growth on 24 hour aerated bacteria. Direct plot. At time zero 2 < 108 phage/cc. were added to 1 cc. of a 24 hour stock culture of bacteria, containing 2 X 10% bacteria/cc. After 5 minutes free phage were determined and suitable high dilutions in broth were further incubated at 37° with aeration. At intervals samples from these mixtures were plated for plaque count. It is seen that the plaque count stays constant for 30 minutes, then increases linearly with time till about 50 minutes when it reaches twenty times the original value. After 50 minutes it stays nearly constant. Phage liberation takes place uniformly during 20 minutes. fies z I = 2. Lysis *. 4 ee A nce O-22@es= Oe eac=s OO ° ul & = Alles ag <_f ae 2 (> oO os f 5 (OMS 20025 «= 30S SaaS Fic. 6. Addition of a great excess of phage to a growing culture of bacteria, at 3% Date P/B log P/B P bound/B’ || Pte a per cent e 9-13 120 2.08 115 4 x 9-22 60 1.78 57 5 Oo 10-26 500 2.70 250 50 A 11-20 700 2.85 270 60 68 M. DELBRUCK 655 (Fig. 4) permits a closer analysis. Since the liberation of phage from the individual bacterium probably occurs quite suddenly when the bacterium is lysed our result means that the infected bacteria represent a mixture of groups with latent periods ranging between 17 and 33 minutes and that there is a uniform distribution of bacteria over this whole range of latent periods. The question arises as to what causes a bacterium to have a shorter or longer latent period. Several hypotheses might be suggested, either by ascribing the cause to statistical fluctuations of reactions involving a small number of particles (10), or by connecting it in one way or another with the bacterial cycle. The latter view seems to the author the more likely one but since it has not yet been worked out, further discussion will be deferred. Multiple Infection It was reported by Ellis and Delbriick (1) that if a bacterial suspension is infected with an excess of phage no changes occur in the latent period or in the burst size. At that time no phage concentrates were available and the maximum ratio of phage to bacteria attained in that work was only four to one. We have repeated this work with our new strains and with the concen- trates and have been able to work with much higher ratios of phage/bac- teria, up to 700 to 1. Fig. 6 shows some of the results obtained with the high ratios. Samples were assayed every 3 or 5 minutes. Since the assays here require several dilution steps these growth curves are less accurate both with respect to assay values and with respect to timing. The results show an initial decrease in plaque count because many phage particles are bound to one bacterium which then gives only one plaque. For instance, starting with 10'° phage/cc. and a hundred times less bacteria, one finds initially 10° plaques/cc. After 10 minutes only 5 per cent of the phage will be left free; these will give 5 x 10° plaques/cc. In addition the 10° bacteria/cc., each having adsorbed on the average 95 phage particles, will give 10% plaques/cc., bringing the total plaques to 6 X 108/cc. If the initial ratio phage/bacteria is greater than a certain critical value the bac- teria show saturation. This saturation value depends on the physiological state of the bacteria. For instance, for rapidly growing bacteria, if the ratio is 500, the plaque count decreases only by a factor two. The satura- tion value is therefore 250. On the other hand, for 24 hour aerated bac- teria, the saturation value is only about 20. (See Table III, last column.) We have indicated in the figure the time during which clearing of the mix- 69 656 GROWTH OF PHAGE AND LYSIS ture occurs. If the initial ratio of phage/bacteria is smaller than the satu- ration value, clearing occurs only slightly earlier than in a one to one mixture, and it is accompanied by a noticeable increase in the plaque count. On the other hand if the initial ratio of phage/bacteria is greater than the saturation value, clearing occurs much earlier and is not accompanied by an increase in plaque count. In both cases the final plaque count is considerably smaller than the in- itial one; we have, in effect, a phage destruction by the adsorption that causes lysis. One can see the difference between the two types of lysis with the naked eye. A culture of rod shaped bacteria, like B. coli, shows flow lines on shaking due to the orientation of the rods under the influence of the shearing forces of unequal flow. In lyses under the influence of great excess of phage these flow lines disappear before the culture clears up, because the rods are transformed into spherical bodies before they disappear, as described in the section on microscopic observations. Growth of Phage and Lysis of Bacteria When Equivalent Numbers Are Mixed It can be predicted that a disturbance must arise when equivalent amounts of phage and bacteria are mixed, due to the fact that the phage that are liberated from the first lysing bacteria will cause an excess of phage over bacteria to be present. These phage will be adsorbed on bacteria that are already infected and will therefore not show up in a plaque count assay. They will moreover interfere with the phage growth in these bacteria and in some of them cause a lysis from without. Qualitatively the following can be predicted. We have seen that the phage will be liberated at a constant rate (after the lapse of the minimum latent period of 17 minutes). They will be adsorbed at a rate that is pro- portional to the phage concentration and to the bacterial concentration. The phage concentration is constantly increasing and the bacterial con- centration is constantly decreasing (due to lysis). The adsorption rate will therefore pass through a maximum and the net free phage production rate will pass through a minimum. ‘The net result is the appearance of a point of inflection, z.e. a secondary step in the phage growth curve, in some cases even a temporary decrease in the free phage if the rate of adsorption at any time exceeds the rate of phage liberation. Because of the loss of phage by adsorption and partial lysis from without the total step size must be smaller than in a one step growth curve where the bacteria are in excess and where multiple adsorption is prevented by extreme dilution, after ad- sorption of the parent phage. 70 M. DELBRUCK 657 These predictions are borne out by the experimental results. Fig. 7 shows three such growth curves where nearly equivalent amounts of phage and bacteria were mixed at time zero. The diminished yield is very pronounced and the secondary step is discernible in two sets of observational points. The condition of single infection of all bacteria at zero time can of course be realized only approximately. Even if exactly equivalent amounts 200 P/B aSioeihe 1.8 ao s a LJ 100 5 decreasing adsorbent in o | = J ae 50 ~ xa a Ba e Gy Oe Constant adsorbent 10 ee ere ae i © | 20 TIME 30 IN MINUTES O Fic. 7. Growth of phage if equivalent high concentrations (~10%/cc.) of phage and bacteria are mixed at time zero. Direct plot. Besides the experimental points from three growth curves three theoretical curves are drawn in the figure. These are 1. A one step growth curve with B in excess, taken from Fig. 4. 2. A calculated growth curve, assuming inactivation of the liberated phage on bacteria not yet lysed. 3. Same, but assuming that the adsorbing power of the bacterial constituents respon- sible for it is unimpaired till the completion of phage liberation and then vanishes abruptly. The time interval from the beginning of clearing to its completion is indicated. It falls well on the ascending part of the one step growth curve. In the one-to-one growth curves this ascending part is soon counter-balanced by the multiple adsorption loss, so that clearing seems to occur during a phase of little phage liberation. were mixed, the phage would not infect all the bacteria, but distribute themselves according to the probability formulas derived by Poisson. If P/B = n there will be a fraction e~” of the bacteria uninfected. On the other hand our phage assays, though fully reliable as far as relative values go, are not as certain with respect to absolute value, because of the difficulty of obtaining an accurate determination of the efficiency of plating (¢. Ellis and Delbriick (1)). It is not possible to make a complete quantitative prediction of the growth curve because it is not known in detail how the adsorption en masse of phage to a bacterium 71 658 GROWTH OF PHAGE AND LYSIS that is already near a lysis from within will interfere with this process. It is also not quite certain whether those parts of the surface of the bacterium that adsorb the phage will lose their capacity of binding phage immediately upon lysis. In the strains used previously a slow decrease of phage assay after lysis could be ascribed to the continued “adsorption” of phage onto those scattered surface elements. No such decrease of phage assay was ever observed with the new strain. But such observations refer only to inactivation long after lysis and do not tell us whether the adsorbent is instantly destroyed upon lysis. We have therefore calculated growth curves on the basis of two extreme assumptions. (a) The amount of adsorbent decreases linearly from its initial value to zero during the 16 minutes in which the bacteria are lysed. (b) The amount of adsorbent stays constant at its initial value throughout the course of lysis. Case (a) is described by the differential equation dP/dt = A — kBy (1—t/T) P In case (b) we have dP/dt = A — kBoP In these equations the first term, A, represents the phage liberation by lysis during the interval 7, as determined in the one step growth curves, the second term is the decrease of phage due to adsorption either on the unlysed bacteria only (case a) or on the unlysed bacteria plus the adsorbent from the lysed bacteria (case 0). These equations can be integrated explicitly. We obtain in case (a) P = 3A a rT)? -8-Y* 1G (T/) — GUT — Al/s)] with r= V2T/kBo and G(x) the Gaussian integral G(x) = eal en?” dx In case (0), with constant adsorbent, the adsorption rate grows continuously with the free phage concentration. In this case we have therefore no point of inflection but a continuous asymptotic approach to the final titre A —kBot P=—(1— 0 Tirade Since all required constants are known from independent experiments, the par- ticular solutions applying to our case can be evaluated quantitatively. These have been plotted in Fig. 6. The experimental values fall between the limits set by these two cases. We have also plotted the curve obtained in an ordinary one step growth curve, with B in excess (taken from Fig. 4). The difference between this a2 M. DELBRUCK 659 curve and the experimental values is the amount of phage lost by adsorption. The data show that about one hundred phage are lost per bacterium. It is clear that this loss depends entirely on the rate of adsorption, which is determined by the product kBy. If we wish to increase the yield per bac- terium we have to decrease either By or k. Reduction of By brings us to the condition employed in the one step growth curves. Here the maxi- mum yield of phage growth per bacterium is obtained, but the actual con- centrations of phage are of course very small. A promising way of increasing the end titre of phage would be to reduce k, the adsorption rate constant. The very interesting experiments of Krueger and his coworkers (8, 9) on the influence of the addition of salts (NaCl and Na2SO,) to a growth mixture of phage and bacteria would seem to be completely in accord with the assumption that the adsorption rate constant is diminished in the presence of salt. In fact, a diminished adsorption rate constant should manifest itself in several ways in a phage growth curve, in which one starts with low concen- trations of both phage and bacteria. Namely 1. Delayed clearing, due to delayed adsorption, and therefore delayed phage growth. 2. Higher maximum concentration of bacteria, due to delayed lysis. 3. Higher end titre of phage, due to (a) higher number of bacteria producing phage (b) reduced loss of phage by multiple adsorption. 4. Higher ratio of free to bound phage during the stationary growth phase, due to the fact, that every phage particle spends a longer time in the free state between liberation and adsorption. 5. A period of constant bacterial concentration preceding lysis, when all bacteria are infected and cease to divide but when the phage concentration is not yet sufficient for lysis. Precisely these five differences from the normal course and no others were noted by Krueger and Strietmann (9) in their study of the influence of the addition of NazSQu. SUMMARY 1. A new strain of B. coli and of phage active against it is described, and the relation between phage growth and lysis has been studied. It has been found that the phage can lyse these bacteria in two distinct ways, which have been designated lysis from within and lysis from without. 2. Lysis from within is caused by infection of a bacterium by a single phage particle and multiplication of this particle up to a threshold value. 73 660 GROWTH OF PHAGE AND LYSIS The cell contents are then liberated into solution without deformation of the cell wall. 3. Lysis from without is caused by adsorption of phage above a threshold value. The cell contents are liberated by a distension and destruction of the cell wall. The adsorbed phage is not retrieved upon lysis. No new phage is formed. 4. The maximum yield of phage in a lysis from within is equal to the adsorption capacity. 5. Liberation of phage from a culture in which the bacteria have been singly infected proceeds at a constant rate, after the lapse of a minimum latent period, until all the infected bacteria are lysed. 6. If the bacteria are originally not highly in excess, this liberation is soon counterbalanced by multiple adsorption of the liberated phage to bacteria that are already infected. This leads to a reduction of the final yield. The author wishes to express his appreciation for the hospitality extended to him by the Biology Department of the California Institute of Technology during the tenure of a Fellowship of The Rockefeller Foundation. In particular he wishes to record his indebtedness to Dr. E. L. Ellis for constant help and advice and to Mr. F. Gardner for technical assistance. REFERENCES . Ellis, E. L., and Delbriick, M., J. Gen. Physiol., 1939, 22, 365. . Bordet, J., Proc. Roy. Soc. London, Series B, 1931, 107, 398. . Krueger, A. P., and Northrop, J. H., J. Gen. Physiol., 1930, 14, 223. . Northrop, J. H., J. Gen. Physiol., 1937, 21, 335. . Northrop, J. H., J. Gen. Physiol., 1939, 28, 59. . Burnet, F. M., and McKie, M., Australian J. Exp. Biol. and Med. Sc., 1929, 6, 277. . Burnet, F. M., and Lush, D., Australian J. Exp. Biol. and Med. Sc., 1936, 14, 27. . Scribner, E. J., and Krueger, A. P., J. Gen. Phystol., 1937, 21, 1. . Krueger, A. P., and Strietmann, W. L., J. Gen. Physiol., 1939, 22, 131. . Delbriick, M., J. Physic. Chem., 1940, 8, 120. . Hershey, A. D., J. Gen. Physiol., 1939, 23, 11. . Bronfenbrenner, J., Muckenfuss, R. S., and Hetler, D. M., Am. J. Path., 1927, 3, 562. . Bayne-Jones, S., and Sandholzer, L. A., J. Exp. Med., 1933, 57, 279. _ SCO ONAN PWN —_ — ao Ww bd 74 THE INTRACELLULAR GROWTH OF BACTERIOPHAGES I. LIBERATION OF INTRACELLULAR BACTERIOPHAGE 14 BY PREMATURE LysIS WITH ANOTHER PHAGE OR WITH CYANIDE By A. H. DOERMANN*: t¢ (From the Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor) (Received for publication, August 20, 1951) Direct studies of bacteriophage reproduction have been handicapped by the fact that the cell wall of the infected bacterium presents a closed door to the investigator in the period between infection and lysis. As a result it was im- possible to demonstrate the presence of intracellular phage particles during this so called latent period, and, much less, to estimate their number or to describe them genetically. This barrier has now been penetrated. It is the purpose of the first two papers of this series to describe two methods for disrupting infected bacteria in such a way that the intracellular phage particles can be counted and their genetic constitution analyzed. The first method used to liberate intracellular bacteriophage depends on the induction of premature lysis in infected bacteria by “lysis from without”’ which occurs when a large excess of phage particles is adsorbed on bacteria (1). It was found by nephelometric tests that T6 lysates are efficient in disrupting cells when moderately high multiplicities are used (2). The further observation was made that the addition of a large number of T6 particles to bacteria previ- ously infected with T4, would, under some conditions, cause liberation of T4 particles before the expiration of the normal latent period of these cells. It therefore seemed hopeful that a method of reproducibly disrupting infected bacteria could be developed on the basis of this preliminary knowledge. * The experiments described here were carried out while the author was a fellow of the Carnegie Institution of Washington. The author is indebted to Dr. M. Demerec and the staff of the Department of Genetics of the Carnegie Institution of Washington for providing facilities for this work. In particular the stimulating discussions with Dr. Barbara McClintock are gratefully acknowledged. The manuscript was prepared while the author held a fellowship in the Department of Biology of the California Institute of Technology. He is grateful to Dr. G. W. Beadle and the staff of that de- partment for their interest in this work, and especially to Dr. Max Delbriick for criticism of the manuscript. t Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee. Reprinted by permission of the author and The Rockefeller Institute from THE JOURNAL OF GENERAL PuysIoLoey, 35 (4), 645-656, March 20, 1952. 73 646 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I The first experiments in devising a method of this kind were made with phage TS (3). It was found that TS is liberated before the end of the latent period if the infected cells are exposed to a high excess of T6. However, the extremely low rate of adsorption of T5 coupled with difficulties in inactivation of unadsorbed phage by specific antisera indicated that this phage was a poor choice. Hence T4 was chosen because of its fast rate of adsorption and because of the availability of high titer antisera against it. The first experiments with T4, along with the TS results, showed conclusively that, by itself, lysis from without is not sufficiently rapid for the purpose of this investigation. It is likely that phage growth continues after the addition of the lysing agent To. There- fore the attempt was made to stop phage growth while T6 was allowed to ac- complish lysis from without. Low temperature could not be used for this purpose TABLE I Composition of the Growth Medium Material Amount | ie r : j Py | gm. per liter KH POR 00 OUR) AY Uae, BOE RL Po | 1.5 Nas HPO, (anhydrous) 425... Lee eRe ane eR Nloe nei 2th 28S ars. 3.0 IN Geo cates FOS 15 Se ln Pa rh a OR Seg Sa cil eon oh | 1.0 1 eas) OVE} (0) b Cee ed copes ae mo CP ad ly Ady hes, Co «ea | 0.2 Gly Cerole dine tency peel Dar Mn aR eM rele oS Wen s 2 a. 10.0 AWeid-=hy drolyzedscaseinie yar. ss ae rae ene i eas 5.0 di-Tryptopbamsas : Mees vue ei Mae ee Ne SOE es EE ee Sy 0.01 Gelatin tat chet ink See ey ee tes A RA TU mek 0.02 TR WEEN=8O by tas5 Bean ee coe aeh Rae De Svar aN os Reset te he) esy 0 ) * To reduce surface inactivation of free phage particles (4). since it also inhibits lysis from without. A search for a suitable metabolic in- hibitor was therefore undertaken, and cyanide was eventually chosen as the most suitable one. Materials and Methods The experiments described here were carried out with the system T4r4g! growing at 37°C. in Escherichia coli, strain B/r/1. The latter is a T1 resistant, tryptophan-de- pendent mutant of B/r obtained from Dr. E. M. Witkin. Two media were used in these experiments, namely the growth medium and the lysing medium. The composition of the growth medium used for both bacterial and phage cultures, is given in Table I. The lysing medium consists of growth medium with 1 The subscript refers to a particular r mutant of T4 which arose by mutation and was numbered after the system of Hershey and Rotman (12) using the high subscript number to avoid confusion with the mutants already described by Hershey and Rotman. 76 A. H. DOERMANN 647 the addition of one part in ten of a high titer T6 phage filtrate (concentration of T6 in lysing medium was ca. 4 X 10% particles per ml.) and cyanide brought to a final concentration of 0.01 m. Specially designed experiments showed that at this concentra- tion the cyanide does not inactivate free phage particles, nor does the amount which reaches the plate affect titration by interference with plaque development. T6 was used as the lysing phage because in several experiments it proved to be a more effective lysing agent than any of the other T phages tested. Since only single stocks of the phages were compared in the early experiments, the superiority of T6 over the other phages may have been due to a difference in the particular stock used, and not to an inherent difference among the phages. In fact, later experiments with different T6 stocks showed marked differences in lysing efficiency, and phage titer proved to be a poor criterion of lysing ability. The experiments described here were made with T6 stocks selected for their ability to induce lysis from without. The se- lections were made on the basis of nephelometric comparisons. Platings were made in agar layer (0.7 per cent agar) poured over nutrient agar plates (1.3 per cent agar), and in order to assay T4 in the presence of high titer T6, the in- dicator strain, B/6, was used. B/6 is completely resistant to T6 (no host range mutants have so far been found which will lyse the strain used here) and gives full efficiency of plating (compared to B) with T4. EXPERIMENTAL Experiments with the Standard Lysing Medium.—The experimental procedure used consisted essentially of a one-step growth experiment (5) with certain modifications. B/r/1 cells in the exponential growth phase were concentrated by centrifugation to about 10° cells per ml. To these concentrated bacteria T4r4g was added and this ad- sorption mixture was incubated for 1 to 2 minutes with aeration, allowing at least 80 per cent of the phage to be adsorbed to the bacteria. Then a 40-fold or larger dilution was made into growth medium containing anti-T4 rabbit serum. The serum inactivated most of the residual unadsorbed phage. After several minutes’ incubation in the serum tube, a further dilution was made to reduce the serum concentration to one of relative inactivity. The resulting culture will be referred to as the source culture (SC). The entire experiment was carried out with the infected bacteria from SC. The titer of infected B/r/1 in this tube was approximately 10° cells per ml. Simultaneously with the dilution into the tube containing serum, another dilution from the adsorption tube was made. From the latter an estimation of the unadsorbed phage was made by assaying the supernatant after sedimentation of the cells. This step permits calculation of the multiplicity of infection (5). From SC a further dilution of 1:20 was made at some time before the end of the latent period. The resulting culture, containing approximately 5 x 10° infected bac- teria per ml., was used for determining the normal end of the latent period and for estimating the average yield of phage per infected cell. It will be called the control growth tube (GT). In addition, a number of precisely timed 20-fold dilutions were made from SC into lysing medium. These were titrated after they had been incubated in the lysing medium for 30 minutes or longer. Serial platings from the lysing medium cultures over a longer period of time have shown that the phage titer remained constant after 30 minutes’ incubation. The titer calculated from these platings, divided by the a7 648 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I titer of infected bacteria given by the preburst control platings, gives the average yield per infected cell. As a working hypothesis, this yield was considered to be the average number of intracellular phage particles per bacterium at the time of dilution into the lysing medium. Dividing these numbers by the control burst size gives the fraction of the control yield found in the experimental lysing medium tubes. The results of several typical experiments are shown in Fig. 1 in which the data are plotted on semilogarithmic coordinates. The fraction of the control yield found in a given experimental culture is plotted against the time at which the dilution is made into lysing medium. Curve 1 shows the results from a single experiment in which the bacteria were infected with an average of 7 phage particles each. Curve 2 is the composite result of four experiments in which the bacteria were infected with single phage particles. Curve 3 is the control one- step growth curve derived from the control growth tube platings in the four experiments of curve 2. Several striking results can be seen in these experiments. First, it is clearly seen that during the early stages of the latent period the virus-host complex is inactivated by the cyanide-T6 mixture, and that not even the infecting particles are recovered. Even when 7 phage particles were adsorbed on each bacterium, less than two are recovered per cell at the earliest stage tested, and the shape of the curve suggests that if earlier stages had been tested, still fewer would have been recovered. In experiments with singly infected bacteria, the earliest tests indicated that less than one infected bacterium in 80 liberated any phage at all. A second point to be noted is that the multiplicity of infection appears to influence slightly the time at which phage particles can be recovered from the cell, and it continues to affect the fraction found in the bacteria at a given time. That this difference is a real one seems clear from the consistency among the points of curve 2. This result has been observed in each experiment, although the effect appeared to be less pronounced in some experiments made at the lower temperature of 30°C. Attention should also be drawn to the fact that the shape of the curves is clearly not exponential. In fact, it parallels with a delay of several minutes the approximately linear DNA increase observed in this system (6). In connection with the preceding experiments a test was made to establish whether the cyanide concentration chosen was maximally effective in inhibiting phage synthesis. Using the described technique, but changing the cyanide con- centration of the lysing medium from 0.01 m to 0.004 M and 0.001 » in parallel aliquots, no difference was detectable in the three lysing media. Thus 0.01 M cyanide is well beyond the minimum concentration necessary and was con- sidered to be adequate for these experiments. Action of Cyanide in the Absence of the Lysing Agent T6.—Cohen and Anderson (7) reported a loss of infectious centers when infected bacteria were incubated in the presence of the antimetabolite 5-methyltryptophan. Although the details 78 "syusWTI0dx9 UOIJDAJUI V[ZUIS BY} WOT 9AINI YZAOIZ daj}s-9UO [OIJUOD JY} SI ¢ AAIND ‘S}UsWedxX9 UOTD9JUT 9[ZUIS INOj a «MoI POATIAP ST Z PAIN] “T]29 rad sapoqaed a8evyd 7¢ YIM payazut oaM BII9}0eq 9} YIYM ul JUDUIIIIdxa a[BuIs & syUasaIdaI J aAIND) 4 ‘aunpasoid sisAy-aprurAd ay} Aq pauturtajap se ‘poued juazey ay} Sulnp uornjejndod aseydorojzoeq srvpnypaovsquI oy, “[ “OT o42 LV WNIGSAW HLMOYS Ni NOILVENON! JO S3SLANIW Ov se O£ Sb S2 ey SI Ol S fe) 100 % 10'0 > G3NIVLNOD B1dwys SOlrli lv ° (eo) ZABND HLMOYD TOYLNOD NIQIZIA TWNIS 40 LN30 Y3d DOERMANN o-__ A. H. 3 2 o2€ Iv Ips 001 79 650 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I of their experiments differed somewhat from those presented here, the loss of infectious centers in their experiments suggested testing whether cyanide could cause a similar loss of infected bacteria in the present procedure. An experi- ment was made which was identical with the standard cyanide lysis experiment 100 3 0 -------- INFECTED BACTERIA @-—CN O-—CN +T6é PER CENT OF FINAL YIELD IN CONTROL GROWTH CURVE 2 0° 5 20 25 MINUTES OF INCUBATION IN GROWTH MEDIUM AT 37° Fic. 2. The comparative effect of cyanide alone and cyanide plus T6 on singly infected bacteria at various stages in the latent period. except that T6 was omitted from one set and included in a parallel set of lysing medium cultures (Fig. 2). As in the case of 5-methyltryptophan, it is seen that cyanide alone caused a loss of infectious centers when added in the early stages of phage growth, although the loss is less than that produced by cyanide and T6 together. Furthermore, in the second half of the latent period, comparison of the two media showed clearly and surprisingly that a definite rise in titer of infective centers occurred even when the lysing agent, T6, was omitted from the 80 A, H. DOERMANN 651 lysing medium. In fact, during the second half of the latent period, phage liberation is identical in the two media. In order to see whether lysis is actually occurring and can account for the liberation of phage, a nephelometric experiment was made introducing CN™ at two points in the latent period. Three cultures of B/r/1 growing exponentially in growth medium were infected with T4r4s (ca. fivefold multiplicity). One culture served as a control for normal lysis. To the second culture cyanide (0.01 m final concentration) was added 7.5 minutes after addition of the virus and to CN” ADDED TO 2 T41,,AT Sime 8 @ {e) b (2) 20 PER CENT OF TURBIDITY OF FIRST READING rea) fe} 0 10 20 30 40 50 60 70 80 90 290 MINUTES AFTER ADDITION OF PHAGE TO BACTERIAL CULTURES Fic. 3. The turbidity of T4-infected bacterial cultures as affected by addition of cyanide at two stages in the latent period. the third tube 17.5 minutes after addition of the T4ras. The turbidities of these three cultures were followed with a nephelometer designed like that described previously by Underwood and Doermann (8), but with four separate units which permit independent readings on the four tubes without removing any of them from the instrument. The results indicate that CN~ added to infected bacteria early during the latent period does not induce lysis (Fig. 3). From the plaque count experiment (Fig. 2) it is seen that a loss of infective centers does occur. This loss must therefore be due to some cause other than lysis of these cells. In the later stages of the latent period, the turbidimetric experiment indicates that lysis occurs promptly upon the addition of CN~ to the culture 81 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I 652 ‘€ GAIN OJ VIP ay} 9AvS YOY sjuourtiedxa ay} Worf AAINI Y}MOIT dojs-aUO pABpU}s BY} SI f AAIND ‘s}UsWZadxo aprueAD ay} UI pasn seM sv avy aes ay} YIM OPPUI S}UIUILIOdX9 UOTJIaJUT 9]SUIS 9914} JO a}ISOdWIOD BSI ¢ BAIND ‘a1BY pequosap s}uawtuedxa Jay}O Jy} UI pasn }eY} URY} JURINUT “PT, WIIYIP B YA JUoWTIadxe UOIJIOJUT BISUIS BZ AAIND pu UOIJasUT (pjoJyaAy) ajdy[nw v syuasosdas [ aamng ‘ueydoydAy[AYyJow-¢ jo aouasoid ay} ul stsd] ainjeuoid Aq poutuajap se ‘posed juaze] ay} Sup uonepndod a8eydouajoeq aypnyfeovsqul ay], “fF “OT eff LY WNIGSW HLMOXS Ni NOILVENONI JO SALNNIW Sb Ov Se ! : OF Sz 02 Ss! ; ) S Oe 3 SAXND HLMOYS TONLNOD NI Q13IA IWNid JO LN39 Yad ° o2e tv Tbs 001 82 A. H. DOERMANN 653 (Fig. 3). The increase of infective centers in comparable cultures (Fig. 2) at these later stages is probably brought about by liberation of phage particles concurrent with this lysis. Experiments Using 5-Methyltryptophan as the Metabolic Inhibitor—In trying to find a suitable metabolic inhibitor for instantaneously stopping phage growth, a large number of experiments was done using the antimetabolite 5-methyltryptophan (SMT)? whose bacteriostatic action is blocked by trypto- phan (9). The technique used was similar to the cyanide lysis procedure except that tryptophan was omitted from the lysing medium and 5MT was used in place of cyanide. The results (Fig. 4) are quite similar to the cyanide results in all respects except one. They are similar in failure to recover any phage particles during the early stages of the latent period, in the difference between single and multiple infection, and in the shapes of the curves. They are different, however, in that both the single and the multiple infection curves are moved to the left along the time scale by 3 to 4 minutes. This indicates that more phage is liber- ated per cell if lysis is induced in the presence of 5MT than if it is brought about in the presence of CN~. This difference may be interpreted on the basis of two alternative hypotheses. First, it might be suspected that CN~ penetrates the cell and reaches its site of inhibition more quickly than 5MT. This would allow more phage repro- duction to go on between the time of exposure to the SMT and the time at which the cell breaks open. In this event, a higher concentration of SMT would enable penetration of an inhibitory amount in a shorter period of time, thus reducing the amount of phage found. To test this, the concentration of 5MT in the lysing medium was increased fivefold. No difference in the amount of phage liberated was found, suggesting that the rate of penetration of the poison is not limiting its effectiveness. A second hypothesis is that the reaction blocked by 5MT may be one of the earlier ones involved in the synthesis of phage constituents. At the time of addition of SMT many individual phages may already have acquired these constituents and thus be able to go on to maturity before lysis disperses the enzyme equipment of the infected cell. Cyanide, on the other hand, may block one of the terminal reactions in phage production, with the result that at a given time fewer individuals will have passed this reaction than will have passed the 5MT-inhibitable step. Consequently, fewer particles will be liberated when using cyanide than when SMT is used. DISCUSSION Earlier experiments (2) and tests made of the lysing efficiency of the T6 stocks used here indicate that rapid lysis occurs when T6 stocks are added in 2 Obtained through the courtesy of Dr. M. L. Tainter, Sterling-Winthrop Research Institute, Rensselaer, New York. 83 654 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I sufficient concentration to bacterial cultures. The very first experiments with bacteria infected with T5 (3) left no doubt that lysis from without by T6 will liberate T5 particles prematurely from infected bacteria. From the evidence contained in the present paper it cannot be definitely established whether the combined action of T6 and cyanide liberates all of the mature phage present in the cells. However, the fact that, during the terminal] stages of intracellular development, the cyanide-lysis method yields as much phage as does spontane- ous lysis, suggests that the cyanide method liberates all of the mature phage. Furthermore, during the second half of the latent period, exactly the same amount of phage is liberated by cyanide alone as by cyanide plus T6. This sug- gests that cyanide acts promptly in arresting phage growth. Otherwise one would expect to find a consistently higher number of phage particles in the cyanide medium than in the medium in which cyanide and T6 are combined. The experiments presented here therefore warrant the working hypothesis that mature intracellular phage is effectively liberated by the treatment described, and that the method gives a true picture of the intracellular phage population. The validity of this working hypothesis will be conclusively demonstrated for the phage T3 in the second paper of this series (10). The bearing of the present experiments on our concept of phage reproduction might be discussed here. The finding that the original infecting particles are not recoverable from the cells during the first stages of the latent period appears at first sight surprising. Nevertheless some indirect evidence indicates that this is to be expected. The discovery that yields from mixedly infected bacteria may con- tain new combinations of the genetic material of the infecting types (11-13) suggests that some alteration of the infecting particles may occur. Furthermore, in mixed infections of bacteria with unrelated phages only one type is repro- duced. The other type, although adsorbed on the cells, it not only prevented from multiplying but the infecting particle of that type is lost (5, 14). On the basis of multiple infection experiments with ultraviolet-inactivated phage particles, Luria (15) has proposed that reproduction of phage occurs by reproduction of subunits which are at some later stage assembled into complete virus particles. The failure to find infective phage particles within the infected cell in the early stages of reproduction agrees with what would have been predicted from these experiments. The results of our experiments agree quite well with the scheme which Latarjet (16) suggested on the basis of x-ray inactivation studies of phage inside infected bacteria. Latarjet differentiated three segments of the latent period of phage growth. Using T2 he found that during the first segment of 6 to 7 minutes’ duration, singly infected bacteria show the same inactivation charac- teristics as do unadsorbed phage particles. In the second period, from time 7 to time 13 minutes, the phage in infected cells became more resistant to x-rays, even during the first 2 minutes of this segment in which the inactivation curves 84 A. H, DOERMANN 655 still retain a single hit character. During the last 4 minutes of this period the curves take on a multiple hit character. In the final segment, from time 13 minutes to the end of the latent period, the curves retain the multiple hit charac- ter, but gradually regain the original x-ray sensitivity characteristic of free phage. These x-ray experiments suggest again that a rather drastic alteration occurs to the infecting particle, and that particles with the original character- istics are not found in the cell until the second half of the latent period. This is precisely what is observed in the results presented here. Our experiments were done with T4, but comparison seems legitimate since the two viruses are quite closely related (17). Results of a similar nature to those discussed here were published by Foster (18). In studying the effect of proflavine on the growth of phage T2, Foster found that the time at which this poison was added influenced the amount of phage liberated by the bacteria. No phage is liberated from T2-infected cells (latent period 21 minutes) if proflavine is added during the first 12 minutes after infection even though lysis of the cells does occur at the normal time. When proflavine is added at later points in the latent period, lysis yields phage particles, the number depending on the time of proflavine addition. When the results of these single infection experiments are compared to the cyanide single infection experiments (Fig. 1) the results are seen to be quite similar. From other experiments Foster concluded that proflavine inhibits one of the final stages in the formation of fully infective phage. These facts, taken together, suggests that proflavine experiments were, in fact, measuring intracellular phage. SUMMARY A method is described for liberating and estimating intracellular bacterio- phage at any stage during the latent period by arresting phage growth and induc- ing premature lysis of the infected cells. This is brought about by placing the infected bacteria into the growth medium supplemented with 0.01 m cyanide and with a high titer T6 lysate. It was found in some of the later experiments that the T6 lysate is essential only during the first half of the latent period. Cyanide alone will induce lysis during the latter part of the latent period. Using this method on T4-infected bacteria it is found that during the first half of the latent period no phage particles, not even those originally infecting the bacteria, are recovered. This result is in agreement with the gradually emerging concept that a profound alteration of the infecting phage particle takes place before reproduction ensues. During the second half of the latent period mature phage is found to accumulate within the bacteria at a rate which is parallel to the approximately linear increase of intracellular DNA in this system. However, the phage production lags several minutes behind DNA production. 85 656 INTRACELLULAR GROWTH OF BACTERIOPHAGES. I When 5-methyltryptophan replaced cyanide as the metabolic inhibitor, similar results were obtained. The curves were, however, displaced several minutes to the left on the time axis. The results are compared with Latarjet’s (16) data on x-radiation of infected bacteria and with Foster’s data (18) concerning the effect of proflavine on in- fected bacteria. Essential agreement with both is apparent. BIBLIOGRAPHY . Delbriick, M., J. Gen. Physiol., 1940, 23, 643. . Doermann, A. H., /. Bact., 1948, 55, 257. Doermann, A. H., Ann. Rep. Biol. Lab., Long Island Biol. Assn., 1946, 22. Adams, M. H., J. Gen. Physiol., 1948, 31, 417. . Delbriick, M., and Luria, S. E., Arch. Biochem., 1942, 1, 111. Cohen, S. S., Bact. Rev., 1949, 13, 1. . Cohen, S. S., and Anderson, T. F., J. Exp. Med., 1946, 84, 525. Underwood, N., and Doermann, A. H., Rev. Scient. Instr., 1947, 18, 665. Anderson, T. F., Science, 1945, 101, 565. Anderson, T. F., and Doermann, A. H., J. Gen. Physiol., 1952, 35, 657. Delbriick, M., and Bailey, W. T., Jr., Cold Spring Harbor Symp. Quant. Buol., 1946, 11, 33. Hershey, A. D., and Rotman, R., Proc. Nat. Acad. Sc., 1948, 34, 89. . Hershey, A. D., and Rotman, R., Genetics, 1949, 34, 44. . Delbriick, M., J. Bact., 1945, 50, 151. wuunia,S. B., Proc, Nat. Acad. Sc: 19477335 253. . Latarjet, R., J. Gen. Physiol., 1948, 31, 529. . Adams, M. H., in Methods in Medical Research, (J. H. Comroe, editor), Chicago, The Yearbook Publishers, Inc., 1950, 2, 1. . Foster, R. A. C., J. Bact., 1948, 56, 795. —_— —_ RK SOoPN AMAR wWN ee IAM RP WH — co 86 INDEPENDENT FUNCTIONS OF VIRAL PROTEIN AND NUCLEIC ACID IN GROWTH OF BACTERIOPHAGE* By A. D. HERSHEY anp MARTHA CHASE (From the Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, Long Island) (Received for publication, April 9, 1952) The work of Doermann (1948), Doermann and Dissosway (1949), and Anderson and Doermann (1952) has shown that bacteriophages T2, T3, and T4 multiply in the bacterial cell in a non-infective form. The same is true of the phage carried by certain lysogenic bacteria (Lwoff and Gutmann, 1950). Little else is known about the vegetative phase of these viruses. The experi- ments reported in this paper show that one of the first steps in the growth of T2 is the release from its protein coat of the nucleic acid of the virus particle, after which the bulk of the sulfur-containing protein has no further function. Materials and Methods ——Phage T2 means in this paper the variety called T2H (Hershey, 1946); T2/ means one of the host range mutants of T2; UV-phage means phage irradiated with ultraviolet light from a germicidal lamp (General Electric Co.) to a fractional survival of 10-5. Sensitive bacteria means a strain (H) of Escherichia coli sensitive to T2 and its h mutant; resistant bacteria B/2 means a strain resistant to T2 but sensitive to its h mutant; resistant bacteria B/2h means a strain resistant to both. These bacteria do not adsorb the phages to which they are resistant. “Salt-poor” broth contains per liter 10 gm. bacto-peptone, 1 gm. glucose, and 1 gm. NaCl. “Broth” contains, in addition, 3 gm. bacto-beef extract and 4 gm. NaCl. Glycerol-lactate medium contains per liter 70 mm sodium lactate, 4 gm. glycerol, 5 gm. NaCl, 2 gm. KCl, 1 gm. NH,Cl, 1 mm MgCl, 0.1 mm CaCl, 0.01 gm. gelatin, 10 mg. P (as orthophosphate), and 10 mg. S (as MgSO,), at pH 7.0. Adsorption medium contains per liter 4 gm. NaCl, 5 gm. K2SO,, 1.5 gm. KH2POs,, 3.0 gm. Na2HPO,, 1 mm MgSOu,, 0.1 mm CaCls, and 0.01 gm. gelatin, at pH 7.0. Veronal buffer contains per liter 1 gm. sodium diethylbarbiturate, 3 mm MgSO,, and 1 gm. gelatin, at pH 8.0. The HCN referred to in this paper consists of molar sodium cyanide solution neutralized when needed with phosphoric acid. * This investigation was supported in part by a research grant from the National Microbiological Institute of the National Institutes of Health, Public Health Service. Radioactive isotopes were supplied by the Oak Ridge National Laboratory on alloca- tion from the Isotopes Division, United States Atomic Energy Commission. Reprinted by permission of the authors and The Rockefeller Institute from THE JOURNAL OF GENERAL PuysIoLoey, 36 (1), 39-56, September 20, 1952. 87 40 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH Adsorption of isotope to bacteria was usually measured by mixing the sample in adsorption medium with bacteria from 18 hour broth cultures previously heated to 70°C. for 10 minutes and washed with adsorption medium. The mixtures were warmed for 5 minutes at 37°C., diluted with water, and centrifuged. Assays were made of both sediment and supernatant fractions. Precipitation of isotope with antiserum was measured by mixing the sample in 0.5 per cent saline with about 10" per ml. of non-radioactive phage and slightly more than the least quantity of antiphage serum (final dilution 1:160) that would cause visible precipitation. The mixture was centrifuged after 2 hours at 37°C. Tests with DNase (desoxyribonuclease) were performed by warming samples diluted in veronal buffer for 15 minutes at 37°C. with 0.1 mg. per ml. of crystalline enzyme (Worthington Biochemical Laboratory). Acid-soluble isotope was measured after the chilled sample had been precipitated with 5 per cent trichloroacetic acid in the presence of 1 mg./ml. of serum albumin, and centrifuged. In all fractionations involving centrifugation, the sediments were not washed, and contained about 5 per cent of the supernatant. Both fractions were assayed. Radioactivity was measured by means of an end-window Geiger counter, using dried samples sufficiently small to avoid losses by self-absorption. For absolute meas- urements, reference solutions of P* obtained from the National Bureau of Standards, as well as a permanent simulated standard, were used. For absolute measurements of S*° we relied on the assays (+20 per cent) furnished by the supplier of the isotope (Oak Ridge National Laboratory). Glycerol-lactate medium was chosen to permit growth of bacteria without un- desirable pH changes at low concentrations of phosphorus and sulfur, and proved useful also for certain experiments described in this paper. 18-hour cultures of sensitive bacteria grown in this medium contain about 2 X 10° cells per ml., which grow ex- ponentially without lag or change in light-scattering per cell when subcultured in the same medium from either large or small seedings. The generation time is 1.5 hours at 37°C. The cells are smaller than those grown in broth. T2 shows a latent period of 22 to 25 minutes in this medium. The phage yield obtained by lysis with cyanide and UV-phage (described in context) is one per bacterium at 15 minutes and 16 per bacterium at 25 minutes. The final burst size in diluted cultures is 30 to 40 per bacterium, reached at 50 minutes. At 2 X 108 cells per ml., the culture lyses slowly, and yields 140 phage per bacterium. The growth of both bacteria and phage in this medium is as reproducible as that in broth. For the preparation of radioactive phage, P® of specific activity 0.5 mc./mg. or S*> of specific activity 8.0 mc./mg. was incorporated into glycerol-lactate medium, in which bacteria were allowed to grow at least 4 hours before seeding with phage. After infection with phage, the culture was aerated overnight, and the radioactive phage was isolated by three cycles of alternate slow (2000 G) and fast (12,000 G) centrifugation in adsorption medium. The suspensions were stored at a concentration not exceeding 4 uc./ml. Preparations of this kind contain 1.0 to 3.0 * 10-” ug. S and 2.5 to 3.5 & 10-1! ug. P per viable phage particle. Occasional preparations containing excessive amounts of sulfur can be improved by absorption with heat-killed bacteria that do not adsorb 88 A. D. HERSHEY AND MARTHA CHASE 41 the phage. The radiochemical purity of the preparations is somewhat uncertain, ow- ing to the possible presence of inactive phage particles and empty phage membranes. The presence in our preparations of sulfur (about 20 per cent) that is precipitated by antiphage serum (Table I) and either adsorbed by bacteria resistant to phage, or not adsorbed by bacteria sensitive to phage (Table VII), indicates contamination by membrane material. Contaminants of bacterial origin are probably negligible for present purposes as indicated by the data given in Table I. For proof that our prin- cipal findings reflect genuine properties of viable phage particles, we rely on some experiments with inactivated phage cited at the conclusion of this paper. The Chemical Morphology of Resting Phage Particles—Anderson (1949) found that bacteriophage T2 could be inactivated by suspending the particles in high concentrations of sodium chloride, and rapidly diluting the suspension with water. The inactivated phage was visible in electron micrographs as tad- pole-shaped “ghosts.” Since no inactivation occurred if the dilution was slow TABLE I Composition of Ghosts and Solution of Plasmolyzed Phage Whole phage labeled with Plasmolyzed | Per cent of isotope] phage labeled bisa ps2 S35 pa | S35 Acid-soluble...... 5 ota hare Oar AA sal Ge aL ra — | —_ 1 — Acid-soluble after treatment with DNase....... tr 1 1 80 1 Adsorbed to sensitive bacteria................. 85 90 2 90 Precipitated by antiphage. .......-.-..:-0.+:- 90 |, 99 5 97 he attributed the inactivation to osmotic shock, and inferred that the particles possessed an osmotic membrane. Herriott (1951) found that osmotic shock released into solution the DNA (desoxypentose nucleic acid) of the phage particle, and that the ghosts could adsorb to bacteria and lyse them. He pointed out that this was a beginning toward the identification of viral functions with viral substances. We have plasmolyzed isotopically labeled T2 by suspending the phage (10% per ml.) in 3 m sodium chloride for 5 minutes at room temperature, and rapidly pouring into the suspension 40 volumes of distilled water. The plas- molyzed phage, containing not more than 2 per cent survivors, was then an- alyzed for phosphorus and sulfur in the several ways shown in Table I. The results confirm and extend previous findings as follows:— 1. Plasmolysis separates phage T2 into ghosts containing nearly all the sulfur and a solution containing nearly all the DNA of the intact particles. 2. The ghosts contain the principal antigens of the phage particle detect- able by our antiserum. The DNA is released as the free acid, or possibly linked to sulfur-free, apparently non-antigenic substances. 89 42 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH 3. The ghosts are specifically adsorbed to phage-susceptible bacteria; the DNA is not. 4, The ghosts represent protein coats that surround the DNA of the intact particles, react with antiserum, protect the DNA from DNase (desoxyribo- nuclease), and carry the organ of attachment to bacteria. 5. The effects noted are due to osmotic shock, because phage suspended in salt and diluted slowly is not inactivated, and its DNA is not exposed to DNase. TABLE II Sensitization of Phage DNA to DNase by Adsorption to Bacteria Non-sedimentable isotope, Phage adsorbed to Phage labeled eee with After DNase No DNase Tuive bacteria... .clsc caus das. ee eee $35 2 1 e¢ OE a ANS uh Be Be ch a citi ie CaO ea a p2 8 Bacteria heated before infection............... S35 15 11 fS a s Eo) AN i ae eR a pz 76 13 Bacteria heated after infection................. S35 12 14 se fs s BE eo ies OE a pz 06 23 Or aimee p2 5 Heated unadsorbed phage: acid- SOFA eas p22 118} soluble P® 90% Ase p2 81 100% oe sa p22 88 Phage adsorbed to bacteria for 5 minutes at 37°C. in adsorption medium, followed by washing. Bacteria heated for 10 minutes at 80°C. in adsorption medium (before infection) or in veronal buffer (after infection). Unadsorbed phage heated in veronal buffer, treated with DNase, and precipitated with trichloroacetic acid. All samples fractionated by centrifuging 10 minutes at 1300 G. Sensitization of Phage DNA to DNase by Adsorption to Bacteria.—The struc- ture of the resting phage particle described above suggests at once the possibil- ity that multiplication of virus is preceded by the alteration or removal of the protective coats of the particles. This change might be expected to show itself as a sensitization of the phage DNA to DNase. The experiments described in Table II show that this happens. The results may be summarized as fol- lows:— 1. Phage DNA becomes largely sensitive to DNase after adsorption to heat-killed bacteria. 2. The same is true of the DNA of phage adsorbed to live bacteria, and then 90 A. D. HERSHEY AND MARTHA CHASE 43 heated to 80°C. for 10 minutes, at which temperature unadsorbed phage is not sensitized to DNase. 3. The DNA of phage adsorbed to unheated bacteria is resistant to DNase, presumably because it is protected by cell structures impervious to the en- zyme. Graham and collaborators (personal communication) were the first to dis- cover the sensitization of phage DNA to DNase by adsorption to heat-killed bacteria. The DNA in infected cells is also made accessible to DNase by alternate freezing and thawing (followed by formaldehyde fixation to inactivate cellu- lar enzymes), and to some extent by formaldehyde fixation alone, as illus- trated by the following experiment. Bacteria were grown in broth to 5 X 10’ cells per ml., centrifuged, resuspended in adsorption medium, and infected with about two P*-labeled phage per bacterium. After 5 minutes for adsorption, the suspension was diluted with water containing per liter 1.0 mm MgSO,, 0.1 mm CaCl, and 10 mg. gelatin, and recentrifuged. The cells were resuspended in the fluid last mentioned at a concentration of 5 X 10° per ml. This suspension was frozen at —15°C. and thawed with a minimum of warming, three times in succession. Immediately after the third thawing, the cells were fixed by the addition of 0.5 per cent (v/v) of formalin (35 per cent HCHO). After 30 minutes at room temperature, the suspension was dialyzed free from formaldehyde and cen- trifuged at 2200 G for 15 minutes. Samples of P*-labeled phage, frozen-thawed, fixed, and dialyzed, and of infected cells fixed only and dialyzed, were carried along as controls. The analysis of these materials, given in Table III, shows that the effect of freezing and thawing is to make the intracellular DNA labile to DNase, without, however, causing much of it to leach out of the cells. Freezing and thawing and formaldehyde fixation have a negligible effect on unadsorbed phage, and formaldehyde fixation alone has only a mild effect on infected cells. Both sensitization of the intracellular P* to DNase, and its failure to leach out of the cells, are constant features of experiments of this type, independently of visible lysis. In the experiment just described, the frozen suspension cleared during the period of dialysis. Phase-contrast microscopy showed that the cells consisted largely of empty membranes, many apparently broken. In another experiment, samples of infected bacteria from a culture in salt-poor broth were repeatedly frozen and thawed at various times during the latent period of phage growth, fixed with formaldehyde, and then washed in the centrifuge. Clearing and microscopic lysis occurred only in suspensions frozen during the second half of the latent period, and occurred during the first or second thaw- ing. In this case the lysed cells consisted wholly of intact cell membranes, appearing empty except for a few small, rather characteristic refractile bodies apparently attached to the cell walls. The behavior of intracellular P” toward DNase, in either the lysed or unlysed cells, was not significantly different from 91 44 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH that shown in Table III, and the content of P* was only slightly less after lysis. The phage liberated during freezing and thawing was also titrated in this experiment. The lysis occurred without appreciable liberation of phage in suspensions frozen up to and including the 16th minute, and the 20 min- ute sample yielded only five per bacterium. Another sample of the culture formalinized at 30 minutes, and centrifuged without freezing, contained 66 per cent of the P* in non-sedimentable form. The yield of extracellular phage at 30 minutes was 108 per bacterium, and the sedimented material consisted largely of formless debris but contained also many apparently intact cell membranes. TABLE III Sensitization of Intracellular Phage to DNase by Freezing, Thawing, and Fixation with Formaldehyde Unadsorbed Infected cells Infected cell ase een, | acted, | Teed Low speed sediment fraction Mota eh s-0. eee so ee cc ctl hep ee eee ee — (pl 86 Acid-solubles.cuuntle daihis scale eee ee eee — 0) 0.5 Acid-soluble'aiters DNase... J. 44 ae ances _ 59 28 Low speed supernatant fraction Rota Pee Au Soe antennae —- 29 14 Weid-solubles ait: asides koe ce Gee Eee 1 0.8 0.4 Acid-soluble atten DINasene .).5. adc aoe ene 11 21 Si5) The figures express per cent of total P® in the original phage, or its adsorbed fraction. We draw the following conclusions from the experiments in which cells infected with P*-labeled phage are subjected to freezing and thawing. 1. Phage DNA becomes sensitive to DNAse after adsorption to bacteria in buffer under conditions in which no known growth process occurs (Benzer, 1952; Dulbecco, 1952). 2. The cell membrane can be made permeable to DNase under conditions that do not permit the escape of either the intracellular P® or the bulk of the cell contents. 3. Even if the cells lyse as a result of freezing and thawing, permitting escape of other cell constituents, most of the P* derived from phage remains inside the cell membranes, as do the mature phage progeny. 4. The intracellular P* derived from phage is largely freed during spon- taneous lysis accompanied by phage liberation. 92 A. D. HERSHEY AND MARTHA CHASE 45 We interpret these facts to mean that intracellular DNA derived from phage is not merely DNA in solution, but is part of an organized structure at all times during the latent period. Liberation of DNA from Phage Particles by Adsorption to Bacterial Frag- ments.—The sensitization of phage DNA to specific depolymerase by adsorp- tion to bacteria might mean that adsorption is followed by the ejection of the phage DNA from its protective coat. The following experiment shows that this is in fact what happens when phage attaches to fragmented bacterial cells. TABLE IV Release of DNA from Phage Adsorbed to Bacterial Debris Phage labeled with S35 p22 Sediment fraction SUBVIVIN Gy DARE: aos - yd nya oye te ls espe ys 16 22 Motalisotopec \oeocaan: ope cea ae weal Ss 87 55 IAGig “soluble ISOLOMEs). fo sop oe ne pee reese ee hee 0 2 Aeiassoluble afters DINases; j.42- 2250+ seo s2e esses: 2 29 Supernatant fraction SLIEVAVAN PAD HAGE ai. 8 staidiee 92. via neges nies pe Ae ee 2s oe 5 5 MipEANSOLONE 6 = noe ita sinks @ eet = 1 Sena is ay es 13 45 cig esGluBlesiISOLepe =. ss25.5 ate och nee ies eek ce cet 0.8 0.5 Ncid=solublevattersDINasenc aciae acs nels ceierie acl erent 0.8 39 S35- and P-labeled T2 were mixed with identical samples of bacterial debris in adsorption medium and warmed for 30 minutes at 37°C. The mixtures were then centrifuged for 15 minutes at 2200 G, and the sediment and supernatant fractions were analyzed separately. The results are expressed as per cent of input phage or isotope. Bacterial debris was prepared by infecting cells in adsorption medium with four particles of T2 per bacterium, and transferring the cells to salt-poor broth at 37°C. The culture was aerated for 60 minutes, M/50 HCN was added, and incubation continued for 30 minutes longer. At this time the yield of ex- tracellular phage was 400 particles per bacterium, which remained unadsorbed because of the low concentration of electrolytes. The debris from the lysed cells was washed by centrifugation at 1700 G, and resuspended in adsorption medium at a concentration equivalent to 3 X 10° lysed cells per ml. It consisted largely of collapsed and fragmented cell membranes. The adsorption of radio- active phage to this material is described in Table IV. The following facts should be noted. 93 46 VIRAL PROTEIN AND NUCLEIC ACID 1N BACTERIOPHAGE GROWTH 1. The unadsorbed fraction contained only 5 per cent of the original phage particles in infective form, and only 13 per cent of the total sulfur. (Much of this sulfur must be the material that is not adsorbable to whole bacteria.) 2. About 80 per cent of the phage was inactivated. Most of the sulfur of this phage, as well as most of the surviving phage, was found in the sediment fraction. 3. The supernatant fraction contained 40 per cent of the total phage DNA (in a form labile to DNase) in addition to the DNA of the unadsorbed surviving phage. The labile DNA amounted to about half of the DNA of the inactivated phage particles, whose sulfur sedimented with the bacterial debris. 4. Most of the sedimentable DNA could be accounted for either as surviving phage, or as DNA labile to DNase, the latter amounting to about half the DNA of the inactivated particles. Experiments of this kind are unsatisfactory in one respect: one cannot tell whether the liberated DNA represents all the DNA of some of the inactivated particles, or only part of it. Similar results were obtained when bacteria (strain B) were lysed by large amounts of UV-killed phage T2 or T4 and then tested with P*-labeled T2 and T4. The chief point of interest in this experiment is that bacterial debris saturated with UV-killed T2 adsorbs T4 better than T2, and debris saturated with T4 adsorbs T2 better than T4. As in the preceding experiment, some of the adsorbed phage was not inactivated and some of the DNA of the inacti- vated phage was not released from the debris. These experiments show that some of the cell receptors for T2 are different from some of the cell receptors for T4, and that phage attaching to these spe- cific receptors is inactivated by the same mechanism as phage attaching to unselected receptors. This mechanism is evidently an active one, and not merely the blocking of sites of attachment to bacteria. Removal of Phage Coats from Infected Bacteria.—Anderson (1951) has ob- tained electron micrographs indicating that phage T2 attaches to bacteria by its tail. If this precarious attachment is preserved during the progress of the infection, and if the conclusions reached above are correct, it ought to be a simple matter to break the empty phage membranes off the infected bacteria, leaving the phage DNA inside the cells. The following experiments show that this is readily accomplished by strong shearing forces applied to suspensions of infected cells, and further that in- fected cells from which 80 per cent of the sulfur of the parent virus has been removed remain capable of yielding phage progeny. Broth-grown bacteria were infected with S**- or P-labeled phage in ad- sorption medium, the unadsorbed material was removed by centrifugation, and the cells were resuspended in water containing per liter 1 mm MgSQ,, 0.1 mm CaClo, and 0.1 gm. gelatin. This suspension was spun in a Waring 94 A. D. HERSHEY AND MARTHA CHASE 47 blendor (semimicro size) at 10,000 r.p.m. The suspension was cooled briefly in ice water at the end of each 60 second running period. Samples were removed at intervals, titrated (through antiphage serum) to measure the number of bacteria capable of yielding phage, and centrifuged to measure the proportion of isotope released from the cells. The results of one experiment with each isotope are shown in Fig. 1. The data for S** and survival of infected bacteria come from the same experiment, in which the ratio of added phage to bacteria was 0.28, and the concentrations Infected bacteria ee Px \2) Extracellular 5 Extracellular P 32 Per cent of total Min. O fi 2 3 4 8 Running time in blendor Frc. 1. Removal of S3° and P® from bacteria infected with radioactive phage, and survival of the infected bacteria, during agitation in a Waring blendor. of bacteria were 2.5 X 10% per ml. infected, and 9.7 X 10% per ml. total, by direct titration. The experiment with P*-labeled phage was very similar. In connection with these results, it should be recalled that Anderson (1949) found that adsorption of phage to bacteria could be prevented by rapid stir- ring of the suspension. At higher ratios of infection, considerable amounts of phage sulfur elute from the cells spontaneously under the conditions of these experiments, though the elution of P® and the survival of infected cells are not affected by multi- plicity of infection (Table V). This shows that there is a cooperative action among phage particles in producing alterations of the bacterial membrane which weaken the attachment of the phage. The cellular changes detected in 95 48 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH this way may be related to those responsible for the release of bacterial com- ponents from infected bacteria (Prater, 1951; Price, 1952). A variant of the preceding experiments was designed to test bacteria at a later stage in the growth of phage. For this purpose infected cells were aerated in broth for 5 or 15 minutes, fixed by the addition of 0.5 per cent (v/v) com- mercial formalin, centrifuged, resuspended in 0.1 per cent formalin in water, and subsequently handled as described above. The results were very similar to those already presented, except that the release of P* from the cells was slightly less, and titrations of infected cells could not be made. The S*-labeled material detached from infected cells in the manner de- scribed possesses the following properties. It is sedimented at 12,000 G, though less completely than intact phage particles. It is completely precipitated by TABLE V Effect of Multiplicity of Infection on Elution of Phage Membranes from Infected Bacteria P22_labeled phage S#5-labeled phage Running time Multiplicity, of in blendor infection = ‘ Isotope eluted eee Isotope eluted mee min. per cent per cent | per cent per cent 0 0.6 | 10 120 | 16 101 25 0.6 21 82 81 78 0 G0 al 13 89 46 90 TG) 6.0 | 24 86 82 85 The infected bacteria were suspended at 10° cells per ml. in water containing per liter 1 mm MgSO,, 0.1 mm CaCh, and 0.1 gm. gelatin. Samples were withdrawn for assay of extracellular isotope and infected bacteria before and after agitating the suspension. In either case the cells spent about 15 minutes at room temperature in the eluting fluid. antiphage serum in the presence of whole phage carrier. 40 to 50 per cent of it readsorbs to sensitive bacteria, almost independently of bacterial concen- tration between 2 X 108 and 10° cells per ml., in 5 minutes at 37°C. The ad- sorption is not very specific: 10 to 25 per cent adsorbs to phage-resistant bac- teria under the same conditions. The adsorption requires salt, and for this reason the efficient removal of S** from infected bacteria can be accomplished only in a fluid poor in electrolytes. The results of these experiments may be summarized as follows:— 1. 75 to 80 per cent of the phage sulfur can be stripped from infected cells by violent agitation of the suspension. At high multiplicity of infection, nearly 50 per cent elutes spontaneously. The properties of the S**-labeled material show that it consists of more or less intact phage membranes, most of which have lost the ability to attach specifically to bacteria. 2. The release of sulfur is accompanied by the release of only 21 to 35 per 96 A. D. HERSHEY AND MARTHA CHASE 49 cent of the phage phosphorus, half of which is given up without any mechan- ical agitation. 3. The treatment does not cause any appreciable inactivation of intracellu- lar phage. 4, These facts show that the bulk of the phage sulfur remains at the cell surface during infection, and takes no part in the multiplication of intracellu- lar phage. The bulk of the phage DNA, on the other hand, enters the cell soon after adsorption of phage to bacteria. Transfer of Sulfur and Phosphorus from Parental Phage to Progeny.—We have concluded above that the bulk of the sulfur-containing protein of the resting phage particle takes no part in the multiplication of phage, and in fact does not enter the cell. It follows that little or no sulfur should be trans- ferred from parental phage to progeny. The experiments described below show that this expectation is correct, and that the maximal transfer is of the order 1 per cent Bacteria were grown in glycerol-lactate medium overnight and subcultured in the same medium for 2 hours at 37°C. with aeration, the size of seeding being adjusted nephelometrically to yield 2 X 10® cells per ml. in the sub- culture. These bacteria were sedimented, resuspended in adsorption medium at a concentration of 10° cells per ml., and infected with S**-labeled phage T2. After 5 minutes at 37°C., the suspension was diluted with 2 volumes of water and resedimented to remove unadsorbed phage (5 to 10 per cent by titer) and S** (about 15 per cent). The cells were next suspended in glycerol- lactate medium at a concentration of 2 X 105 per ml. and aerated at 37°C. Growth of phage was terminated at the desired time by adding in rapid suc- cession 0.02 mm HCN and 2 X 10" UV-killed phage per ml. of culture. The cyanide stops the maturation of intracellular phage (Doermann, 1948), and the UV-killed phage minimizes losses of phage progeny by adsorption to bac- terial debris, and promotes the lysis of bacteria (Maalge and Watson, 1951). As mentioned in another connection, and also noted in these experiments, the lysing phage must be closely related to the phage undergoing multiplica- tion (e.g., T2H, its # mutant, or T2L, but not T4 or T6, in this instance) in order to prevent inactivation of progeny by adsorption to bacterial debris. To obtain what we shall call the maximal yield of phage, the lysing phage was added 25 minutes after placing the infected cells in the culture medium, and the cyanide was added at the end of the 2nd hour. Under these condi- tions, lysis of infected cells occurs rather slowly. Aeration was interrupted when the cyanide was added, and the cultures were left overnight at 37°C. The lysates were then fractionated by centrifuga- tion into an initial low speed sediment (2500 G for 20 minutes), a high speed supernatant (12,000 G for 30 minutes), a second low speed sediment obtained by recentrifuging in adsorption medium the resuspended high speed sediment, and the clarified high speed sediment. 97 50 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH The distribution of S*° and phage among fractions obtained from three cul- tures of this kind is shown in Table VI. The results are typical (except for the excessively good recoveries of phage and S**) of lysates in broth as well as lysates in glycerol-lactate medium. The striking result of this experiment is that the distribution of S** among the fractions is the same for early lysates that do not contain phage progeny, and later ones that do. This suggests that little or no S* is contained in the mature phage progeny. Further fractionation by adsorption to bacteria con- firms this suggestion. Adsorption mixtures prepared for this purpose contained about 5 X 109 heat-killed bacteria (70°C. for 10 minutes) from 18 hour broth cultures, and TABLE VI Per Cent Distributions of Phage and S* among Centrifugally Separated Fractions of Lysates after Infection with S*°-Labeled T2 Lysis at Lysis at Maximal yield Fraction t+=0 #=10 |_ 2 Se sas S35 Phage istilow/speedisediment..,. semaaeea eee ase e 79 81 82 19 Phaxal 8 Bede eRe ae ee CN OL ata eda toe | 2.4 | Det 2.8 14 High speed Cy ll A PAM mt AR Pel 2S. Oh. . 6.9 71 61 2 USIIDELN ACAI ears ne ere et eeRa ect ah eee 10 We 0) ee 7.0 Recs verde Dubin clu nripamyen toni aT ie i) 100 100° |. 1.96 100 | | Infection with S**-labeled T2, 0.8 particles per bacterium. Lysing phage UV-killed k mutant of T2. Phage yields per infected bacterium: <0.1 after lysis at ¢ = 0; 0.12 att = 10; maximal yield 29. Recovery of S** means per cent of adsorbed input recovered in the four fractions; recovery of phage means per cent of total phage yield (by plaque count before fractionation) recovered by titration of fractions. about 10" phage (UV-killed lysing phage plus test phage), per ml. of adsorp- tion medium. After warming to 37°C. for 5 minutes, the mixtures were diluted with 2 volumes of water, and centrifuged. Assays were made from supernatants and from unwashed resuspended sediments. The results of tests of adsorption of S** and phage to bacteria (H) adsorbing both T2 progeny and /i-mutant lysing phage, to bacteria (B/2) adsorbing lysing phage only, and to bacteria (B/2/:) adsorbing neither, are shown in Table VII, together with parallel tests of authentic S*°-labeled phage. The adsorption tests show that the S** present in the seed phage is adsorbed with the specificity of the phage, but that S*® present in lysates of bacteria infected with this phage shows a more complicated behavior. It is strongly adsorbed to bacteria adsorbing both progeny and lysing phage. It is weakly adsorbed to bacteria adsorbing neither. It is moderately well adsorbed to bac- 98 A. D. HERSHEY AND MARTHA CHASE Si teria adsorbing lysing phage but not phage progeny. The latter test shows that the S* is not contained in the phage progeny, and explains the fact that the S* in early lysates not containing progeny behaves in the same way. The specificity of the adsorption of S**-labeled material contaminating the phage progeny is evidently due to the lysing phage, which is also adsorbed much more strongly to strain H than to B/2, as shown both by the visible re- duction in Tyndall scattering (due to the lysing phage) in the supernatants of the test mixtures, and by independent measurements. This conclusion is further confirmed by the following facts. TABLE VII Adsorption Tests with Uniformly S*°-Labeled Phage and with Products of Their Growth in Non-Radioactive Medium Per cent adsorbed Uniformly labeled BesArerarar Adsorbing bacteria 85 phage lyaistat Phage progeny “s hae 1 =10 (Maximal yield) + UV-k | No UV-A | S35 S35 S35 S35 | Phage Bensitive (D9 aii: tos! has. sas: 84 | 86 79 78icii\ae 96 Resistant (2) err vsusae reste 4c ors cen ts: 15 | 11 46 49 10 Resistant: (B/2/2) Parse So esc ce: 13 | 12 29 28 8 The uniformly labeled phage and the products of their growth are respectively the seed phage and the high speed sediment fractions from the experiment shown in Table VI. The uniformly labeled phage is tested at a low ratio of phage to bacteria: +UV-h means with added UV-killed # mutant in equal concentration to that present in the other test materials. The adsorption of phage is measured by plaque counts of supernatants, and also sedi- ments in the case of the resistant bacteria, in the usual way. 1. If bacteria are infected with S** phage, and then lysed near the midpoint of the latent period with cyanide alone (in salt-poor broth, to prevent read- sorption of S** to bacterial debris), the high speed sediment fraction contains S** that is adsorbed weakly and non-specifically to bacteria. 2. If the lysing phage and the S**-labeled infecting phage are the same (T2), or if the culture in salt-poor broth is allowed to lyse spontaneously (so that the yield of progeny is large), the S*® in the high speed sediment fraction is adsorbed with the specificity of the phage progeny (except for a weak non- specific adsorption). This is illustrated in Table VII by the adsorption to H and B/2h. It should be noted that a phage progeny grown from S**-labeled phage and containing a larger or smaller amount of contaminating radioactivity could not be distinguished by any known method from authentic S*°-labeled phage, 99 52 VIRAL PROTEIN AND NUCLEIC ACID JN BACTERIOPHAGE GROWTH except that a small amount of the contaminant could be removed by adsorp- tion to bacteria resistant to the phage. In addition to the properties already mentioned, the contaminating S* is completely precipitated with the phage by antiserum, and cannot be appreciably separated from the phage by further fractional sedimentation, at either high or low concentrations of electrolyte. On the other hand, the chemical contamination from this source would be very small in favorable circumstances, because the progeny of a single phage particle are numerous and the contaminant is evidently derived from the parents. The properties of the S**-labeled contaminant show that it consists of the remains of the coats of the parental phage particles, presumably identical with the material that can be removed from unlysed cells in the Waring blen- dor. The fact that it undergoes little chemical change is not surprising since it probably never enters the infected cell. The properties described explain a mistaken preliminary report (Hershey et al., 1951) of the transfer of S** from parental to progeny phage. It should be added that experiments identical to those shown in Tables VI and VII, but starting from phage labeled with P*, show that phosphorus is transferred from parental to progeny phage to the extent of 30 per cent at yields of about 30 phage per infected bacterium, and that the P® in prema- turely lysed cultures is almost entirely non-sedimentable, becoming, in fact, acid-soluble on aging. Similar measures of the transfer of P* have been published by Putnam and Kozloff (1950) and others. Watson and Maalge (1952) summarize this work, and report equal transfer (nearly 50 per cent) of phosphorus and adenine. A Progeny of S*°-Labeled Phage Nearly Free from the Parental Label.—The following experiment shows clearly that the obligatory transfer of parental sulfur to offspring phage is less than 1 per cent, and probably considerably less. In this experiment, the phage yield from infected bacteria from which the S*°-labeled phage coats had been stripped in the Waring blendor was assayed directly for S*. Sensitive bacteria grown in broth were infected with five particles of S**-labeled phage per bacterium, the high ratio of infection being necessary for purposes of as- say. The infected bacteria were freed from unadsorbed phage and suspended in water containing per liter 1 mm MgSOu,, 0.1 mm CaCl», and 0.1 gm. gelatin. A sample of this suspension was agitated for 2.5 minutes in the Waring blendor, and centrifuged to remove the extracellular S**. A second sample not run in the blendor was centri- fuged at the same time. The cells from both samples were resuspended in warm salt-poor broth at a concentration of 10® bacteria per ml., and aerated for 80 min- utes. The cultures were then lysed by the addition of 0.02 mm HCN, 2 xX 10" UV- killed T2, and 6 mg. NaCl per ml. of culture. The addition of salt at this point causes S*5 that would otherwise be eluted (Hershey et al., 1951) to remain attached to the 100 A. D. HERSHEY AND MARTHA CHASE 53 bacterial debris. The lysates were fractionated and assayed as described previously, with the results shown in Table VIII. The data show that stripping reduces more or less proportionately the S**- content of all fractions. In particular, the S**-content of the fraction contain- ing most of the phage progeny is reduced from nearly 10 per cent to less than 1 per cent of the initially adsorbed isotope. This experiment shows that the bulk of the S** appearing in all lysate fractions is derived from the remains of the coats of the parental phage particles. Properties of Phage Inactivated by Formaldehyde-—Phage T2 warmed for 1 hour at 37°C. in adsorption medium containing 0.1 per cent (v/v) com- mercial formalin (35 per cent HCHO), and then dialyzed free from formalde- TABLE VIII Lysates of Bacteria Infected with S*°-Labeled T2 and Stripped in the Waring Blendor Cells stripped Cells not stripped Per cent of adsorbed S*5 or of phage yield: S35 Phage S35 Phage Elutedianeblendor fluideacey 2.2.08. .2 4. 2.) 86 = 39 —- iistelow-speedisediment(e yee se -patis.o 42 Ae aoe 3.8 9.3 31 13 2nd “ e Seam Ri cece torino hott ic pyran he (0.2) 11 Dail 11 High-speed Cem eae ited OMA RR Lan (Oe7)olleass 9.4 89 ue SS SUDEENALAN Un errr lane ace es oe (2.0) iV gal (ep) 1.6 RECOVERY SSR ene OER ee eee hans eM Cee ek 93 79 84 115 All the input bacteria were recovered in assays of infected cells made during the latent period of both cultures. The phage yields were 270 (stripped cells) and 200 per bacterium, assayed before fractionation. Figures in parentheses were obtained from counting rates close to background. hyde, shows a reduction in plaque titer by a factor 1000 or more. Inactivated phage of this kind possesses the following properties. 1. It is adsorbed to sensitive bacteria (as measured by either S** or P® labels), to the extent of about 70 per cent. 2. The adsorbed phage kills bacteria with an efficiency of about 35 per cent compared with the original phage stock. 3. The DNA of the inactive particles is resistant to DNase, but is made sensitive by osmotic shock. 4, The DNA of the inactive particles is not sensitized to DNase by adsorp- tion to heat-killed bacteria, nor is it released into solution by adsorption to bacterial debris. 5. 70 per cent of the adsorbed phage DNA can be detached from infected cells spun in the Waring blendor. The detached DNA is almost entirely re- sistant to DNase. 101 54 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH These properties show that T2 inactivated by formaldehyde is largely in- capable of injecting its DNA into the cells to which it attaches. Its behavior in the experiments outlined gives strong support to our interpretation of the cor- responding experiments with active phage. DISCUSSION We have shown that when a particle of bacteriophage T2 attaches to a bacterial cell, most of the phage DNA enters the cell, and a residue contain- ing at least 80 per cent of the sulfur-containing protein of the phage remains at the cell surface. This residue consists of the material forming the protective membrane of the resting phage particle, and it plays no further role in infec- tion after the attachment of phage to bacterium. These facts leave in question the possible function of the 20 per cent of sul- fur-containing protein that may or may not enter the cell. We find that little or none of it is incorporated into the progeny of the infecting particle, and that at least part of it consists of additional material resembling the residue that can be shown to remain extracellular. Phosphorus and adenine (Watson and Maalge, 1952) derived from the DNA of the infecting particle, on the other hand, are transferred to the phage progeny to a considerable and equal ex- tent. We infer that sulfur-containing protein has no function in phage multi- plication, and that DNA has some function. It must be recalled that the following questions remain unanswered. (1) Does any sulfur-free phage material other than DNA enter the cell? (2) If so, 1s it transferred to the phage progeny? (3) Is the transfer of phosphorus (or hypothetical other substance) to progeny direct—that is, does it remain at all times in a form specifically identifiable as phage substance—or indirect? Our experiments show clearly that a physical separation of the phage T2 into genetic and non-genetic parts is possible. A corresponding functional separation is seen in the partial independence of phenotype and genotype in the same phage (Novick and Szilard, 1951; Hershey et al., 1951). The chemi- cal identification of the genetic part must wait, however, until some of the questions asked above have been answered. Two facts of significance for the immunologic method of attack on problems of viral growth should be emphasized here. First, the principal antigen of the infecting particles of phage T2 persists unchanged in infected cells. Second, it remains attached to the bacterial debris resulting from lysis of the cells. These possibilities seem to have been overlooked in a study by Rountree (1951) of viral antigens during the growth of phage TS. SUMMARY 1. Osmotic shock disrupts particles of phage T2 into material containing nearly all the phage sulfur in a form precipitable by antiphage serum, and capable of specific adsorption to bacteria. It releases into solution nearly all 102 A. D. HERSHEY AND MARTHA CHASE 5 the phage DNA in a form not precipitable by antiserum and not adsorbable to bacteria. The sulfur-containing protein of the phage particle evidently makes up a membrane that protects the phage DNA from DNase, comprises the sole or principal antigenic material, and is responsible for attachment of the virus to bacteria. 2. Adsorption of T2 to heat-killed bacteria, and heating or alternate freezing and thawing of infected cells, sensitize the DNA of the adsorbed phage to DNase. These treatments have little or no sensitizing effect on unadsorbed phage. Neither heating nor freezing and thawing releases the phage DNA from infected cells, although other cell constituents can be extracted by these methods. These facts suggest that the phage DNA forms part of an organized intracellular structure throughout the period of phage growth. 3. Adsorption of phage T2 to bacterial debris causes part of the phage DNA to appear in solution, leaving the phage sulfur attached to the debris. Another part of the phage DNA, corresponding roughly to the remaining half of the DNA of the inactivated phage, remains attached to the debris but can be separated from it by DNase. Phage T4 behaves similarly, although the two phages can be shown to attach to different combining sites. The inactiva- tion of phage by bacterial debris is evidently accompanied by the rupture of the viral membrane. 4. Suspensions of infected cells agitated in a Waring blendor release 75 per cent of the phage sulfur and only 15 per cent of the phage phosphorus to the solution as a result of the applied shearing force. The cells remain capable of yielding phage progeny. 5. The facts stated show that most of the phage sulfur remains at the cell surface and most of the phage DNA enters the cell on infection. Whether sulfur-free material other than DNA enters the cell has not been determined. The properties of the sulfur-containing residue identify it as essentially un- changed membranes of the phage particles. All types of evidence show that the passage of phage DNA into the cell occurs in non-nutrient medium under conditions in which other known steps in viral growth do not occur. 6. The phage progeny yielded by bacteria infected with phage labeled with radioactive sulfur contain less than 1 per cent of the parental radioactiv- ity. The progeny of phage particles labeled with radioactive phosphorus con- tain 30 per cent or more of the parental phosphorus. 7. Phage inactivated by dilute formaldehyde is capable of adsorbing to bacteria, but does not release its DNA to the cell. This shows that the inter- action between phage and bacterium resulting in release of the phage DNA from its protective membrane depends on labile components of the phage particle. By contrast, the components of the bacterium essential to this inter- action are remarkably stable. The nature of the interaction is otherwise un- known. 8. The sulfur-containing protein of resting phage particles is confined to a 103 56 VIRAL PROTEIN AND NUCLEIC ACID IN BACTERIOPHAGE GROWTH protective coat that is responsible for the adsorption to bacteria, and functions as an instrument for the injection of the phage DNA into the cell. This pro- tein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented. REFERENCES Anderson, T. F., 1949, The reactions of bacterial viruses with their host cells, Bot. Rev., 15, 464. Anderson, T. F., 1951, Tr. New York Acad. Sc., 13, 130. Anderson, T. F., and Doermann, A. H., 1952, J. Gen. Physiol., 35, 657. Benzer, S., 1952, J. Bact., 63, 59. Doermann, A. H., 1948, Carnegie Institution of Washington Yearbook, No. 47, 176. Doermann, A. H., and Dissosway, C., 1949, Carnegie Institution of Washington Year- book, No. 48, 170. Dulbecco, R., 1952, J. Bact., 63, 209. Herriott, R. M., 1951, J. Bact., 61, 752. Hershey, A. D., 1946, Genetics, 31, 620. Hershey, A. D., Roesel, C., Chase, M., and Forman, S., 1951, Carnegie Institution of Washington Yearbook, No. 50, 195. Lwoff, A., and Gutmann, A., 1950, Ann. Inst. Pasteur, T8, 711. Maalge, O., and Watson, J. D., 1951, Proc. Nat. Acad. Sc., 37, 507. Novick, A., and Szilard, L., 1951, Science, 113, 34. Prater, C. D., 1951, Thesis, University of Pennsylvania. Price, W. H., 1952, J. Gen. Physiol., 35, 409. Putnam, F. W., and Kozloff, L.,1950, J. Biol. Chem., 182, 243. Rountree, P. M., 1951, Brit. J. Exp. Path., 32, 341. Watson, J. D., and Maalge, O., 1952, Acta path. et microbiol. scand., in press. 104 NUCLEIC ACID TRANSFER FROM PARENTAL TO PROGENY BACTERIOPHAGE by J. D. WATSON": ** anp O. MAAL@E Institute for Cytophysiology and State Serum Institute, Copenhagen (Denmark) INTRODUCTION In recent years several investigators have used isotopic markers to determine the transfer of P or N atoms from parental to progeny virus particles. PUTNAM AND KozLoFF* using the bacteriophage T6 labelled with **P, found that 20-40% of the label appeared in material identified as progeny phage by differential centrifugation. LESLIE ef al.? found similar, or lower, values with %2P-labelled T2 bacteriophage. KozLorF* extended his original observations by studying P and N transfer from normal as well as radiation damaged phage particles. All these experiments show incomplete transfer varying greatly from one experiment to the other. To determine the true transfer values, two technical problems must be solved: (x) adsorption of the labelled phage must be complete within about 2 minutes because later adsorbing particles are broken down before entering the cells (LESLIE ef Gl.) (2) means must be found to prevent progeny particles from adsorbing onto bacterial debris or unlysed cells. Failure to control either of these processes will result in under- estimation of the transfer values. In our first experiments (MAALGE AND Watson‘), although the second factor was well controlled, the first was not, and our #?P transfer values of about 30% were, there- fore, like those of Purnam AND KoztorrF!, LESLIE e¢ al.?, and KozLorF?, too low. In this paper, we present experiments in which both factors are controlled, and which indicate that T2 and T4 phages transfer about 50%, T3 phages about 40% of their phosphorus to the progeny. Identical values are found when, instead of 32P, 14C-labelled adenine is used to label the parental phage. The **P experiments show that the trans- ferred material goes predominantly to the early formed phages, and that the transfer values of about 50% are maximum values. Our finding that the early completed phages receive most of the parental material confirms similar observations by DoERMANN (personal communication) and WEED AND COHEN? (unpublished). In agreement with KozLorF’, we have also demonstrated that considerable amounts of ®2P may be transferred from labelled particles which do not participate in repro- duction, either because of radiation damage or because they are excluded by another phage. These ‘‘abnormal’” cases show that infecting particles may be broken down * Merck Fellow in the Natural Sciences of the National Research Council at the Institute for Cytophysiology, Copenhagen. ** Present address; Cavendish Laboratory, Cambridge, England. References p. 442. Reprinted by permission of the authors and Elsevier Publishing Co. from Brocuimica ET Bropuysica Acta, 10, 432-442 (1953). 105 voL. 10 (1953) NUCLEIC ACID TRANSFER 433 extensively before*their constituents are used for synthesis of new phage particles. We do not, however, think that this evidence excludes the possibility that under normal conditions the infecting and reproducing particle remains essentially unbroken. We shall return to this important point in the discussion. MATERIALS AND METHODS The phages T2, T3 and T4, and their common host E. coli, strain B/1, have been used: the latter because contamination of cultures with phage Tr occurs in our laboratory. Most of the techniques used in this study have been described in detail by Apams®. All cultures were grown at 37° C: cen- trifugations were done in a Servall Angle centrifuge at 10° C. Media. The nutrient broth is an aqueous extract of minced meat enriched with 1% peptone and containing 0.02% Tween 80 and 0.5% NaCl: pH is adjusted to 7.4. For experiments with T3, the concentration of NaCl was reduced to 0.05% to obtain rapid adsorption. In this medium, at 37° C the latent period of T4r is 22-23 minutes. and the burst size about 150. **P-labelled phage was prepared using the synthetic g-medium described earlier (MAALGE AND WATSON‘). Anti-sera. Rabbit anti-phage sera were prepared, using highly purified and concentrated phage suspensions as antigens. All sera had k-values (ADams®) of 500-1000 when tested against phage suspended in broth (cf. JERNE’). These sera showed no agglutination of F. coli, strain B/1, in dilution 1 to 10. Samples of anti-phage sera were absorbed with large amounts of live B/1, and used in parallel with unabsorbed serum for phage precipitation: no evidence for the presence of antibacterial anti- bodies was found. Serum against E. coli, strain B/1, was obtained after series of subcutaneous and intravenous injections of heat-killed and subsequently of live cultures. This serum had no anti-phage activity. X-ray technique. The X-ray source was a Holbeck-Beaudouin tube operating at 33 kv and 36 mA. A cooled molybdenum target produced radiation with an average wave-length of 0.9 A. On the surface of the samples the intensity of radiation was 66,000 r.p.m. The irradiation was done by Dr R. LatarJET on samples sent to Paris by airmail. No decrease in titer was observed in control samples as a result of the shipment. Isotope technique. Carrier-free orthophosphoric acid was obtained from the Isotope Division of the Oak Ridge National Laboratory, United States Atomic Energy Commission. Adenine labelled with 14C in position 8 was also used: this preparation was synthesized by CLARK AND KatckKar’, and had a specific activity of 0.8 mC per mMole. All samples containing C were evaporated to dryness and self-absorption due to solids in the suspension medium was made uniform by diluting into nutrient broth before counting. Variation between counts on duplicate *C samples was less than 10%. The counting equipment was the same as previously described. Preparation of ®2P-labelled T4vy. Washed B/1 bacteria from a 24-hour broth culture were in- oculated into 10 ml of g-medium containing 20 wC **P. After two and a half hours of aeration, the bacterial density was about 108 cells per ml, and the culture was then infected with about 10 T4r particles per cell. Aeration was continued, and 2-3 minutes before the onset of lysis 0.5 ml of un- diluted antibacterial serum was added. If antibacterial serum is not added, the titer of a crude T4r lysate will drop appreciably during the first 24 hours and a fraction of the remaining phages will adsorb slowly. Both effects are presumably due to phage particles absorbing on bacterial debris (MAALGE AND STENT). The antibacterial serum proved completely effective in blocking adsorption of T3 and T4 on B/1, but was not fully effective for T2r+. T2r+ stocks may, therefore contain inactive as well as slowly adsorbing particles even when antibacterial serum is used. It is possible to restore infectivity and full adsorbability to these particles by diluting into distilled water for several hours at 37° C: presumably because adsorbed particles dissociate from the debris at low salt concentrations (Puck, GAREN, AND CLINE! and HERSHEY, personal communication). This treatment was first used by BERTANI (personal communication) to raise the titer of Tzr* broth lysates. The crude radioactive lysates were centrifuged at 5000 g for 5 minutes to remove bacterial debris and at 12,000 g for one hour to sediment the phage. Three cycles of low and high speed centrifugation reduced the concentration of inorganic **P by a factor of about 10°. Further puri- fication was achieved by adding heat-killed resistant bacteria (B/3, 4, 7 heated to 58° C for 1 hour) at a concentration of 5-108 per ml. After 30 minutes at 37° C, about 5% of the radioactivity had adsorbed to the resistant cells, which were removed by centrifugation. A similar number of sensitive B/r cells adsorbed 95-98% of the activity. An additional test of the purity of the virus stock was obtained by precipitation with antiphage serum, as previously described; with anti-T4 serum, 94% of the radioactivity was precipitated, while in a control sample in which T3 phage was precipitated with anti-T3 serum, the precipitate contained less than 2% of the activity. Assuming that about 95 % of the #*P in the final preparation was present as phage phosphorus, the initial specific activity was 1o-® counts per minute per particle. From this we can calculate References p. 442. 106 434 J. D. WATSON, 0. MAALGE voc. 10 (1953) that each virus particle contained an average of 0.25 **P atoms. Since the inactivation efficiency of nuclear decay is only 1/12 (HERSHEY, KAMEN, KENNEDY AND Gest!!), the fraction of labelled particles which will lose infectivity during an experiment is negligible. With minor variation indicated by adsorption requirements, etc., the procedure just described was also used to prepare %2P-labelled stocks of the phages T2r+, T3, and T4rt+. Phage labelled with 14C adenine was grown on a purine requiring mutant of E. coli, strain B, which was obtained from Dr A. H. DoERMANN of the Oak Ridge National Laboratory. For this purpose the g-medium was supplemented with 5 y 14C adenine per ml. The specific activity of phages grown on the purine requiring strain was ten times that of phages grown similarly on B/1. EXPERIMENTAL The basic experimental procedure is unchanged throughout this study. It will, therefore, suffice to describe one typical and simple experiment in detail; more complex experiments can then be introduced briefly and the results summarized in tables. Distribution of 32P following infection of unlabelled B/1 with labelled Tar: Exponentially growing bacteria from an unlabelled broth culture were collected by centrifugation and resuspensed in unlabelled broth at 37°C at a concentration of 2-10° cells per ml. 3?P-labelled T4r phage was added at a ratio of 4.5 particles per bac- terium and one and one-half minutes allowed for adsorption. The culture was then chilled and centrifuged at 5000 g for 5 minutes. The supernatant was carefully siphoned off and samples for phage assay and radioactivity measurement were taken. The pellet was resuspended in broth at 37° C to give a suspension of infected bacteria with about 108 cells/ml; this figure was determined by a separate assay. Aeration was then started, and 25 minutes after infection one volume of undiluted antibacterial serum was added to 19 volumes of culture to prevent adsorption of progeny phage particles to bacterial debris. The cooling and centrifugation retarded phage growth by about 8 minutes. The assays and radioactivity measurements showed that in this experiment over 99.5 % of the phages and 96% of the input radioactivity were adsorbed on the bacteria. About 30 minutes after lysis, the culture was centrifuged at 5000 g for 5 minutes to remove bacterial debris and then at 12,000 g for one hour to sediment the progeny phage. The material collected during these centrifugations will be referred to as the “low speed pellet” and the “‘high speed pellet”’, respectively. The latter was resuspended in broth and again centrifuged at low speed to remove remaining bacterial debris. The TABLE I DISTRIBUTION OF 32P AFTER INFECTION OF B/I WITH LABELLED T4r Growing bacteria were concentrated to 2-109 cells per ml and infected with labelled T4r at a concentration of 9-109 particles per ml. Following an adsorption period of 1 ¥ minutes the bacteria were centrifuged for 4 minutes at 5000g to remove unadsorbed phage and then resuspended in nutrient broth at a concentration of 1.1-108 cells per ml. Approximately 99.5% of the phage and 96% of **P adsorbed to the bacteria. 32 P distribution Material Phage titer |mh (% of radioactivity adsorbed on bacteria) Crude lysate 155-1010 100% Low Speed Pellets .027* 1010 8.4% High Speed Pellet 1.45:1010 42.1% High Speed Supernatant Bday 49.5% References p. 442. 107 voL. 10 (1953) NUCLEIC ACID TRANSFER 435 two low speed pellets were resuspended in broth and all fractions assayed for 32P and phage. Table I shows that 42.1% of the 8?P which initially adsorbed to the bacteria and about 95% of the progeny phage was recovered in the high speed pellet. The radioactivity of the high speed pellet was characterized as belonging to phage particles by precipitation with an excess of anti-T4 serum and by adsorption to sensitive bacteria. Native as well as B/r adsorbed serum was used and in both cases 93% of the **P was precipitated with the phage. Adsorption tests showed that over 90% of the radioactivity adsorbed on B/I cells while less than 5% adsorbed on the resistant strain B/3, 4,7 on which T4 does not itself adsorb. These tests show that 90-95% of the radioactivity in the high speed pellet is somehow associated with the phage particles. Before asserting that this 3*P is truly incorporated into the progeny phages, the following possibilities must be considered: (1) degraded parental nucleic acids might stay attached to the surface of the progeny particles, or (2) non-infective parental particles might be adsorbed to the bacteria initially, released during lysis, and later sediment together with the progeny particles. Both these possibilities are ruled out by the fact that the progeny particles from an experiment like the one just described transfer their *2P in exactly the same way as did their uniformly labelled parents (MAALGE AND Watson‘). We, therefore, conclude that about 95% of the 32P in the high speed pellet is incorporated into the progeny particles. Using this estimate in correcting for the phage present in the low speed pellets and in the high speed supernatant, the fraction of the parental *2P which has become incorporated into the progeny particles is 45% in this experiment. Table II shows a series of similar experiments involving different stocks of labelled T4r; it is notable that the differences in transfer are very small, usually less than 5%. These values are not changed by varying the number of infective particles from I to 10 (the highest number tested). The amount of *#*P remaining attached to bacterial debris is uniformly about 5 to 10%. If the different phage stocks had contained greatly varying fractions of adsorbing but non-infecting phages, this would have caused the low speed pellet values to fluctuate greatly. TABLE II EXPERIMENTAL VARIATION IN 92P TRANSFER VALUES FOR T4r % of parental radioactivity in Experiment Burst size “pelins —ProBeny Phage — I 122 6 47 47 2 141 7 46 47 3 150 6 45 49 4 126 3 45 52 5 160 9 43 48 6 155 IO 42 48 32P transfer from secondarily adsorbed phage The experiments given in Table III show that partieles which adsorb on a bacterium more than two minutes after a primary infection with T4r transfer insignificant amounts of *?P. This was demonstrated by infecting bacteria with an average of 5 non-labelled phages per cell and at various times later reinfecting with labelled phage. Column I References p. 442. 108 436 J. D. WATSON, O. MAALGE vot. 10 (1953) of Table III further shows that with increasing intervals between primary and secondary infection, the amount of 32P which stays attached to the bacteria decreases. Since the labelled phages adsorb at the normal rate, most of the **P in the supernate of the infected cells must initially have been adsorbed onto the cells. The observed increase in unadsorbed material is probably an expression of the breakdown of secondarily adsorbed particles described by LESLIE, FRENCH, GRAHAM, AND VAN RooyveEn?. These authors observed that a primary infection stimulates within a few minutes the infected cellin such a way that, if a new phage particle adsorbs, it is broken down extensively on the surface of the bacterium, releasing about 50% of its phosphorus into the medium in the form of material soluble in 5% trichloroacetic acid. This stimulation phenomenon is of further importance for transfer experiments, since we must assume that early released progeny particles which adsorb on unlysed cells will be broken down and release half their phosphorus into the medium. In most of the previous work adsorption of progeny particles was not prevented and the break- down effect presumably decreased the transfer values. Since in our experiments ad- sorption was blocked by antibacterial serum, the 50% transfer values can be taken as a good estimate of the transfer to all the progeny particles. TABLE II DISTRIBUTION OF 32P FOLLOWING SECONDARY INFECTION OF B/I BY LABELLED T4r Growing bacteria concentrated to 1.3-10° cells/ml in broth were infected at ¢ = 0, with an average of 5 unlabelled T4r particles. At various intervals. labelled T4r was added at a ratio of I particle per bacterium. Several minutes after the addition of the labelled phage, unadsorbed 3p was removed by low speed centrifugation and the infected bacteria resuspended in nutrient broth at a concentration of 108 cells/ml. Wien % of parental radioactivity in : Unadsorbed * primary and sccondary 32 p Low speed High speed infection pellet Progeny phage supernatant ry 5 6 44 45 0.5 6 4 48 42 2 23 15 18 44 4 48 23 4 25 6 45 20 3 3? * This material must initially have been adsorbed but has been released again because of the breakdown effect described by LEsSLEy et al.’. Isotope transfer from parental to progeny T3 At the salt concentration usually employed in growth media, T3 phage adsorbs rather slowly. To ensure the necessary rapid adsorption of the labelled T3 particles, the NaCl concentration in the adsorption tube had to be lowered to 0.05%. At this lowsalt concentration, over 99% of the infecting particles adsorbed within two minutes. Except for this modification, the experiments were like the T4 experiments and included characterization of the progeny by serum precipitation and adsorption on sensitive bacteria. Table IV shows that about 40% of the parental °?P was transferred. Since nearly simultaneous adsorption of the infecting phage particles and isolation of the entire progeny were achieved, we may consider the transfer value of about 40% as a maximum value. As in the case of the phages T2 and T4, the transfer of phosphorus is incomplete. References p. 442. 109 VoL. 10 (1953) NUCLEIC ACID TRANSFER 437 TABLE IV DISTRIBUTION OF 82P FOLLOWING INFECTION OF B/I BY LABELLED T3 % of parental radioactivity in Experiment Burst size 3 Low speed High speed pellet Progeny phage supernatant I 195 9 46 45 2 234 8 38 54 Purine transfer from parental to progeny phage Over 95% of the phage phosphorus is located in DNA, and it is therefore desirable to know whether other nucleic acids constituents such as the purine bases are transferred incompletely like the phosphorus. Phages T2, T3 and T4 were grown in purine requiring bacteria in the presence of adenine labelled with 14C in position 8 (see page 433 et seq.). Paper chromatography shows that in this way both the phage adenine and guanine is labelled with 14C. Table V presents a series of experiments with the purine labelled phages. They all show incomplete transfer with values not significantly different from those obtained with **P; T3 again seems to transfer a little less than do T2 and T4. TABLE V DISTRIBUTION OF }4C FOLLOWING INFECTION OF B/I WITH PURINE LABELLED PHAGE % of parent radioactivity in Pee ten TLE EROS AL Low speed Progeny High speed pellet phage supernatant I Tart 322 12 55 33 2 Tart 238 16 54 30 33 Darth 162 18 43 39 4 2r 360 14 48 38 5 T3 180 14 38 48 6 Ne) 276 TE 38 51 Uf T3 210 14 32 54 8 T4r 115 12 44 44 9 T4r 122 14 40 46 Isotope transfer as a function of burst size In experiments with T3 and T4r spontaneous lysis always occurs when the burst size is relatively low. It is known, however, that the r+-phages behave differently, and that lysis can be delayed by a secondary infection (DOERMANN?2). At the time of normal lysis the infected cells have not exhausted their capacity for phage production. During the last ten minutes before normal lysis, large amounts of phosphorus-containing phage material is produced in the cells which is not developed into mature phage before lysis (MAALGE AND STENT®). After normal lysis, this material cannot be recovered by centrifugation. The incompleteness of the phosphorus and purine transfers might, therefore, be due to our failure to detect parental material transmitted to the immature phage particles formed late in the latent period. This hypothesis was tested by infecting bacteria with labelled T4r+-phage and inhibiting lysis artificially by reinfection with unlabelled phage. The burst size in this References p. 442. 110 438 J. D. WATSON, O. MAALGE VoL. 10 (1953) experiment was 350, or 2 or 3 times greater than in the experiment with T4r. Despite this increase in phage yield, the transfer was again about 50%, showing that the late formed particles received very little, if any, of the parental phosphorus. The experiment reported in Table VI shows directly that the transferred **P goes predominantly to the early formed particles. In this experiment, bacteria infected with labelled Tzr+-phage were lysed prematurely by addition of M/1000 KCN and a large number of ultraviolet inactivated phage, as previously described‘. The excess of added phage served as carrier material during centrifugation, and insured that the labelled progeny particles were effectively isolated even when the yield was low. Several identical experiments were carried out, all showing the same trend. Thus the material which an infecting particle transfers to the progeny usually goes to one or more of the early formed particles, while the later formed ones virtually never receive any of it. We therefore conclude that our transfer values of about 50% are true maximum values. Table VI also shows that bacteria lysed before the appearance of the first progeny particles contain insignificant amounts of radioactivity sedimentable at high speed. This is additional evidence that our results are not affected by spurious measurements of non-infective but still sedimentable parental particles released upon lysis. TABLE VI DISTRIBUTION OF PARENTAL 32P AMONG PROGENY PARTICLES FROM PREMATURELY LYSED BACTERIA Concentrated B/1 were infected with an average of 5 **P-labelled Tart particles per bacterium. Two minutes after infection the culture was chilled and centrifuged at 5000 g for 4 minutes to remove unadsorbed radioactivity. The pellet was resuspended in broth at 37° C ata bacterial concentration of 108 cells/ml. The progress of phage growth was retarded about 8 minutes by the cooling and cen- trifugation. Ten minutes after infection ultraviolet (UV) inactivated Tart at the average multiplicity of 5 particles was added to inhibit lysis. Samples of the infected bacteria were then broken open at various times by the addition of M/1000 KCN and approximately 2000 UV inactivated Tart particles per cell. Readsorption of the progeny particles was prevented by saturation of the bacterial surface with the UV treated phage (MAALGE AND Watson’). % of parental 32P in Time of premature lysis Burst sexe Low speed High speed High speed pellet pellet supernatant Nee ee ee I9 minutes fo) 13 2 85 25 34 15 15 70 28 100 14 26 60 34 210 15 38 47 48 275 16 37 47 Isotope transfer in the absence of genetic transfer The ‘“‘second generation experiment” (MAAL@E AND WATSON’) referred to earlier in this paper permits the conclusion that the transmitted phosphorus is distributed in the progeny particles in the same uniform way as in the parental particles. It leaves open the question whether transfer occurs via large blocks carrying biological specificity or via highly degraded material. In this study we have tried to answer a complementary question: Does isotope transfer occur under conditions where no genetic transfer is possible? i.¢., can material from an infecting particle be broken down to genetically unspecific structures which are then incorporated into new phage particles ? References p. 442. 111 voL. 10 (1953) NUCLEIC ACID TRANSFER 439 TABLE VII TRANSFER OF 82P FROM X-RAY INACTIVATED T4r Bacteria were mixedly infected with unlabelled T4r+ and X-ray irradiated %2P-labelled Tar The adsorption of **P to bacteria was similar in the mixtures containing irradiated phage and in control mixtures containing non-irradiated phage, being approximately 93 % in both cases. a a ss % of parental 2 P in Tube contents Low speed Progeny High speed pellet phage supernatant Eg ee Unlabelled T4rt + labelled T4r (non-irradiated) II 43 46 Unlabelled Tart + labelled T4r (e~$ survival) 31 24 45 Unlabelled T4rt + labelled T4r (e-* survival) 45 22 33 Unlabelled T4rt+ + labelled T4r (e1°survival) 42 30 28 To answer this question we first carried out experiments with labelled phage heavily irradiated with X-rays. Such particles retain the ability to adsorb to bacteria while the majority of them have lost not only their infectivity, but also the ability to kill the host cell and to transfer genetic specificity (Watson}8). The experiments pre- sented in Table VII show, however, that heavy X-ray damage reduces the transfer values only from the normal 40 to 50% to about 25%. Thus, substantial amounts of phosphorus may be transferred from particles which do not participate in genetic ex- change. It should be noted that nearly 50% of the phosphorus of the irradiated particles remain attached to the bacterial debris. This value is significantly greater than the values of 5 to 15% found when active phage reproduces. It is possible that as many as 50% of the irradiation damaged phages remains passively attached to the cell surfaces: if so, the transfer value per transferring particle is again about 50%. Transfer of parental isotope without simultaneous genetic transfer can also be demonstrated in bacteria in which one of the infecting phages does not multiply because of the presence of an unrelated phage. This ‘mutual exclusion” (DELBRUCK#4) is well illustrated by the unrelated phages T3 and T4; if a bacterium is infected simultaneously by both phages, T3 multiplication is completely suppressed and only T4 progeny particles appear ; even the infecting T3 particles are lost. In Table VIII are shown the results of an experiment in which bacteria were simultaneously infected with 32P-labelled T3 particles and unlabelled T4 particles. After lysis, the progeny particles were isolated and tested for radioactivity, the specificity of which was shown by the extent of its precipitation with anti-T3 and anti-T4 serum. It can be seen that approximately 25% of the **P originally present in the T3 particles was transferred to the T4 progeny, another 25% was associated with bacterial debris, while the remaining 50% cannot be sedimented at high speed. The excluded phage thus does not sit passively on the bacterial surface but must penetrate to the interior of the cell where it is broken down into its simpler components. Our experiment, therefore, supports the conclusion of WEIGLE AND DELBRUCK® that mutual exclusion must involve some mechanism other than the establishment of a barrier to penetration. References p. 442. 112 440 J. D. WATSON, O. MAALGZE voL. 10 (1953) TABLE VIII TRANSFER OF 32P FROM EXCLUDED PHAGE Bacteria were mixedly infected with an average of 5 particles of T4r and 1 particle of labelled T3 per bacterium. Following lysis the progeny particles were isolated and tested for radioactivity. a % of parental (T3) radioactivity in Experiment ESTE % of high speed pellet radioactivity No. T Tar Low speed High speed precipitated by High speed 3 t pellet pellet ss —___ Supernatant anti T3 serum anti T4 serum I 2 145 20 21 — —_— 59 2 4 162 24 27 4 92 49 3 5 148 19 16 = = 65 DISCUSSION We shall now discuss the results of the seven different types of experiments de- scribed in the preceding section in relation to the problem of virus reproduction. First it must be stressed, however, that in all our experiments only the nucleic acid portion of the phage has been labelled; entirely different results are obtained if a specific protein labelling isotope, like ®°S, is used, (HERSHEY AND CuHasE}*), 1. Infection of unlabelled bacteria with *2P-labelled T4r phage results in the transfer of 40 to 50% of the label to the progeny particles. The transmitted phosphorus is truly incorporated into the new phage. When the infected bacteria lyse, 5 to 10% of the parental ®2P remain associated with the bacterial debris; this may mean that 5 to 10% of the adsorbing particles stay passively attached to the bacterial surfaces. In our experiments the transfer value is an extremely reproducible figure and must be close to the maximum value since (a) nearly all the adsorbed particles participate in the reproduction process, and (b) all the progeny particles are recovered. The transfer value is constant for different preparations of labelled phage. 2. For maximum *?P transfer to occur, all the phage particles which adsorb on a bacterium must do so within about two minutes. Particles which adsorb more than two minutes after the primary infection of the cell do not transfer significant amounts of 32P. Half the 32P of such late adsorbing particles remains attached to the bacterial debris; the rest appears within a few minutes in the medium as material soluble in 5% trichloroacetic acid. In contrast, over 95% of the **P of the early adsorbing phage is retained in the infected cells until lysis. Under conditions of almost simultaneous infection, the transfer value per infecting particle is constant for multiplicities of infection up to ten. 3. 32P-labelled Tz phage shows the same transfer as T4, and, according to PUTNAM AND KozLorF! and Koz.orr’, similar maximum values are obtained with the related phage TO. The unrelated phage T3 transfers about 40% of its phosphorus to the progeny. Thus an incomplete ®2P transfer of 40 to 50% may be a general characteristic of phage reproduction in E£. colt. 4. If the purines of Tz, T3 and T4 phages are labelled with 14C, transfer values are obtained which do not differ from those obtained with *2P. This suggests that the purine bases and the phosphorus are not transferred independently but as the constituents of nucleotides or larger units. References p. 442. 113 vot. 10 (1953) NUCLEIC ACID TRANSFER 441 5. Most of the transmitted %*P is incorporated into the early finished phage. An increase by a factor 2 to 3 in the phage yield per infected cell can be obtained in lysis inhibited cultures; such an increase in yield does not significantly increase the transfer. 6. The “‘second generation experiments’? previously published (MAALgE AND Watson?) show that uniformly labelled parental particles transmit phosphorus to all parts of the progeny particles which in turn become uniformly labelled. 7. Phage heavily damaged by X-rays or excluded from growth by the simultaneous presence of an unrelated phage transfer part of their nucleic acid. Isotope transfer is, therefore, not necessarily connected with the transfer of genetic specificity. It is well known that infecting particles cannot be recovered by artificial lysis at any time during the first half of the latent period (DOERMANN!’), and it is now firmly established that the nucleic acid components, phosphorus, adenine and guanine, are incompletely transferred from the infecting particle to the progeny. The transfer experi- ments, and especially the observation that chemical transfer may occur in the absence of genetic transfer, might, therefore, be viewed as evidence for an obligate and extensive breakdown of the infecting particle soon after adsorption. There is no doubt that such a breakdown can occur as evidenced by the unspecific transfer from radiation damaged or excluded phage (7). In both these cases, however, transfer takes place under abnormal conditions where the transferring particle does not participate in the reproduction processes. It is therefore unwarranted to conclude from these experiments that phos- phorus is always transferred via genetically unspecific structures. As they have turned out, the existing data on nucleic acid transfer, including the second generation experiments, do not decide whether transfer occurs via extensively degraded parental material or via large, genetically specific units. The data even fit with the assumption that nucleic acid structure of the infecting phage remains intact during replication and with a probability of about 50% becomes infective again and reappears among the progeny. ACKNOWLEDGEMENTS This investigation has been generously assisted by funds from the Danish Society for Infantile Paralysis and by the William Waterman Fund for the Combat of Dietary Diseases. The synthesis of the *C-adenine by Drs. M. CLARK AND H. M. KALCKAR was made possible by a grant to Dr KALcKar from the Lederle Laboratories Division of the American Cyanamid Company. We are also grateful to Dr A. H. DoERMANN for supplying us with the purine requiring strain of E. coli, to Dr DEAN FRASER for a most generous gift of anti-T3 rabbit serum, and to Dr R. Latarjet for putting the X-ray facilities of the Laboratoire Pasteur de l’Institut du Radium, Paris, at our disposal. SUMMARY a. When 2P-labelled phage reproduce in unlabelled coli bacteria a maximum of 40-50% of their label is transmitted to the phage progeny. Only 5-10 % of the label stay associated with bacterial debris after lysis: the remaining about 40% appear as non-sedimentable material in the lysate. b. The maximum transfer-values are very reproducible provided that all phages adsorbing on a given cell do so within about 2 minutes, and that the entire progeny is accounted for. c. Experiments with Tz, T3 and T4 all show a maximum *P transfer of 40-50%. The same phages labelled with 4C in the purines yield identical transfer-values. References p. 442. 114 442 J. D. WATSON, O. MAALGE VoL. 10 (1953) d. The transmitted °2P is found predominantly in the early formed phages. The latest formed progeny particles receive no **P from the parental particles. e. Damaged or excluded phage particles which do not participate in reproduction or in genetic exchange nevertheless transmit considerable amounts of **P to the phage progeny. RESUME a. Lorsque des phages marqués par “P se reproduisent dans des colibactéries non marquées, le 40-50% au maximum de leur *P est transmis aux phages nouveaux. Le 10% du *#P seulement reste dans les débris bactériens apres la lyse: le reste, 409% environ, apparait comme matiére non sédimentable dans le lysat. b. Les valeurs maxima de transfert sont bien reproductibles pourvu que l’adsorption de tous les phages d’une méme cellule ait lieu en moins de 2 minutes environ et que l’on tienne compte de tous les phages nouveaux. c. Des expériences faites avec T2, T3 et T4 donnent toutes, pour le transfert de *P, un maximum de 40-50%. Les mémes phages, marqués par /4C dans les purines, donnent des valeurs de transfert identiques. d. Le #2P transmis se trouve surtout dans les phages formés les premiers. Les phages de la nouvelle génération formés les derniers ne re¢oivent pas de **P des particules méres. e. Des particules de phages endommagées ou “‘exclues’’ (c.a d., dont la croissance est empéchée par un autre phage) qui ne prennent pas part a la reproduction ou a l’échange génétique, transmettent tout de méme des quantités considérables de **P a la nouvelle génération. ZUSAMMENFASSUNG a. Bei der Vermehrung von mit °2P markierten Phagen in nicht markierten Colibakterien wird ein Maximum von 40-50% des markierten Phosphors auf die Phagennachkommenschaft tibertragen. Nur 5-10% des markierten Phosphors bleiben mit den Bakterienresten nach der Lysis verbunden, die iibrigen 40% erscheinen als nicht sedimentierbares Material im Lysat. b. Die Maximumiibertragungswerte sind sehr gut reproduzierbar, vorausgesehen, dass die Adsorption bei allen Phagen einer gegebenen Zelle innerhalb von zwei Minuten stattfindet und dass die gesamte Nachkommenschaft beriicksichtigt wird. c. Versuche mit Tz, T3 und T4 zeigen alle ein Maximum der #2P-Ubertragung von 40-50%. Die gleichen, mit ##C in den Purinen markierten Phagen ergeben identische Ubertragungswerte. d. Der iibertragene *?P findet sich iiberwiegend in den zuerst gebildeten Phagen wieder. Die zuletzt gebildeten Nachkommenteilchen erhalten keinen **P von den Elternteilchen. e. Beschadigte oder von der Vermehrung ausgeschlossene Phagenteilchen, die nicht an der Vermehrung oder dem genetischen Austausch teilnehmen, tibertragen trotzdem betrachtliche Mengen 22P an die Phagennachkommenschaft. REFERENCES 1 F, W. PutNnaM AND L. Koztorr, J. Biol. Chem., 182 (1950) 243. 2S. M. Lesig, R. C. FRENCH, A. F. GRAHAM AND C. E. VAN ROOYEN, Can. J. Med. Sci., 29 (1951) 128. 3L.M. Koztorr, J. Biol. Chem., 194 (1952) 95. 40. MaaLgE AND J. D. Watson, Proc. Natl. Acad. Sci., 37 (1951) 507. 5 L. WEED AND S. S. COHEN, 1951, unpublished. 6 M. H. Apams, In Methods in Medical Research, Vol. ii, Year Book Publishers, Chicago, 1950. 7N. K. JERNE, Nature, 169 (1952) 117. 8 M. M. CLarK AND H. M. Katcxar, J. Chem. Soc., (1950) 1029. 9 O. MaaLoE AND G. S. STENT, Acta Path. Microbiol. Scand., (in press). 10 T. T. Puck, A. GAREN AND J. CLINE, J. Exptl Med., 93 (1951) 65. 11 A. D. HersHey, M. D. KaMEN, J. W. KENNEDY AND H. Gest, J. Gen. Physiol., 23 (1951) 643. 12 A. H. DoERMANN, J. Bact., 55 (1948) 257. 13 J. D. Watson, J. Bact., 60 (1950) 697. 14M. DELBRUCK, J. Bact., 50 (1945) 137, I5I. 1 J. J. WEIGLE AND M. DELBRUCK, J. Bact., 62 (1951) 301. 16 A. D. HERSHEY AND M. Cuase, J. Gen. Physiol., in press. 17 A. H. DoERMANN, Carnegie Inst. Year Book, 47 (1948) 176. Received April 24th, 1952 115 THE SYNTHESIS OF BACTERIAL VIRUSES* II. THE ORIGIN OF THE PHOSPHORUS FOUND IN THE DESOXYRIBO- NUCLEIC ACIDS OF THE T, AND T, BACTERIOPHAGES By SEYMOUR 8. COHEN (From the Children’s Hospital of Philadelphia (Department of Pediatrics) and the Department of Physiological Chemistry, School of Medicine, University of Pennsylvania, Philadelphia) (Received for publication, August 28, 1947) It has been shown that Escherichia coli B infected by T, bacteriophage in a synthetic medium, I’, synthesizes protein-bound constituents containing phosphorus and nitrogen at a constant rate (1). Evidence was presented that suggests that the virus-infected cell synthesizes virus constituents solely. This was most clearly indicated in the phosphorus metabolism, since the only phosphorylated protein-bound constituent synthesized in the infected cell was that characteristic of virus; 7.e., desoxyribonucleic acid (DNA). The ribonucleic acid (RNA) content of infected cells did not increase. Although the data presented suggested that the materials formed during infection were derived from the medium as in normal cells, it had not been proved that the nucleic acid phosphorus found in virus was not derived from cellular substance existing prior to infection, or had not been in normal host constituents during infection prior to appearance in virus. It seemed un- likely that the virus nucleic acid was derived from the host DNA, since sev- eral times more DNA was synthesized after infection than was originally present. However, it was possible that the RNA of the infected cell pos- sessed an active turnover yielding its phosphorus to DNA nucleotides, while the RNA was continually replaced with nucleotides whose phosphorus was derived from the medium. Two basic problems were therefore posed. (1) Was the DNA-P of virus derived from the phosphorus in the host prior to infection? (2) Was RNA a precursor of DNA? These hypotheses were tested by means of radio- active phosphorus, P??. EXPERIMENTAL Conditions for Optimal Virus Yield—A single generation of virus multiph- cation was studied under conditions of multiple infection. The possibility was therefore eliminated that the generations of virus produced after the first generation in a medium of known composition would be affected by the *The work described in this paper was aided by the Office of Naval Research. Reprinted by permission of the author and the American Society of Biological Chemists, Inc. from Tur JouRNAL OF BIOLOGICAL CueEmistry, 174 (1), 295-303 (1948). 116 296 BACTERIAL VIRUSES. II appreciable amounts of complex products, radioactive and otherwise, liber- ated by lysed cells. Some of the chemical phenomena and basic biological aspects of this system have been described in Paper I (1). The yield of virus from infected cells under conditions of multiple infec- tion in the aerated I’ medium at 37° was studied at concentrations of 5 X 107, 108, 2 X 108, and 5 X 10% bacteria per cc. It was found that with both T, and Ts maximal titers were obtained with 2 X 10* bacteria per cc. in about 4 to 6 hours. After this time, titers generally decreased markedly, and to a greater extent in T. lysates than in Ty lysates. This was due probably to a combination of several factors, readsorption to cellular débris, spontaneous thermal inactivation, surface denaturation, etc. In view of this marked loss of titer, it was considered desirable to prepare virus lysates by a single generation in infected cells for a 6 hour period, to minimize the destruction of virus activity. Distribution of DNA and Protein-Bound P in T, and T, Lysates—Two types of experiments were done. (1) Bacteria were grown in media contain- ing radioactive P, washed several times, and infected in media containing non-radioactive P; (2) bacteria were grown in media free of P*? and in- fected in the presence of P*?. The following typical control experiments were performed to indicate whether (1) the experimental conditions employed would permit the isola- tion of sufficient virus for analytical purposes, (2) virus isolated by these procedures would be very low in inorganic P and otherwise possess the proper chemical properties, (3) the newly synthesized DNA appeared in the virus fraction. Bacteria were grown to 2 X 108 per cc. in Ff medium. Two 125 cc. aliquots were sedimented and the bacteria washed twice with 0.85 per cent NaCl. Each aliquot was resuspended in 125 ce. portions of I me- dium to which were added an adsorption cofactor, 12 ec. of 5 X 10-7 M tryp- tophan in F (2). To these bacterial suspensions (A and C) were added small volumes of purified concentrates of Tyrt-F and T.rt-F to give cultures T.A and T.C containing 6 X 108 virus particles per cc. The virus concen- trates were also added to unwashed bacterial cultures (B and D) in F me- dium at the same concentrations to yield T,;B and T,.D; B contained 5 X 10-4 m tryptophan. The four infected cultures were assayed periodically ; the titers followed the course described previously. After 5 hours at 37°, the T, and T, lysates were stored for 13 hours at 4°. Very little inactivation occurred; the final titers were T;A 2.0 X 10”, T.B 4.0 X 10”, T.C 1.5 X 10", T.D 1.5 X 10” active virus particles per ec. The lysates were analyzed for total protein-bound P and DNA (1). They were sedimented at 4000 r.p.m. for 30 minutes and the sediments were washed twice with cold 0.85 per cent NaCl. The supernatant fluids were sedimented at 10,000 r.p.m. for 2 hours, and the supernatant fluids from 117 Ss. S. COHEN 297 this high speed centrifugation, containing more than 5 per cent of the activity of the lysate, were analyzed for protein-bound P and DNA. Virus was isolated from the high speed sediments as described previously (3, 4). The distribution of these substances in the four lysates is given in Table I. It may be seen that the largest amounts of DNA and P appear in the virus-containing fraction; 7.e., the high speed sediment. When this fraction was resuspended in 5 ce. of 0.85 per cent NaCl and centrifuged at 4000 R.P.M. for 30 minutes, approximately 25 per cent of the DNA of the fraction had become insoluble. After dialysis of this supernatant fluid against running water overnight, the once sedimented virus concentrate had DNA-P to total P ratios which were very close to 1.0. These values for isolated dialyzed virus after one differential centrifugation cycle were TABLE | Distribution of Protein-Bound P and DNA in Lysates of Infected Bacteria Low speed High speed | High speed Virus lysate Analyses Initial total | Total lysate sediment supernatant |sediment con- fluid taining virus mg. mq. per cent per cent per cent AAV an eCC: iP 0.210 31 29 40 DNA 0.213 1.49 25 37 38 i BeOh iP 0.141 0.263 19 21 60 DNA 0.220 1.80 17 21 62 eC OGie 12 0.161 0.275 27 22, 51 DNA 0.275 1.63 29 20 51 sD HO2b es) |e 0.148 0.260 33 1%, 50 DNA 0.221 1.36 35 16 49 T,A 1.05, TiB 1.00, T.D 1.02. Thus the conditions described above yielded fractions with the characteristics of virus whose inorganic P contents were so low as to be undetectable. Infected cells may synthesize as much as 7 to 8 times as much DNA as was originally present. In many cases, at least 3 times as much DNA was isolated in the virus as was present in the bacteria at the onset of infection. It is considered probable that DNA formed after infection and not recov- ered in isolated virus was nevertheless originally part of the virus and was lost either by adsorption to débris or by becoming insoluble as a result of high speed centrifugation. No indication has been obtained in prepara- tions of either Tor* or Tyrt bacteriophage from F lysates that a phosphoryl- ated compound other than DNA is present in either virus. Furthermore, in contrast to the observations of Taylor (5), preparations of these viruses from nutrient broth lysates prepared from infected cells as described above also possessed DNA-P to total P ratios of 1.0 (6). Hence no evidence has 118 298 BACTERIAL VIRUSES. II been obtained to confirm the report of Taylor that T.r* virus contains ribonucleic acid (5). It is considered possible that the method of isolating virus from 5 to 6 hour lysates described above assists in the removal in the low speed sediment of bacterial components containing RNA. Since pro- teolysis in r+ systems is relatively weak, the prolonged period used by Hook et al. (7) in the preparation of lysates may have assisted the degra- dation of bacterial débris to a size which would not sediment at a low speed but would at high speeds. Proteolysis in r* systems in F medium seems even less pronounced than in broth. Synthesis of Virus in Labeled Host Cells—Small inocula of bacteria were grown to2 X 10% per ce. in 125 aliquots of F medium containing 0.02 to 0.05 millicurie of P?? in 13.7 mg. of inorganic phosphate. More than 99 per cent TaBLeE II Radioactivity of Virus Isolated after Synthesis in P%?-Labeled Cells in Media Free of P** Relative radioac- DNA-P tivity, Virus isolated Total P Bacteria Virus virus P in virus host P counts per 10 y P per | counts per 10 y P per min. min. per cent ore 1.00 222 33 14 Tora 1.00 293 53.5 18 Un ere 1.01 37.8 5.9 16 Shri | 1.06 293 53.3 18 *Virus to cell ratio 3.0. tVirus to cell ration 5.0. of the P of the bacteria was derived from the P of the medium. The cells were washed twice with 0.85 per cent NaCl. The second washing con- tained less than 0.1 per cent of the radioactivity of the original medium, as determined on a Geiger-Miiller counter, kindly loaned by Dr. H. D. Brun- ner of the Department of Pharmacology of the University of Pennsylvania. The radioactive cells were resuspended in 131 cc. of F medium containing 2.5 X 10-4 m tryptophan. Purified Tort or Tirt virus was added and the infected cultures were incubated for 5 hours and regularly assayed for virus during this interval. The lysates were stored at 4° overnight and virus was isolated as previously described. The DNA contents of the initial cultures, final lysates, and isolated virus were determined. The radioactivity of the virus was compared with the radioactivity of the uninfected host cell per unit weight of P. Samples of known P content were digested in 1.2 cc. of 60 per cent perchloric acid and diluted to 10 ec. after the solutions were neutralized to pH 4. The counting 119 Ss. S. COHEN 299 chamber was filled by perfusion and held identical aliquots of sightly more than 3 cc. The background was determined between samples and yielded 8 to 11 counts per minute. This was subtracted from the count on the sample. Samples and the time of counting were adjusted to yield 100 to 1000 counts. Comparisons of samples were made at similar total counts. In Table II are presented data on four experiments of this type, in which the increments in DNA in the infected bacteria and the yields of isolated virus were comparable to the data presented previously. The radioactivity of the virus isolated after synthesis under these conditions was far lower than the radioactivity of the host P. It has therefore been concluded that most of the P organized into virus is derived from the medium after in- fection. TaBLe III Radioactivity of Virus Synthesized in Unlabeled Cells in Media Containing P?? Relative radioac- ) DNA-P b : tivity Virus isolated Total P F medium Virus virus P in virus medium P counts per 10 y P per | counts per 10 y P per min. min. per cent AU ers 1.04 443 315 71 earish 1.00 840 633 75 ais 422 291 69 *Virus to cell ratio 5.0. tVirus to cell ratio 3.0. Synthesis of Virus in Unlabeled Host Cells in Media Containing Radio- active Phosphate—Bacteria were grown in the absence of radioactive P. Virus was added immediately after the addition of 0.02 to 0.05 millicurie of inorganic phosphate and tryptophan for the adsorption of Ty. The cul- tures were incubated, and the DNA increments, rates of virus liberation, and isolated virus were studied as described previously. In Table III are presented some data on experiments of this type. The radioactivity of the virus isolated after synthesis under these conditions was 70 to 75 per cent of that of the inorganic phosphate of the medium. These data confirm the previous conclusion that most of the P organized into virus is derived from the medium after infection. Nucleic Acid Turnover after Infection—Since the P*? added to the F me- dium was assimilated in DNA after infection, the distribution of the radio- activity in the infected cell was studied. In a typical experiment bacteria were grown to 2 X 108 per ce. in 1 liter of F medium. The cells were centri- fuged and resuspended in 100 ce. of F medium. To the culture were added 120 300 BACTERIAL VIRUSES. II 4.5 cc. of Ter? at 2.2 X 10!! per cc. and 1.5 mg. of P in inorganic phosphate containing about 0.05 millicurie of P**. The infected culture was aerated vigorously at 37° for 1 hour. At zero time and at 1 hour, aliquots were analyzed for protein-bound P, DNA, and radioactivity. The counts per minute of the medium were 93 per microgram of P. In 1 hour, the bacterial protein-bound P increased from 0.0109 mg. per cc. to 0.0163 or 5.4 y per cc. Newly assimilated P had a radioactivity of 98 counts per microgram. The DNA had increased from 24 to 72 y per cc., or the ratio of newly formed DNA to original DNA was 2:1. Following the removal of aliquots after 1 hour, the entire culture was pre- cipitated with 5 per cent trichloroacetic acid (TCA) and the sediment was washed twice in the centrifuge with TCA. The supernatant fluids were pooled. The sediment was washed twice with 95 per cent alcohol and twice with ether. These supernatant fluids were combined with lipide extracts prepared by three extractions of this sediment with boiling alcohol-ether (1:1). The pooled lipide extracts were taken to dryness in vacuo and dis- solved in 10 ec. of CHCls. The dried lipide-free sediment was fractionated according to Schmidt and Thannhauser (8). It was incubated with 2 ce. of N KOH at 37° for 20 hours and chilled. To the solution containing a very slight flocculent precipitate were added 0.4 ec. of 6 N HCl and 2 cc. of 5 per cent TCA. After 30 minutes at 0°, the sediment, containing DNA, was removed by centrifugation and washed with several ec. of water. The DNA fraction was dissolved in 0.1 N NaOH. The combined supernatant fluids were precipitated according to Delory (9); essentially no P was found in this sediment and this fraction was discarded. The supernatant fluid contained ribose-3-phosphate nu- cleotides and comprised the RNA fraction. Both the DNA and RNA fractions prepared above were analyzed for DNA. The former contained 6.9 mg. and the latter 0.6 mg. of DNA, or a total of 7.5 mg. of DNA was recovered. The total DNA content of the culture was 7.4 mg. Radioactivity measurements of the RNA fraction were corrected for the radioactivity of the DNA it contained. This com- prised 10 per cent of the P of this fraction. The radioactivities of the nucleic acid fractions are summarized in Table IV. One-half of the P in the RNA fraction consisted of ribose-3-phosphate nucleotides reactive in the Bial reaction (10). Pyrimidine nucleotides de- rived from RNA and comprising one-half of the total P would not be expected to react. Thus the RNA fraction with the exception of a small amount of DNA contained ribose nucleotides of the expected character- istics. It may be seen from Table IV that the newly synthesized DNA contained P of essentially similar radioactivity to that in the medium, while the RNA 121 S. S. COHEN 301 fraction contained essentially no radioactive P. It may be concluded from this experiment and others which yielded the same result that not more than 2 per cent of the RNA could have been undergoing synthesis involving P of the medium. It is highly improbable that the turnover of this much RNA, if any synthesis actually occurred, could have produced the synthesis of the DNA found. Therefore it appears that RNA nucleotides are not precursors to DNA synthesis in this system. Since special precautions were not taken to purify the nucleotides of the RNA fraction before estimating the radioactivity, it is possible that even the low radioactivity found (2 per cent) was due to some contaminant, and was not truly significant. In this experiment the lipide P was found to have a radioactivity of about 26 per cent of the P of the medium. Repre- cipitation of phospholipide in the presence of carrier sphingomyelin derived TABLE IV Inclusion of Radioactive P into Nucleic Acids of Escherichia coli B Infected with T ort Bacteriophage Nucleic acid | Total Exec peund Lipide-free bacterial | Counts per 7 P per | Corrected counts per residue min. vy P per min. per cent per cent 7 DNA 48 13.8 60 90 RNA 36.5 10.5 8.8 2 DNA, counts corrected for presence of original bacterial DNA; RNA, counts cor- rected for presence of traces of DNA in RNA fraction. from beef lung readily reduced the radioactivity sufficiently to suggest the inactivity of the phospholipide fraction. Fractionation of Non-Protein P—It was not found possible to isolate or- ganic phosphate from the TCA supernatant fluids in this experiment, owing to the large excess of inorganic P of the medium. In another experiment, the bacteria were first sedimented to remove the inorganic P of the medium before fractionation. 10 ec. of TCA extract contained 66 y of organic P and 183 y of inorganic P. To this solution, adjusted to pH 8.3 with NH,OH, were added 1 cc. of 2.5 per cent CaCl, and 2 cc. of a 0.5 per cent suspension of MgCO3;. After being chilled, the mixture was centrifuged and the sediment was discarded. The supernatant fluid was precipitated with 4 volumes of alcohol and stored overnight in the ice box. The alcohol precipi- tate was placed in 3 cc. of water. It contained 44 y of organic P, of which ribose-5-phosphate in some form comprised 75 per cent of the total organic P of the fraction (10). The water-soluble fraction of the alcohol precipi- tate contained 16.5 y of pentose P in 25 y of organic P. The radioactivity per microgram of P of this fraction was 37 per cent of that of the inorganic 122 302 BACTERIAL VIRUSES. II P of the medium. It is not known in what compounds the radioactivity resided. DISCUSSION It may be seen from the data presented in this paper that the hypotheses suggested in Paper I have been verified by means of the isotope technique. These are (1) that phosphorylated virus constituents are synthesized in the main from P assimilated from the medium after infection, and (2) that RNA after infection has a very low, if any, turnover rate, and is not a precursor of virus DNA. Nevertheless the radioactivity of the virus P was significantly different from that of the P of the medium. It has been observed that a very small amount of virus multiplication may occur in the absence of external P (11). Therefore it appears that some of the P of the host may be incorporated into virus. It is considered likely that this small amount of P is derived from the intracellular pool of inorganic P or low molecular weight organic P which can equilibrate with P assimilated after infection. This intracellular metabolic pool would be used for the synthesis of DNA after infection and possibly even before the P assimilated after infection. Thus in short term experiments involving r strains the percentage of host P appearing in virus may conceivably be greater than that observed in these long term experi- ments with rt type virus. This remains to be tested. Many workers have found that protein and DNA syntheses are accom- panied by a vigorous RNA metabolism in a wide variety of tissues. In addition it has been reported that there is apparently a conversion of ribose nucleotides to desoxyribonucleotides in the early stages of cleavage of the fertilized sea-urchin egg (12). These data are ably reviewed by Brachet (13). In the system described in these papers, none of these phenomena are to be observed. It appears possible to study the precursors of DNA in this system uncomplicated by the metabolism of RNA. I am indebted to Dr. Samuel Gurin of this University and Dr. M. Kamen of Washington University for their advice and assistance in the course of these studies. I wish to thank Miss Catherine Fowler for her technical assistance. SUMMARY The preparation and properties of bacterial lysates after multiple infec- tion have been described. As a result certain isotope experiments requiring viral isolation could be performed. The distribution of the bacterial and viral components in these lysates has been studied. It has been found pos- sible to recover in a purified concentrate of virus much of the P assimilated 123 Ss. S. COHEN 303 and the DNA synthesized after infection. The newly synthesized DNA greatly exceeded the starting DNA of the bacterial culture. With P*? in the labeled host or labeled medium, the DNA of the virus has been found to be built in the main from the inorganic P of the medium. Ribonucleic acid was not a precursor in the synthesis of this DNA and was essentially inert in infected cells. BIBLIOGRAPHY . Cohen, 8. S., J. Biol. Chem., 174, 281 (1948). . Anderson, T. F., J. Cell. and Comp. Physiol., 25, 17 (1945). . Cohen, 8. S., and Anderson, T. F., J. Exp. Med., 84, 511 (1946). . Cohen, 8. 8., J. Biol. Chem., 168, 511 (1947). . Taylor, A. R., J. Biol. Chem., 165, 271 (1946). . Cohen, 8. 8., Cold Spring Harbor symposia on quantitative biology, Cold Spring Harbor, 12, 35 (1947). 7. Hook, A. E., Beard, D., Taylor, A. R., Sharp, D. G., and Beard, J. W., J. Biol. Chem., 165, 241 (1946). 8. Schmidt, G., and Thannhauser, S. J., J. Biol. Chem., 161, 83 (1945). 9. Delory, G. E., Biochem. J., 32, 1161 (1938). 10. Albaum, H. G., and Umbreit, W. W., J. Biol. Chem., 167, 369 (1947). 11. Fowler, C. B., and Cohen, 8. 8., J. Exp. Med., in press. 12. Brachet, J., Arch. Biol., 48, 520 (1937). 13. Brachet, J., Embryologie chimique, Liége (1944). oor WW Fe 124 ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. VI. INFLUENCE OF BACTERIOPHAGE T2 ON THE SYNTHETIC PATHWAY IN HOST CELLS* By ArrHuR KornBerG, STEVEN B. ZimMerMAN,'S. R. KornNBERG, AND Joun Jossz* DEPARTMENT OF MICROBIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS Communicated April 20, 1959 Information now available about DNA! synthesis by Escherichia coli enzymes? ~4 has encouraged an inquiry into the biochemical basis for the observation that a phage-infected LF. coli cell ceases to produce its own DNA and makes instead the DNA characteristic of the infecting phage.’ This general problem poses several rather specific questions which may be summarized as follows: 1. T2, T4, and T6 DNA differ from the DNA of £. coli, as well as from that of other sources which have been examined, in containing hydroxymethyleytosine (HMC) but no cytosine.6 Flaks and Cohen’ have already shown that within several minutes after infection by phage T2, T4, or T6, a new enzyme which hy- droxymethylates deoxycytidine 5’-phosphate is produced. Is there an enzyme for converting the resulting dHMC-5-P to the triphosphate level in order to provide a functional substrate for DNA synthesis? 2. With respect to the exclusion of cytosine from the DNA of phage T2, T4, and T6, is there a mechanism in the infected cell for removal of deoxycytidine triphos- phate from the site of polymerase action? 3. The DNA’s of T2, T4, and T6 contain glucose linked to the hydroxymethyl groups of the HMC in characteristic ratios,*® * !° although it is clear that in T2 and T6 some of the HMC groups contain no glucose.? According to our present understanding of DNA synthesis,‘ it is difficult to conceive how these constant ratios are achieved if the incorporation were to occur via glucosylated and non- glucosylated HMC nucleotides. Is there an alternative mechanism involving direct glucosylation of the DNA even though direct substitutions on intact DNA have been hitherto unknown? 4. Following phage T2 infection there is a temporary halt followed by a resump- tion of DNA synthesis at about 5 times the rate shown by the uninfected cell."! However, measurements with extracts of infected cells, using standard substrates, revealed much diminished rather than the anticipated augmented levels of DNA- synthesizing activity.!2. What are the altered conditions for assay of DNA syn- thesis in infected cell extracts which would elicit the high levels of activity ex- pected from the physiologic studies? We have explored these questions and have found that following infection with phage T2 several new enzymes appear.!* These are (1) an enzyme which phos- phorylates dHMC-5-P, Jeading to the synthesis of hydroxymethyldeoxycytidine triphosphate (€dHMC-TP), (2) an enzyme which removes the termina] pyro- phosphate group from dCTP, and (3) an enzyme which transfers glucose from UDPG directly to the HMC in DNA. Measurements of DNA synthesis, using dHMC-TP in place of dCTP, revealed about a 12-fold increase in activity in extracts of infected cells over the levels observed in uninfected cell extracts. Reprinted by permission of the authors and the National Academy of Sciences from the PROCEEDINGS OF THE NATIONAL ACADEMY OF Sciences, 45 (6), 772-785, June, 1959. 125 Vou. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. Sy Sa | Os METHODS AND MATERIALS Preparation of Cell Extracts.—E. coli B was grown at 37° with vigorous aeration in M-9 medium™ modified to contain per liter: KH,PO,, 3 gm, NasHPQ,, 6 gm, NHC, 1 gm, MgSO,-7H20, 0.49 gm, glucose, 5.0 gm, FeSO,-7H20, 0.5 mg, CaCle, 55 mg. Growth at a logarithmic rate continued to 4-5 X 109 cells per ml with a generation time of about 50 min. Cell extracts were prepared in two ways: Method I: Cultures grown to 2 X 108 cells per ml were chilled, centrifuged, and the cells resuspended at 4 X 10° cells per ml in cold growth medium. Five T2rt+® or T5 per cell were added and after a 4-min adsorption period at 0°, the culture was diluted twenty-fold into fresh growth medium at 37° and aeration continued. The time of dilution was taken as “zero minutes.” 50-ml aliquots were pipetted rapidly onto crushed ice at intervals. The cells were sedimented by centrifuging for 5 min at 10,000 X g, were resuspended in 1 ml. of 0.5 M glycylglycine buffer, pH 7.0, containing 0.001 M glutathione, and were stored for 1 to 3 days at —15°. The cells were disrupted in a 10 ke Raytheon sonic oscillator. After removal of a small amount of debris by centrifugation, the extracts contained about 2 mg of protein per ml. Method II: Cultures were grown to 2 X 10° cells per ml, in the modified medium without CaCl, and four T2r* per cell added (‘‘zero minutes”). 50-ml aliquots were pipetted rapidly onto crushed ice at intervals. The cells were sedimented and resuspended in 4 ml of 0.05 M glycylglycine buffer, pH 7.0, containing 0.001 M glutathione and disrupted as above. Extracts containing about 6 mg of protein per ml were obtained after centrifugation. All results refer to extracts prepared by Method I unless otherwise stated. While Method ITI was less effective for phage multiplication (see below), a description of this method is included since it provided an alternative and efficient technique for obtaining concentrated extracts in kinetic studies. The activities per mg protein for the several enzymes studied were found to be at levels similar to those obtained by Method I. Bacteriophage Determinations.—Bacteriophage was assayed by standard tech- niques.'® Intracellular phage was measured after “lysis from without’ essentially as described by Doermann.” The formation of infectious units in both T2r+ and T5 infected cells (Fig. 1) was found to proceed in normal fashion in cells infected as described in Method I. T2r+-infections produced by Method II yielded only about 2 phage per original cell at 25 min when measured after “lysis from without,” although after clearing of the culture a yield of several hundred phage per original cell was obtained. Enzyme Assays and Preparations.—Phosphorylation of the deoxynucleoside monophosphates (kinase activities) was measured, as described before,? by using a 5’-P*-labeled mononucleotide as substrate and assaying the amount of label which becomes resistant to the action of semen phosphatase.'8 Formation of dHMC-5-P from dC-5-P (hydroxymethylase) was assayed according to Flaks and Cohen.” The assay of DNA synthesis (“polymerase”) was measured by the con- version of a C'-labeled deoxynucleoside triphosphate into an acid-insoluble product.” “Polymerase” fraction VII from uninfected H. coli was prepared as previously described.? 126 774 BIOCHEMISTRY: KORNBERG ET AL. Proc. N. A. S. Substrates —Deoxynucleotides and samples of native and enzymatically syn- thesized DNA were prepared as in earlier studies.2 4 dHMC-5-P was synthesized according to Flaks and Cohen’ using a 230-fold purified hydroxymethylase!®; C1- dHMC-5-P was obtained by using C!4-formaldehyde (Volk Radiochemical Co.) in the hydroxymethylation reaction, and P*-dHMC-5-P was prepared by using P?2-dC-5-P. The E,, value determined for the nucleotide in this preparation was 13.5 X 103 at 284 mu at pH 1. Since this value conflicts with that of 11.7 103 given by Flaks and Cohen,’ it is regarded as provisional and requires further pe) Os 4 (eo) oO io) phage per bacterium fe) O 10 20 30 40 50 TIME - minutes Fic. 1.—Appearance of phage in T2- or T5-infected cells. In- fected cultures were prepared by Method I. Infectious units were measured after “lysis from without.” checking. C!-glucose 6-phosphate was prepared by hexokinase action on uni- formly labeled C!4-glucose (Isotope Specialties Co.). C!-UDPG was prepared from C4-glucose 6-phosphate and uridine triphosphate by the action of phospho- glucomutase and UDPG pyrophosphorylase as outlined by Glaser and Brown.” Unlabeled UDPG was a product of the Sigma Chemical Company. RESULTS An Enzyme which Phosphorylates dHMC-5-P.—At about 4 min after infection of E. coli with phage T2, it was possible to detect in the extracts an enzyme which catalyzes the phosphorylation by ATP of dHMC-5-P (Fig. 2A). This reaction was 127 Vou. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 775 undetectable (<0.5 per cent of the maximal value after infection with T2) in normal cells or in extracts of cells infected with T5 (Fig. 2 A), a phage without HMC in its DNA. The maximal level of phosphorylating (‘‘kinase’’) activity for dHMC-5- P was of the same order as that of the kinases for the other deoxynucleotides in- femoles/hr. /mg.protein -5$ 10 20 3040 -54 10 20 3040 -5# 10 20 3040 TIME-minutes Fig. 2.—Deoxynucleotide-phosphorylating enzymes (“kinases”) and hydroxymethylating enzyme levels before and after infection with phage T2 or T5. The arrow indicates the start of infection (‘‘zero minutes,’’ see Method I). Assays were as referred to in Methods. 128 776 BIOCHEMISTRY: KORNBERG ET AL. Proc. N. A.S. corporated into viral DNA (see below) and was essentially similar in extracts pre- pared by either Method I or Method II. The product of the dHMC-5-P kinase action (with the presumed participation of nucleoside diphosphate kinase in the preparation) was shown to be the triphos- phate. Using a 20-fold purified kinase preparation,!® 25 wmoles of dHMC-TP were prepared and isolated by ion-exchange chromatography, a yield of 92 per cent based on the starting dHMC-5-P. Theoretical specific radioactivity values were found in the isolated dHMC-TP when P*- or C'-labeled dHMC-5-P was the otein moles /hr./mg. pr Sect Unio! P20" «or MaNaS TIME - minutes Fic. 3.—dCTPase levels before and after infection with phage T2 or T5. The arrow indicates the start of infection (‘‘zero minutes,’ see Method I). The incubation mixtures (0.25 ml) contained: 6.0 mumoles of dCTP labeled with P*? in the terminal pyrophosphate group (103 cpm per mumole), glycine buffer, pH 9.2 (0.04 M), MgCl, (0.008 M), 2-mercaptoethanol (0.01 M/) and a quantity of extract containing about 0.3 ug of protein. After 20 min of incubation at 37°, the reaction was terminated by add- ing 0.5 ml of 0.1 N HCl, followed by 0.2 ml of a mixture (contain- ing per ml: 5 mg of crystalline bovine serum albumin, 25 umoles of sodium pyrophosphate and 25 umoles of potassium phosphate, pH 7), and 0.1 ml of a Norit suspension (20 per cent packed volume). The Norit was removed by centrifugation and the Supernatant fluid was assayed for radioactivity. starting material. After concentration with Norit, analysis revealed a ratio of HMC: total P: acid-labile P of 1.0: 3.2: 2.2, using an E,, value of 13.5 X 10° at 284 my at pH 1. Kinase Levels for the Other Deoxynucleotides and Levels of the Hydroxymethylating Enzyme.—It is noteworthy that after T2 infection, the kinase levels for dT-5-P and dG-5-P were increased approximately 20 and 45 times, respectively, while that for dA-5-P was essentially unaltered (Fig. 2 B, C, D). Bessman has made similar observations independently.2!_ As mentioned above, the kinase levels for 129 VoL. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 777 each of the four deoxynucleotides incorporated into phage T2 DNA reach values of about the same magnitude. By contrast, only traces of dC-5-P kinase were detected (Fig. 2 #). Furthermore, extracts of T2-infected cells were found to inhibit the dC-5-P kinase activity of normal cell extracts when equal amounts of infected and non-infected extracts were mixed. As will be described below, this inhibitory effect is due to an enzyme (dCTPase) which splits dCTP. By use of fluoride (8 X 10~3 MW), which inhibits dCTPase more than 98 per cent, but the dC- 5-P kinase by 15 per cent or less, it was possible to show that there was actually little or no change in the dC-5-P kinase levels upon infection. Extracts prepared by Method II showed the same kinase patterns after T2 infection. Infection with phage T5, which contains cytosine rather than HMC, showed in- creased kinase activities for the four deoxynucleotides which are present in its DNA (Fig. 2 B, C, D, FE). The 10-fold increase in dC-5-P kinase activity in the extracts of T5-infected cells may be contrasted with the absence of any increase in this activity in the extracts of T2-infected cells. Hydroxymethylating activity was first detected at 4 min after T2 infection, and, as predicted from the results of Flaks and Cohen,’ was absent from T5-infected cell extracts (Fig. 2 F). An Enzyme which Destroys Deoxycytidine Triphosphate (dCT Pase).—The in- hibitor of dC-5-P kinase that develops upon T2 infection has been identified as an enzyme which splits dCTP by removal of the terminal pyrophosphate group (Fig. 3). Extracts of uninfected cells have 1 per sent or less of the dCTPase activity observed in extracts of T2-infected cells; the level of dCTPase activity of T5-infected cells was the same as in normal cells. The relative insensitivity to fluoride of the dCTPase activity in normal cells makes it doubtful that it represents the same enzyme found in the T2-infected cells. After partial purification of the dCTPase,'® which reduced the level of an inor- ganic pyrophosphatase to 2 per cent of the dCTPase, it was demonstrated that the complete splitting of 0.92 umole of dCTP was accompanied by the appearance of 0.74 umole of inorganic pyrophosphate identified as Norit-nonadsorbable, acid- labile P and 0.15 umole of orthophosphate. With the purified enzyme preparation, the rates of cleavage of cytidine triphosphate and ATP were less than 1 per cent of that of dCTP; the splitting of dHMC-TP was 1-2 per cent of that of dCTP. The K,, of dCTP for the enzyme is in the region of 10-* M. In view of this high affinity for dCTP and of the high level of enzyme activity compared to the dC-5-P kinase in the extract (cf. Fig. 2 E and Fig. 3), it is reasonable to suppose that the dCTP concentrations in the infected cell are reduced to an extremely low level. An Enzyme which Glucosylates the HMC of DNA.—An enzyme which transfers glucose from UDPG to DNA containing HMC was observed in extracts of T2- infected but not T5-infected or normal cells (Fig. 4). With the partially purified enzyme!® UDPG could not be replaced by glucose, glucose 1-phosphate or glucose .6-phosphate (Table I); similar results were also obtained with the crude extracts. The reaction requires HMC-containing DNA, which for these experiments was enzymatically synthesized from dHMC-TP, dATP, dGTP, dTTP, purified poly- merase, and primer DNA derived from any one of several sources (calf thymus, E. coli, phage T2, phage ¢X17422). When DNA enzymatically synthesized with dCTP in place of dHMC-TP, or when the glucosylated DNA from phage T2 itself 130 778 BIOCHEMISTRY: KORNBERG ET AL. Proc. NiALS: were used, no glucose fixation in DNA was detectable (Table 1). dHMC-5-P and dHMC-TP failed to substitute for HMC-containing DNA as glucose acceptors. For example, in an incubation mixture containing these three types of HMC com- pounds with 30-fold purified enzyme, the HMC-DNA fixed 46 per cent of the glucose of the UDPG, while dHMC-5-P and dHMC-TP fixed none (<0.5 per cent). 60 ol (e) oO (e) moles /hr./mg. protein ua eo) fo) TIME - minutes Fic. 4.—DNA-glucosylating enzyme levels before and after infection with phage T2 or T5. The arrow indicates the start of infection (‘‘zero minutes,’”’ see Method I). The incubation mix- tures (0.20 ml) contained: 10 myumoles of UDPG labeled uni- formly with C!4in the glucose residue (2 X 10% cpm per myumole), Tris buffer, pH 7.5 (0.1 M), glutathione (0.02 17), DNA synthe- sized enzymatically (using thymus or phage T2 DNA as primer) and containing about 1 mumole of HMC, and extract containing 10-50 wg of protein. After 15 min of incubation at 30°, the mix- ture was treated as in the “‘polymerase’’ assay of incorporation of a labeled deoxynucleotide into an acid-insoluble product (see Methods). In the presence of an excess of HMC-DNA, the glucose of UDPG is transferred completely to the DNA. With an excess of UDPG and enzyme, the fixation of glucose in DNA reaches a limiting value, which is a function of the HMC-DNA present (Fig. 5). The number of glucose residues fixed in this experiment was approximately 60 per cent of the number of HMC residues in the added DNA. At this point it may be premature to regard the glucosylation limit observed with 131 Vou. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 719 TABLE 1 SPECIFICITY OF THE ENZYME WHICH GLUCOSYLATES DNA Expt. Glucose fixed in No. Conditions DNA, myumoles 1 Complete system (10 mumoles of C'4-UDPG) 69 Add C!-UDPG (10 mumoles) . Add C!2-glucose (200 mumoles) Add C}2-glucose 1-P (200 mymoles) Add C}2-glucose 6-P (250 mumoles) Replace UDPG with C!*-glucose (10 mumoles) Replace UDPG with C!*-glucose 6-P (12 myumoles) 2 Complete system (DNA containing 0.46 mumole of HMC) Replace HMC-DNA with cytosine-DNA (containing 0.75 myumole cytosine) Replece HMC-DNA with T2 DNA (containing 2 mymoles ) 00 S i=) S i=) The complete system had the composition and was treated as described in Figure 4, using a 30-fold purified enzyme, 0.3 pg in Expt. 1 and 1.5 wg in Expt. 2. In each case the DNA was glucosylated to its limit (see Fig. 5). * The glucose fixed is calculated on the basis of the specific radioactivity of the UDPG after dilution of the C'# sample with C!2-UDPG. oles m ‘@) on ixed in DNA— my M ro) Oz nie enzyme protein Atal ug enzyme protein Glucose F e) oO 0.0 1.0 20 3.0 4.0 HMC-DNA — my Moles of HMC Fia. 5. —Limit of glucose fixation in DNA as a function of the amount of DNA added. The experimental details were as in Fig. 4, using the partially purified enzyme. The HMC-DNA was prepared with thy- mus DNA as primer. a given sample of HMC-DNA as distinctive for the type of primer used in the enzymatic synthesis of the HMC-DNA. Further studies are required to determine how conditions of enzymatic polymerization, as well as the isolation of the DNA, may influence the glucose/HMC ratios obtained. 132 780 BIOCHEMISTRY: KORNBERG ET AL. Proc. N. A. §. The C1*-glucose fixed in DNA was rendered acid-soluble by crystalline pan- creatic deoxyribonuclease. When 52 per cent of the nucleotides were no longer acid-precipitable, 88 per cent of the glucose had been made acid-soluble. When the C'-glucosylated HMC-DNA was digested to completion under conditions which Lehman has found?’ to yield a quantitative conversion of phage T2 DNA to 5’- mononucleotides, over 95 per cent of the radioactivity was found in the HMC deoxynucleotide fractions of the ion-exchange chromatogram. Increase Rate of DNA Synthesis upon Infection—DNA synthesis, measured by the standard assay,” but with dHMC-TP instead of dCTP, is increased 12-fold in extracts prepared 19 min after infection with T2 (Fig. 6). Little or no DNA syn- thesis can be measured in these extracts when dCTP replaces dHMCTP. However, Fic. 6—DNA “‘polymerase”’ levels before and after infection with phage T2 with dHMC-TP as substrate in place of dCTP. The arrow indicates the start of infection (“zero minutes,” see Method I). The incu- bation mixtures (0.30 ml) contained: 10 myumoles each of dGTP, dATP, dTTP and dHMC-TP (C!4, 1 < 103 cpm per mumole), Tris buffer, pH 7.5 (0.07 M), MgCl. (0.007 M), 2-mercaptoethanol (0.001 M), 0.04 ml. of “heated DNA” and sonic extract con- taining 10-50 wg of protein. The “heated DNA” was prepared by heating at 100° for 5 min, at pH 9.2, an extract of T2- infected cells; this preparation could be purified without loss of activity by treat- ment with ribonuclease, Norit, dialysis and precipitation with acid; it was inactivated by treatment with deoxyribonuclease. Fur- ther details of procedure were as referred to in Methods. mu moles/hr./mg. protein -5 4 lo 20 TIME- minutes using 0.005 M F~ to inhibit dCTPase, DNA synthesis was elevated to levels near those observed in assays with dHMC-TP. It should be emphasized that these measurements of rates of DNA synthesis with dHMC-TP were made with heated DNA as primer; when unheated DNA was used, there was no demonstrable increase in rate in the infected cell extracts. Heated DNA of phage T2 or calf thymus origin served as well as that used in Figure 6. The basis for this requirement for heated DNA with infected cell extracts requires further investigation. DISCUSSION In addition to the interest inherent in understanding the nature of viral infection of a cell, studies of the pathway of DNA synthesis in infected cells provide a means of testing and expanding our conceptions about the mechanism of DNA replication in normal cells. 133 Vou. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 781 Control of DNA Synthesis at the Level of Deoxynucleotide Phosphorylation.— It appears from studies with various analogues of the naturally occurring purines and pyrimidines* that an analogue in the form of a deoxynucleoside triphosphate is incorporated into DNA when its structure has the hydrogen-bonding properties of the base it replaces. For example, uracil and 5-bromouracil are both effective substitutes for thymine. Yet, uracil is never found in DNA* while bromouracil, when supplied to the cell, readily replaces thymine in the DNA.” The reason ap- pears to be the absence of any mechanism for phosphorylating deoxyuridylate as contrasted with the availability of an enzyme for phosphorylating 5-bromodeoxyuri- dylate or 5-methyldeoxyuridylate (thymidylate).’ The present studies of phage-infected cells provide additional examples of control at the phosphorylation level. Earlier observations with 5-methyldeoxycytidylate* had suggested the lack of an enzyme for phosphorylation of a 5-substituted cytosine deoxynucleotide, and posed the problem of how 5-hydroxymethyleytosine deoxy- nucleotide becomes a substrate for phage T2 DNA synthesis. This problem seems to be solved by the development of a new enzyme after phage T2 infection of the cell, According to our studies, and those of Flaks and Cohen,’ the synthesis of a compound novel for the cell is carried out by a new enzyme, the formation of which is presumably induced by the phage DNA. Another example of control at the phosphorylation level is provided by the device which appears to be responsible for the absence of cytosine in T2 DNA. For lack of a mechanism to eliminate the dCTP-synthesizing enzyme system, the evolution of an enzyme to destroy the dCTP provides an effective alternative. Direct Substitution on DN A.—Genetic studies indicate that the glucose contents of DNA of phages T2, T4, and T6, and strains derived from them by recombination, are a fixed property resembling other phenotypic characters of these phages.® 1° It appears plausible, therefore, that the precise arrangement of these glucosylated HMC residues in the DNA may be a source of genetic information, and insight into the nature of the replication of these types of phage DNA would clearly be of con- siderable value. The incorporation of a fixed proportion of non-, mono-, and_ polyglucosylated derivatives of dHMC-TP during the polymerization of the DNA chain is difficult to conceive because it is likely that these derivatives would all behave similarly in hydrogen-bonding to guanine. It becomes even more difficult to conceive the in- corporation of these various HMC residues in a precise sequence in DNA on the basis of selection of the triphosphate derivatives. The existence of an enzymatic system for direct glucosylation of DNA offers an alternative which seems to circum- vent these difficulties. At this stage, the information is still too fragmentary to determine whether this glucosylating enzyme, and perhaps an additional one for polyglucosylation, will be sufficient to explain the replication of various phage DNA’s. It is apparent that further studies with T4- and T6-, as well as T2-in- fected cell enzyme systems are essential. Superficially analogous to the partial glucosylation of the HMC residues in phage DNA is the partial methylation of the cytosine residues in certain plant and animal DNA’s, such as wheat germ and calf thymus.”* In the light of our findings, it would be interesting to look for an enzymatic mechanism for direct methylation of DNA in these tissues. 134 782 BIOCHEMISTRY: KORNBERG ET AL. Proc. N. A. 8. Kinetics of Enzyme Development and Enzyme Reactions.—The temporal pattern of development of all the enzymes studied in this report that were either “new,” or the levels of which were significantly raised, was similar. The first traces of change were apparent at four minutes after T2 infection and about ten minutes after T5 infection. While the precision of measurement of these time intervals is not great, it is clear that there is a time lag before significant levels of these enzyme activities appear. Several groups of investigators?’ 27 8 have shown that 5- methyltryptophan and chloramphenicol administered to cells at levels sufficient to inhibit protein synthesis also blocked DNA synthesis when given before or up to about 5 min after infection. When given 10 min or later following T2 infection, the inhibition of protein synthesis had only a small or even no effect on the rate of DNA synthesis. It seems reasonable to consider that the time lag we have ob- served may be related to the chloramphenicol-sensitive interval required for development of the enzymatic machinery for DNA synthesis, as well as other protein components vital to virus information. There have been indications of non-phage protein synthesis immediately upon infection? and it remains to be determined what fraction of this protein can be identified with the enzymes de- scribed here. The multiplicity of enzyme changes described in this study along with the findings on the hydroxymethylating’ and deoxyuridylate-methylating® enzymes, are all directly related to DNA synthesis. Enzyme measurements related to other metabolic pathways in infected cells have as yet disclosed few significant changes.*! It would be surprising if further exploration of the phage-infected cell failed to reveal additional examples of new or augmented enzyme activities related to the requirements imposed by rapid phage synthesis. In view of the pitfalls inherent in assaying the level of an enzyme activity in a cell extract, let alone in the cell itself, a detailed evaluation of the various enzyme values in terms of virus DNA synthesis does not seem warranted. However, it is interesting to note that the rates of the kinases and the glucosylating enzyme are all greater than that found for the DNA polymerizing activity and, further, that the increase in the latter activity over levels found in uninfected cell extracts is about the same as the increase in DNA synthesis in whole cells following T2 infection. Also it is remarkable that the dA-5-P kinase activity (very likely identical to adenylate kinase) is about 10 times that of the other deoxynucleotide kinases in the uninfected cell extract and does not change upon T2 infection, while after infection the other kinases and hydroxymethylase reach levels com- parable to the dA-5-P kinase. Finally, it is noteworthy that the dC-5-P kinase activity does not increase upon T2 infection but remains at the relatively low level observed in the uninfected cell, whereas it increases about 10-fold upon T5 infection. References to increases in level of a preexisting enzyme carry no implication that the additional enzyme activity is identical to the old or even that more enzyme has been synthesized ‘‘de novo.”? To resolve this important point, it will be necessary to characterize isolated preparations of the normal- and infected- cell enzymes and to establish by tracer techniques that the enzymes developed after infection have, like induced enzymes,*? been synthesized from the amino acid pool. Control of DNA Synthesis by the Nature of the DNA Primer.—Perhaps the most 135 Vou. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL. 783 interesting question regarding T2 infection of HF. coli is how the T2 DNA seems to preempt from the host DNA the primer function for the polymerase system. The present studies can explain why DNA synthesized by the phage T2-Z. coli system contains hydroxymethyleytosine rather than cytosine, but not why the base composition is that of phage rather than that of host DNA. An observation reported in this paper which may bear on this question is the dependence of the augmented polymerase activity of the infected cell extracts on a physically altered DNA primer. When “native” calf thymus or phage T2 DNA was used, the polymerase values of infected and normal cell extracts were similar. However, with DNA samples heated at pH 9, the values for dHMC-TP incorporation with infected cell extracts were increased 12-fold, while the normal cell values were unaffected or even depressed. It is not clear as yet whether infection has resulted in formation of a new type of polymerase or in an increase of the normal poly- merase and new factors which modify it. Further studies to clarify this question may lead to a better insight into how the injected phage DNA proves to be the chosen primer for this system. SUMMARY 1. Extracts of EH. coli infected with bacteriophage T2 have been shown to contain three enzymes which are undetectable in extracts of uninfected or in T5- infected cells. These are: (a) an enzyme which phosphorylates hydroxymethylde- oxycytidine 5-phosphate, leading to the synthesis of the triphosphate, (6) an enzyme which removes the terminal pyrophosphate group specifically from deoxycytidine triphosphate, and (c) an enzyme which transfers glucose from uridine diphosphate glucose directly to the hydroxymethylcytosine of certain DN A’s. 2. These new enzymes ean account for (a) the availability of the triphosphate of hydroxymethyldeoxycytidine for the enzymatic synthesis of T2 DNA, (6) the absence of deoxycytidylate from T2 DNA, and (c) the presence of glucose on a fixed fraction of the hydroxymethylcytosine residues in DNA. 3. The DNA-polymerizing enzyme assayed with hydroxymethyldeoxycytidine triphosphate in place of deoxycytidine triphosphate reveals about a 12-fold increase in activity after T2 infection. Increases, after T2 infection, in the levels of thymine and guanine deoxynucleotide phosphorylating enzymes (by 20-45 fold) bring their activities up to the level of the adenine deoxynucleotide phosphorylating enzyme which is unchanged; the level of the cytosine deoxynucleotide phosphorylat- ing enzyme remains at a low level. 4. After T5 infection, levels of the thymine, guanine, and cytosine deoxynucleo- tide phosphorylating enzymes increase by 10—40 fold, bringing their activities up to the level of the adenine deoxynucleotide phosphorylating enzyme, which in- creases about 2-fold. 5. The new enzymes and the increases in level of the enzymes occurring in normal cells are first detectable about 4 min after infection with phage T2 and about 10 min after infection with phage T5. These results are consistent with previously published studies which have indicated with the use of inhibitors of protein synthesis that viral DNA synthesis requires a preliminary period of pro- tein synthesis. 136 784 BIOCHEMISTRY: KORNBERG ET AL. Proc. iN. Avs. * This investigation was supported by research grants from the National Institutes of Health of the Public Health Service and the National Science Foundation. + National Science Foundation Predoctoral Fellow. t Fellow of the National Foundation. 1The abbreviations used in this report are: cpm, counts per minute; ATP, adenosine tri- phosphate; dA-5-P, deoxyadenosine 5’-phosphate; dC-5-P, deoxycytidine 5’-phosphate; dG-5-P, deoxyguanosine 5’-phosphate; dHMC-5-P, hydroxymethyldeoxycytidine 5’-phosphate; dT-5-P, deoxythymidine 5’-phosphate; dATP, deoxyadenosine triphosphate; dCTP, deoxycytidine tri- phosphate; dGTP, deoxyguanosine triphosphate; dHMC-TP, hydroxymethyldeoxycytidine tri- phosphate; dTTP, deoxythymidine triphosphate; DNA, deoxyribonucleic acid; HMC, 5- hydroxymethyleytosine; UDPG, uridine diphosphate glucose; Tris, tris-(hydroxymethyl)amino- methane; KPO,, potassium phosphate buffer; PRPP, 5-phosphoryl a-D-ribofuranose 1-pyro- phosphate; E,,, molar extinction coefficient. 2 Lehman, I. R., M. J. Bessman, E. S. Simms, and A. Kornberg, J. Biol. Chem., 233, 163 (1958). 3 Bessman, M. J., I. R. Lehman, J. Adler, 8. B. Zimmerman, E. S. Simms, and A. Kornberg, these PROCEEDINGS, 44, 633 (1958). 4 Bessman, M. J., I. R. Lehman, E. 8S. Simms, and A. Kornberg, J. Biol. Chem., 233, 171 (1958); Adler, J., I. R. Lehman, M. J. Bessman, E. 8. Simms, and A. Kornberg, these PROCEEDINGS, 44, 641 (1958); Lehman, I. R., S. B. Zimmerman, J. Adler, M. J. Bessman, E. S. Simms, and A. Kornberg, these ProcEEDINGS, 44, 1191 (1958); Kornberg, A., Harvey Lectures, 53, 83 (1957- 1958). *’ Hershey, A. D., J. Dixon, and M. Chase, J. Gen. Physiol., 36, 777 (1952-1953). 6 Wyatt, G. R., and S. 8. Cohen, Biochem. J., 55, 774 (1953). 7 Flaks, J. G., and 8. 8. Cohen, Biochim. et Biophys. Acta, 25, 667 (1957); Federation Proc., 17, 220 (1958). 8 Sinsheimer, R. L., Science, 120, 551 (1954); Volkin, E., J. Am. Chem. Soc., 76, 5892 (1954). 9 Sinsheimer, R. L., these PRocEEDINGs, 42, 502 (1956); Jesaitis, M. A., J. Exp. Med., 106, 233 (1957); Federation Proc., 17, 250 (1958). 10 Streisinger, G., and J. Weigle, these PROCEEDINGS, 42, 504 (1956). 11 Cohen, 8. 8., J. Biol. Chem., 174, 281 (1948). 12 Kornberg, A., I. R. Lehman, and E. 8. Simms, Federation Proc., 15, 291 (1956), and more recent unpublished observations. 13 An abstract of this work has appeared [Zimmerman, S. B., 8. R. Kornberg, J. Josse, and A. Kornberg, Federation Proc., 18, 359 (1959)], as have abstracts regarding an enzyme which phos- phorylates dHMC-5-P (R. Somerville and G. R. Greenberg, [bid., 327), dHMC-TP incorporation into DNA, and a suggested dCTP-degrading enzyme (J. F. Koerner and M. 8. Smith, /bid., 264) in T2-infected EF. colt. 14 Anderson, E. H., these PROCEEDINGS, 32, 120 (1946). 1 We are indebted to Dr. Helen Van Vunakis for a generous gift of phage T2r*. 16 Adams, M. H., in Methods in Medical Research, ed. J. H. Comroe, Jr. (Chicago: Yearbook Publishers, Inc., 1950), 2, 1. 17 Doermann, A. H., J. Gen. Physiol., 35, 645 (1952). 18 When fluoride was present in the kinase assays, the nucleotides were adsorbed to and eluted from Norit before phosphatase treatment in Stage II of the assay. 19 For large-scale enzyme preparations, 50-liter cultures at the late exponential phase (2 x 10° cells/ml), in modified M-9 medium (see Methods) but lacking CaCl, were treated with 3-4 T2r* per cell and 10 min later with 50 y of chloramphenicol per ml. The culture was then chilled to 0° with ice over a 10-min period and harvested by centrifugation. The initial steps in the purifi- cation of the dHMC-5-P kinase, dCTPase, glucosylating enzyme, and hydroxymethylating enzyme were the same. Sonic extracts, prepared in 5 volumes of 0.05 M glycylglycine buffer, pH 7.0, were centrifuged and the supernatant fluid diluted with buffer to contain 10 mg of pro- tein per ml. Streptomycin sulfate (5 per cent solution), equal to 0.3 volume of the diluted ex- tract, was added; the supernatant fluid collected after centrifugation contained 3 to 4 mg of pro- tein per ml. It was adjusted to pH 8 with KOH and applied to a diethylaminoethylcellulose (Brown Co.) column [Peterson, E. A., and H. A. Sober, J. Am. Chem. Soc., 78, 751 (1956)], equi- 137 VoL. 45, 1959 BIOCHEMISTRY: KORNBERG ET AL 785 librated with 0.02 M KPO,, pH 8.0, containing 0.01 M 2-mercaptocthanol. Linear gradients were applied and the purifications obtained are expressed relative to t'1e supernatant fluid of the sonic extract; all solutions contained 0.01 M 2-mercaptoethanol. Glucosylating enzyme: gradient of 0.08 to 0.32 M NaCl in 0.02 M KPO,, pH 8.0-30 purification; dCTPase: gradient of 0.08 M to 0.32 M NaCl in 0.02 M KPO,, pH 8.0-24 purification, rechromatographed with a gradient of 0.06-0.32 M KPO,, pH 6.5-85 x purification; dHMC-5-P kinase: gradient of 0.06 to 0.32 M KPO,, pH 6.5-20 purification; hydroxymethylating enzyme: gradient of 0.3 M KPO,, pH 6.5 to 1.0 M KPO,, pH 6.1-230 purification. Fractions containing 2-mercapto- ethanol were not frozen. Active fractions were concentrated by precipitation with solid (N H,4)2- SO,. Further details of the purification procedures will be published elsewhere. 20 Glaser, L., and D. Brown, these ProcrEpinGs, 41, 253 (1955); Glaser, L., J. Biol. Chem., 232, 627 (1958). 21 Personal communication from Dr. M. J. Bessman. 22 We are indebted to Dr. R. L. Sinsheimer for the sample of ¢X174 DNA. 23 Personal communication from Dr. I. R. Lehman. 24 Chargaff, E., in Nucleic Acids, ed. E. Chargaff and J. N. Davidson (New York: Academic Press, Inc., 1955), 1, 307. 2% Dunn, D. B., and J. D. Smith, Nature, 174, 305 (1954); S. Zamenhof and G. Griboff, Nature, 174, 306 (1954). 26 Burton, K., Biochem. J., 61, 473 (1955). 27 Tomizawa, J., and 8. Sunakawa, J. Gen. Physiol., 39, 553 (1956). 28 Hershey, A. D., and N. E. Melechen, Virology, 3, 207 (1957). 29 Watanabe, I., Biochim. et Biophys. Acta, 25, 665 (1957); Watanabe, I., and Y. Kiho, Proc. Int’l. Symp. Enzyme Chemistry (Tokyo-Kyoto, 1958), p. 418; Hershey, A. D., A. Garen, D. K. Fraser, and J. D. Hudis, Carnegie Insittution of Washington Year Book, 53, 210 (1953-1954). 3% Cohen, 8. S., Abstracts of the American Chemical Society, Meeting, September, 1958, p. 22C. 31 Pardee, A. B., and R. E. Kunkee, J. Biol. Chem., 199, 9 (1952). Dr. Fred Bergmann has found no changes, following T2 infection of EZ. coli B, in amino acid “‘activation”’ levels for valine, isoleucine, methionine, leucine, phenylalanine, tryptophan, and tyrosine. We have observed that levels of the following activities did not change (+20 per cent) within 15 min after E. coli B infec- tion with phage T2: PRPP synthetase, orotate + PRPP conversion to uridylate, inorganic pyro- phosphatase, adenine + PRPP conversion to adenylate, and adenylate kinase. 32 Hogness, D. 8., M. Cohn, and J. Monod, Biochim. et Biophys. Acta, 16, 99 (1955); Rotman, B., and S. Spiegelman, J. Bacteriol., 68, 419 (1954). 138 THE FREQUENCY DISTRIBUTION OF SPONTANEOUS BACTERIOPHAGE MUTANTS AS EVIDENCE FOR THE EXPONENTIAL RATE OF PHAGE REPRODUCTION' S. E. Luria University of Illinois, Urbana, Illinois The phage geneticist is faced with the task of constructing a satisfactory model of phage reproduction, in the absence of direct morphological evidence similar to the one available to the macro-geneticist. Cell division, mitosis, meiosis, fertiliza- tion have a solid basis of morphological observation that the modern geneticist takes for granted. The virologist, on the other hand, begins where the cyto- geneticist ends; in a sense, he deals directly with the units of genetic material whose existence the macro-geneticists (including the bacterial geneticists) must infer. Here lies his weakness, since little is known of the performance of such units — and also his strength, since he can manipulate this subcytological world. He is not limited to dealing with integrated units of reproduction at the cellular level, but can control to a certain extent what goes into his cells. Because of this, virology’s methods may lead more directly to solving the problem of the mode of replication of genetic material. Penetration of one phage particle into a susceptible bacterium leads to pro- duction of a large number of similar particles. The intervening steps are un- known. We conjecture a reorganization of the viral material, because of its nonrecoverability in infectious form early after penetration (Doermann, 1948). We conjecture an integration of the viral material into the cell machinery at the genetic level (Luria and Human, 1950), because cell syntheses are redirected toward the production of virus specific substances. The genetic complexities of bacteriophage tell us that the viral specificities to be replicated are multiple (Hershey, 1946b) and that the new virus may receive imprints from more than one viral ancestor within the same cell (Delbriick and Bailey, 1946; Hershey, 1946b). There is evidence suggesting a discrete and regularly assorted nature of the material determinants of these specificities (Hershey and Rotman, 1948). 1Aided by grants from the American Cancer Society (upon recommendation of the Committee on Growth) and from the Research Board, University of Illinois. The competent assistance of Miss Martha R. Sheek is gratefully acknowledged. Reprinted by permission of the author and the Long Island Biological Association from CoLtp Spring HAarBor SYMPOSIA ON QUANTITATIVE BroLoey, 16, 463-470 (1951). 139 S. E. LURIA A theory proposed by this writer (Luria, 1947) assumed independent replication of these determinants (or groups of determinants) followed by their assembly into mature virus particles. This theory originally aimed at accounting for the reactivation of ultraviolet inactivated phage inside multiple-infected bacteria (Luria and Dulbecco, 1949) and was extended to account for some features of genetic recombination (Hershey and Rotman, 1949). The main ground for proposing the theory, namely, the belief that the reactivation resulted from genetic exchange, has been weakened by new evidence (Dulbecco, 1952), and the theory has as little left to support it as to disprove it. Yet, all this concerns the organization of virus material during reproduction, not the elementary process of replication. The latter cannot yet be attacked at the chemical level by any tool except speculation. It can be attacked on a limited front, however, by strictly genetic means. The experiments described in this paper were done to investigate the rate of replication of individual genetic determinants of the virus. They indicate that reproduction is exponential, each replica acting as a source of new replicas. THEORY Phage mutations occur only in the intracellular state, presumably during replication. If a phage mutation occurs in a bacterium, that bacterium will liberate one or more mutants (assuming that no loss occurs intracellularly). Delbriick pointed out several years ago that the actual numbers of mutants would depend on the mode of phage replication — more specifically, on the mode of replications of the determinant or ‘‘gene”’ involved (see Luria, 1945b). We shall analyze a few possible mechanisms; other mechanisms may be proposed, but do not seem to lead to any simple picture. I. Exponential Reproduction One gene produces n genes, each of which in turn gives n genes, and so on. After r generations, the number will be n'. For n = 2 (duplication mechanism), the situation is analogous to bacterial reproduction. Let us assume this to be the case. N gene copies will derive from one gene by N-1 acts of replication. Let the last generation have the order number 0, the second last the order number 1, the third last 2, and so on. Suppose a mutation occurs at generation k, either as an “error of replication” affecting one of the two products of duplication, or as a change in one gene during interphase. If the replication process is com- pletely synchronized (the consequences of nonsynchronization can, if necessary, be analyzed), the resulting clone of mutants will at generation 0 consist of 2* individuals. If the total number of individuals (at generation 0) in the popula- tion is N, at generation k there were N/2* individuals. Assuming a constant probability m of mutation per individual, the number of mutations occurring at generation k is mN/2*. The relation between the number x of mutants in a clone and the frequency yx of such clones will then be obtained as follows: i ye — mN _ mN (1) Gas ae x Vootas 140 FREQUENCY OF SPONTANEOUS BACTERIOPHAGE MUTANTS Synchronization is expressed by the requirement that for x # 2*, yx = 0. The frequency distribution (1) is, of course, identical to that of bacterial mutants in a series of similar populations (Luria and Delbriick, 1943; Lea and Coulson, 1949). If we limit our observation to one intracellular cycle of phage production, the maximum clone size will be the maximum ‘“‘burst size.’”’ Each phage burst with 2* particles represents a population. For m > 2°, there will practically never be more than one mutation per bacterium; the frequency dis- tribution (1) will then be that of the mutant clones among a large number of bacterial yields, with limitations imposed by the inequality of burst size from cell to cell. II. Independent Successive Replications Let us suppose that each new replica of a gene is produced independently of the preceding one, for example, by a series of successive acts of replication controlled by the initial gene brought in by the infecting phage particle. (a) If one of the copies mutates (at the time of its formation or later) the probability of mutation in other copies produced in the same cell should not be affected. Assuming a uniform mutation probability m, the mutants will be distributed at random among phage bursts (Poisson distribution). (b) If the initial gene, the pattern, mutates while turning out replicas, we may assume that afterwards it produces only mutants. If the mutation rate is con- stant in successive replications, there will occur as many cases of mutation just before the production of the last viable gene copy as before the production of the second last, the third last, and so on. The mutants will be in clones, and the frequency of clones of different sizes will be uniform, at least up to the value of the minimum burst size. EXPERIMENTAL WORK Previous data on the number of host range mutants in individual phage cultures (Luria, 1945a; Hershey, 1946a) did not allow the desired type of analy- sis, because the quantitative detection of the mutants was uncertain and because their low frequency made it necessary to look for them in mass cultures, where more than one cycle of intrabacterial growth of some mutants could take place. The experiments here reported consisted in making single infection of H’scheri- chia coli B with phage T2L (Hershey, 1946a; for the experimental methods used, see Adams, 1950) and counting the mutants r or w produced in phage bursts from individual bacteria. These mutant phenotypes (Hershey, 1946b) were chosen because they occur with suitable frequency, can be recognized and scored efficiently, and can be tested for genetic allelism or nonallelism (Hershey and Rotman, 1948). Previous extensive tests by Dulbecco (1949) and other tests made in the course of this work established the following technical points. 1. Mutant plaques r and w can be detected and scored without difficulty after six to eight hours of incubation at 37°C on nutrient agar plates under standard conditions (plating in a 0.6% agar layer over 24 hour old 1% agar plates, with about 2 X 10* young bacteria per plate). Over 100 single plaque isolations and replatings confirmed the scorings made by plaque type. 141 S. H. LURIA 2. A plaque of r type can be distinguished without difficulty from a “mottled plaque” stemming from a bacterium infected with a mixture of r and wild-type, even when r is in moderate excess. Thus, it is possible to distinguish a plaque originating from an r particle from one resulting from a mutation that occurs on the plate during the development of a wild type plaque, unless the r mutation occurred in the first bacterium infected after plating and produced within that bacterium a large majority of r mutants — an improbable occurrence, as the results of the present work will show. 3. As for the w mutants, mixed plaques of w and wild type closely resemble the plaques of wild type, so that the chances of mistaking a mixed plaque for a pure w one are rather small. Several single burst experiments of T2L on B, with single infection in Difco nutrient broth, gave average yields per bacterium around 60-100; the burst size distributions are shown in Table 1. The reasons for the lower average yield of T2L in Difco nutrient broth, as compared with the yields obtained several years ago in the same system (Delbriick and Luria, 1942), are unknown; they may have to do with changes in medium composition, in the bacterial host, or in the virus itself. Mass phage lysates often contain more phage particles than plaque-forming units; most of the particles can be caused to form plaques by treatment with distilled water or Zn** before plating (Bertani, unpub.). Electron micrographic counts give, for carefully assayed lysates of T2L, ratios “particles/plaques” between 1.0 and 2.0 (Luria, Williams and Backus, 1951 and unpub.). The failure of some phage particles to form plaques is apparently a peculiarity of phage in mass lysates, probably due to a combination between phage and inhibitors of bacterial origin. Repeated attempts to reveal, by various treat- ments, any increase in the plaque count of dilute lysates similar to those used in the experiments here reported, constantly failed. It is likely that plaque counts reveal nearly 100 per cent of the phage particles plated. In our experiments, we plated the full content of tubes in which one or several bacteria had lysed. The loss of phage remaining in each tube after plating is of the order of five per cent or less. Thus, we feel that we recovered and examined practically the totality of the active phage produced by the bacteria. Preliminary experiments indicated that mutants were present in about one burst out of 200. This made it possible to examine on each plate the phage yields from several infected bacteria, and yet to have almost never more than one mutant clone per plate. As many as 20 bursts per plate were examined in some experiments, particularly when only the r mutants were scored, since these are more easily recognized than the w mutants. Independently isolated 7 mutants are generally found to be nonallelic, yielding wild-type recombinants in mixed infection (Hershey and Rotman, 1948). To test for allelism among r mutants isolated in our experiments, stocks were pre- pared from individual mutant plaques and used, separately or in mixtures, to infect bacteria. The yields were examined for wild-type plaques. Similar tests with w mutants are technically more difficult and were therefore not attempted. 142 FREQUENCY OF SPONTANEOUS BACTERIOPHAGE MUTANTS TABLE 1. BURST SIZE DISTRIBUTION, T2L ON B, SINGLE INFECTION Average Number number Average Date of of bursts yield Median Range* plates per per plate burst 12/7/50 96 0.93 69 101 13-472 12/8/50 105 0.89 71 84 5-347 4/10/51 96 0.73 92 106 9-477 4/23/51 48 0.85 87 111 10-254 *The maximum values are almost certainly too high, since they probably represent plates with two or more bursts. THE FREQUENCY DISTRIBUTION OF MUTANTS A total of 16 experiments, done between April 1949 and April 1951, yielded the data to be reported. In five experiments, only the r mutants were scored. The results are presented in Tables 2 and 3. A total of 90 plates with mutants r and 103 plates with mutants w were observed. The expected number of plates with both r and w mutants (calculated from the assumption of independent incidence of r mutations and w mutations, either in the same bacterium or in different bacteria whose bursts were pooled on one plate) was seven; six were found. TABLE 2. SYNOPSIS OF EXPERIMENTS ON DISTRIBUTION OF r AND w MUTANTS Average Number Number Stock Number Number number of _ of plates of plates Date phage, of of bursts per with r with w number plates bursts plate mutants mutants 4/26/49 33 144 660 4.6 3 10 5/6/49 3 144 660 4.6 7 9 3/9/50 44 142 1120 7.9 12 7 3/14/50 ie 143 1180 8.25 4 15 3/31/50 ie 144 2020 14 6 18 5/2/50 ce 143 250 1.75 2 3 5/19/50 46 144 1060 5) 9 12 5/26/50 ee 144 1800 13 15 9 12/18/50 51 605 540 0.89 2 1 1/11/51 a 144 800 5.5 7 10 2/12/51 a 144 820 BY 4 9 3/1/51 om 192 1500 ae) 1 Be 3/8/51 ? 192 2400 IPs) 1 3/20/51 180 3400 19 7 3/29/51 aa 173 4400 25.5 9 4/10/51 ae 96 70 0.73 1 Re Totals 22620 90 103 IED aS ia a SE OUT 8 tt > vo EE Be ee A Ue ie *Mutants w not scored. 143 S. HE. LURIA Let us observe the frequency distributions of the numbers of mutants (Table 3). The total number of mutants observed in all experiments (Table 3; total clone frequency) was 766, distributed among 2874 plates containing about 1.8 X 10® normal phages. The proportion of mutants is about 4 X 107‘, and their average number per plate is about 2.5 X 10°'. If the mutants were dis- tributed at random, there would be about 550 plates with one mutant, about 90 with two or three mutants, and only four with four mutants or more. There is no doubt that the distributions are not random, but clonal. Asa test of the nature of the mutants appearing on the same plate, 11 pairs of r mutants were isolated from 11 plates, which contained between 2 and 59 mutants. In all cases, the mutants in each pair proved allelic (probably iden- tical); no wild-type recombinant was observed among at least 1000 plaques of the yield from mixed infected bacteria. In 11 out of 12 crosses between mutants isolated from different plates we observed wild-type recombinants; the twelfth cross failed to show recombinants. It may have represented either a case of repeated occurrence of the same mutation or a case of two mutations with recombination frequency lower than 0.2 per cent, the lowest frequency detectable in our rather crude tests. These results, then confirm the clonal nature of the mutants produced within a given bacterium. We will now consider the clonal distributions. Inspection of Table 3 shows that the mutant distribution, though clonal, fails to fit the uniform frequency predicted for small clones of various sizes by the hypothesis of independent gene replication, with mutations occurring in the pattern. Let us turn next to the distribution predicted by the hypothesis of exponential duplication. Inspection of the data shows: (a) there are clones with two mutants; therefore, if exponential reproduction occurs, the elementary process is probably one of duplication (from 1 to 2) rather than triplication or quadruplication; (b) there are clones with 3, 5, 6, 7, ... in addition to clones with 1, 2, 4, 8, ... mutants. Thus, exponential reproduction, if present, must be nonsynchronized, a conclu- sion also suggested by the well-known distribution of the total burst size. For a quantitative test of the hypothesis of exponential reduplication we shall use, instead of Equation (1), the following expression (accumulated distribution) suggested by Dr. Howard Levene: mN mN 1 mN 2mN P oe = ah => eee = ay = mND> Ok es 5k 1 = 2 (for N = 92 SS x); (2) Y, is the number of clones with x or more mutants. The product Yx X x is constant and a plot of log Y versus log x gives a straight line with slope — 1; the vertical intercept for x = 1 is the logarithm of the total number of mutant clones. This plot has the advantage that it is hardly affected by nonsynchronization. A clone with three mutants can be considered either as a clone that should have had two mutants and underwent one extra reduplication, or as a four-mutant clone 144 FREQUENCY OF SPONTANEOUS BACTERIOPHAGE MUTANTS that lagged one reduplication behind. In either case, the clone will contribute to the value Y2 and will be deducted from the value Y ,. A correction should be made before comparing the results with the theoretical expectation. The stocks of phage used to infect bacteria contained small, known TABLE 3. THE DISTRIBUTION OF MUTANTS IN INDIVIDUAL PLATES Clone frequency, 7 mutants Clone frequency, w mutants Clone frequency, Clone Corrected for Corrected for total, corrected size All plates full mutant All plates full mutant for full mutant clones clones clones 1 47 47 46 46 93 2 8) ) 18 18 27 3 11 11 11 11 22 4 2 2 4 4 6 5 2 2 1 1 3 6 " a tt a 2, ”y 2, 2 4 8 ] 1 1 1 2 9 10 2 2 1 1 3 11 1 1 12 1 il 1 i) 14 1 1 1 1 2 15 1 1 1 1 2 16 1 ] 1 20 2 1 1 D2, 2 PE 2 25 1 26 1 1 1 30 1 I 34 1 ] 1 37 1 39 1 40 1 1 41 2 1 1 47 1 1 1 1 53 1 59 1 1 1 100 1 Total 90 85 103 98 183 numbers of r and w mutants. Since we plated more than one burst per plate, some of the mutant clones observed may have stemmed from bacteria infected solely with one mutant particle. These clones ought to be eliminated (since they derive from mutant particles originated in a mass lysate, where different condi- tions obtain) but they cannot be recognized. The expected number of such “full mutant” clones was 10. Assuming that a “full mutant’’ clone would be at least as large as the minimum burst size, we eliminated from the experiments where 145 S. BE. LURIA such clones were expected to be present an appropriate fraction of the largest clones (see Table 3). This correction is indeed a small one. Figure 1 compares the experimental distribution of clones with one or more mutants with the expected distribution from the hypothesis of exponential reduplication. The data corresponding to the “corrected” columns in Table 3 are plotted for 7 mutants, for w mutants and for the two together. They fit well the expected relationship (linear relation between log Y, and log x with slope — 1) for low clone size, up to mutant clone sizes of the order of 10-15. Above that point, the frequency of mutant clones falls below the theoretical values. This behavior is precisely what we should expect. The linear relation between log y and log x — see Equation (1) — should only obtain for clones so small that they have equal chances to be formed in all bacteria. For clone sizes of the order of the burst size, a limitation is placed on the frequency with which these clones can be observed. In the curve log Y, versus log x this limitation will manifest itself as a downward concavity, which becomes appreciable around the value corre- sponding to the lowest class of frequent burst sizes (about 20 phages per bacteri- um in our experiments) and progressively more pronounced as the median burst size is approached. Of course, there cannot be any clone larger than the maximum burst size. An additional factor (suggested by Dr. 8. Dancoff) that works in the same direction is nonsynchronization itself; in fact, this results in the existence, within each burst, of subclones that have originated at the same generation but have different sizes, thus producing effects similar to those of the burst size differences. No closer analysis of the concave portion of the distribution frequency curve is feasible beyond these qualitative considerations, since the clones in this region are few and fluctuations affect the results strongly. Altogether, our results fit quite well the hypothesis that the genes responsible for the investigated pheno- types reproduce exponentially by successive reduplications. Let us now analyze some of the factors that might affect the experimental results. 1. Failure to recognize mutants. This cause of error is difficult to assess; we believe the error to be very small. All plates were scored by the same observer after the optimum incubation period, and every plate that might have presented difficulties in scoring, because of crowding or of faulty layering, was discarded before examination. All the doubtful plaques were picked and replated for the phenotype test. Any residual error from this source would probably result in underestimation of the frequency of clones with one mutant, since the finding of the first mutant on a plate might sharpen the alertness of the observer, thereby increasing the chances of detecting other mutants on the same plate; the classes of clones with more than one mutant might thus have been favored in our observations. 2. A more definite and more easily evaluated source of error is the coincidence of more than one clone of a given mutant type (7 or w) on the same plate because of coincidence of two mutations, either in the same bacterium or in the group of bacterial bursts examined on one plate. The expected coincidences (‘‘doubles”’) were calculated to be 2.6 7 and 4.3 w. It isnot easy to correct for these ‘“doubles,” 146 FREQUENCY OF SPONTANEOUS BACTERIOPHAGE MUTANTS 128 64 32 FIG. 1. The distribution of mutant clones. x = number of mutants in a clone. Y = number of clones with x or more mutants. X:7 mutants. C: w mutants. @: 7 and w mutants. The solid lines represent the theoretical distributions, with slope —1. 147 S. E. LURIA which cannot be recognized by inspection. A first approximation can be made by assuming that all “doubles” include a clone of one mutant, and that they all occur among the most frequent clone size classes observed (plates with 2, 3, 4 mutants). The resulting corrected distribution has a slight excess of ones, but does not deviate significantly from Equation (2), in spite of the fact that the correction is an extreme one, which concentrates all the distortion in the initial, most critical portion of the frequency distribution curve. 3. Failure of the plaque count. method to reveal the full number of mutants is unlikely to result from technical reasons, but might be due to intrinsic properties of phage reproduction. For example, only a fraction of the gene copies produced might appear in active phage particles because of loss or inactivation of a certain proportion of phage within the bacteria before liberation. Such “sampling losses,’’ superimposed on a logarithmic distribution, would make the initial slope of log Y versus log x steeper; a sampling loss of 50 per cent would give a quite appreciable deviation from the initial slope of —1. This is in conflict with our results. Sampling losses could be superimposed on any other distribution, but it is hard to visualize how the results could simulate those of an exponential distribution. A variety of alternate hypotheses was considered in an attempt to find one, besides that of exponential reproduction, that could lead to the results found experimentally. No sensible hypothesis could be devised. Altogether, the hypothesis of mutations occurring at a constant rate in the course of exponential nonsynchronized gene reduplication appears adequate to account for our results. Mutation RAtTEs Our results permit fairly accurate estimations of mutation rates. A total of 87.6 r mutations (85 mutant clones plus 2.6 coincidences) occurred in 23,000 bursts, producing approximately 1,850,000 active phage particles. The mutation frequency per reduplication is around 5 X 10°°. 102.3 w mutations in 11,000 bursts, or 880,000 particles, correspond to a mutation frequency of 1.2 K 1074. These mutation rates, of course, are the sums of the mutation rates at all the individual loci that can mutate to give either the r or the w phenotype. INCIDENTAL OBSERVATIONS 1. The mutants of T2L classified as r or w are generally clearly recognizable as such. The r phenotype, however, is not uniform; different r mutations give plaque types often distinguishable from one another. The w phenotype, though generally sharply distinct from r and from wild-type, iseven more variable. Only one clone (consisting of one plaque), even after repeated replatings, could not be classified with certainty as either w or 7; since at any rate it seemed to represent a novel phenotype, it was excluded from the analysis. 2. Several other types of mutants were observed in the course of our experi- ments, mainly “minute” or “sharp’’ plaque types. These were not included in the results. 148 FREQUENCY OF SPONTANEOUS BACTERIOPHAGE MUTANTS 3. Mottled plaques were often seen on our plates. They generally derive from mutations occurring during plaque formation. In several cases, mottled plaques were present on the same plate with r mutants. Three such mottled plaques were replated and the r component strain was isolated and crossed with a strain from a pure r plaque on the same plate. In two cases wild-type recombinants appeared, indicating that the mottled plaque probably stemmed from an independent mutation that occurred on the plate; in the third case no recombinant was found. The latter type of mottled plaque could appear in the yield of a bacterium containing a clone of r mutants, owing to some limitations to the complete segregation of virus particles or of their genetic components (Hershey and Chase, 1951). DISCUSSION The exponential rate of gene reproduction in phage, suggested by our analysis of the clonal distribution of spontaneous phage mutants, simply means that the initial gene copy brought in by the infective virus does not possess the monopoly of replication. Its copies act in turn as sources for new replications and the elementary replication process is a reduplication. This conclusion, if correct, represents a step further in our analysis of phage reproduction. Our results do not contribute any information as to whether the mutations occur only at reduplication or between reduplications, in the “interphase.” They are compatible both with production of phage particles as such and of individual genetic components, although the apparent lack of ‘sampling losses’ is more easily reconciled with the former. There is some interest in comparing the clonal distribution of spontaneous mutants with the almost random distribution of recombinant phage from mixed-infected bacteria (Hershey and Rotman, 1949). The very first active phage particles that appear inside mixed-infected bacteria include recombinants (Doermann and Dissosway, 1949), whose distribution is also intermediate between a clonal, reduplicational one and a random one. The facts suggest the following conclusions: 1. Recombination occurs late in reproduction of the genetic material of phage. This may be due either to the coincidence of recombination with some terminal step in phage maturation or to an increased probability of recombination when large numbers of phage elements are present in a cell. 2. Recombinants detectable as mature particles around the middle of the intracellular growth period do not reduplicate as such; otherwise, by giving rise to clones, they would cause the later population of recombinants to be distributed more and more like spontaneous mutants, contrary to experimental finding (terminal distribution of recombinants almost random). 3. Reproduction of the genetic material of phage, therefore, takes place mainly by reduplication of elements that are not yet in the form of mature phage particles. 149 S. EH. LURIA REFERENCES Apams, M. H., 1950, Methods of study of bacterial viruses. In: Methods in Medical Research. 2: 1-73. Detprick, M., and Baitny, W. T., Jr., 1946, Induced mutations in bacterial viruses. Cold Spring Harb. Symposium Quant. Biol. //: 33-37. Devsriick, M., and Luria, 8. E., 1942, Interference between bacterial viruses. I. Arch. Biochem. /: 111-141. DorrmMaNn, A. H., 1948, Intracellular growth of bacteriophage. Yearb. Carneg. Instn. 47: 176-182. DorrMANN, A. H., and Dissosway, C. F.-R., 1949, Intracellular growth and genetics of bacteriophage. Yearb. Carneg. Instn. 48: 170-176. Dutsecco, R., 1949, The number of particles of bacteriophage T2 that can participate in intracellular growth. Genetics 34: 126-132. 1952, A critical test of the recombination theory of multiplicity reactivation. J. Bact. (in press). Hersuey, A. D., 1946a, Mutations of bacteriophage with respect to type of plaque. Genetics 31: 620-640. 1946b, Spontaneous mutations in bacterial viruses. Cold Spring Harb. Symposium Quant. Biol. 17: 67—76. Hersuey, A. D., and Cass, M., 1951, Genetic recombination and heterozygosis in bacteriophage. Cold Spring Harb. Symposium Quant. Biol. /6. Hersuey, A. D., and Rorman, R., 1948, Linkage among genes controlling inhibition of lysis in a bacterial virus. Proc. Nat. Acad. Sci. Wash. 34: 89-96. 1949, Genetic recombination between host-range and plaque-type mutants of bacterio- phage in single bacterial cells. Genetics 34: 44-71. Lea, D. E., and Coutson, C. A., 1949, The distribution of the numbers of mutants in bacterial populations. J. Genet. 49: 264-285. Luria, S. E., 1945a, Mutations of bacterial viruses affecting their host range. Genetics 30: 84-99. 1945b, Genetics of bacterium-bacterial virus relationships. Ann. Missouri Bot. Gard. 32; 235-242. 1947, Reactivation of irradiated bacteriophage by transfer of self-reproducing units. Proc. Nat. Acad. Sci. Wash. 33: 253-264. Luria, S. E., and Desriicx, M., 1943, Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491-511. Luria, 8. E., and Dusecco, R., 1949, Genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics 84: 938-125. Luria, S. E., and Human, M. L., 1950, Chromatin staining of bacteria during bacterio- phage infection. J. Bact. 59: 551-560. Luria, 8. E., WitutaMs, R. C., and Backus, R. C., 1951, Electron micrographic counts of bacteriophage particles. J. Bact. 67: 179-188. DISCUSSION Aurensura: I suggest that the conclusion to be drawn from Dr. Luria’s experiments is that the reproduction cycle of viruses conforms with that of organisms in general, and that therefore viruses are to be considered organisms. 150 GENETIC RECOMBINATION BETWEEN HOST-RANGE AND PLAQUE-TYPE MUTANTS OF BACTERIOPHAGE IN SINGLE BACTERIAL CELLS!:? A. D. HERSHEY anp RAQUEL ROTMAN? Department of Bacteriology and Immunology, Washington University Medical School, St. Louis, Missouri Received June 28, 1948 E HAVE previously shown that any two of several independently arising plaque-type (7) mutants of the bacterial virus 72H interact with each other, in bacterial cells infected with both, to give rise to wild type and double mutant genetic recombinants (HERSHEY and Rorman 1948). In this paper we describe comparable interactions between host-range and 7 mutants of the same virus. The experiments furnish new information because it has proved possible to count the numbers of all four types of virus found in yields from the mixedly infected bacteria. MATERIALS AND METHODS The types of viral mutant to which we shall refer in this paper may be summarized in terms of the mutational pattern illustrated in fig. 1. In this diagram, / refers to a host-range mutant, 7 to any one of the rapidly lysing mutants (HERSHEY and Rorman 1948), and m (“minute”) to a mutant not previously described which is characterized by a very small haloless plaque. The A mutant is one which forms plaques identical in appearance and number on typically sensitive strains of Escherichia coli, and on an indicator strain (No. 2 B/2H, 2K) resistant to k+ forms of the virus (HERSHEY 1946a). All the steps indicated in the diagram by arrows can be observed either as spontaneous mutations, or by making the appropriate crosses. Only one example of the mutant m, obtained by crossing wild type with an 7m arising in a stock of the mutant r/3, has been studied. The plaques of m and rm are different, but are not easily distinguishable, as shown in the photograph (fig. 2). In principle, the experimental technique we have to describe is very similar to that of genetic crossing, and will be referred to in this paper in genetic terms. One starts with a pair of mutants, each corresponding to a mutant haploid germ cell differing from wild type by a different unit change. Bacterial cells are infected with both members of the pair, and during viral growth the pair interact to produce viral progeny corresponding to germ cells of a new 1 Aided by a grant from the U. S. Pusitic HEALTH SERVICE. 2 The manuscript was prepared while the senior author held a temporary appointment in the Department of Biology of the CaLirornia INSTITUTE OF TECHNOLOGY. It is a pleasure for him to acknowledge material and intellectual aid received from members of the staff of that department. 3 Present address: University of Minnesota, Minneapolis, Minn. Reprinted by permission of the authors and Genetics, Inc. from GENETICS, 34, 44-71, January, 1949. 151 RECOMBINATION IN BACTERIOPHAGE 45 generation, but now including some individuals differing from wild type by both unit changes, and other individuals differing from wild type not at all. wild type oo™ Ficure 1.—Mutational pattern of the bacterial virus 72H. The analogy to other genetic recombinations is obvious, and it is natural to look for a common mechanism. The procedure of making a cross consists essentially in infecting a measured number of growing bacteria with larger measured numbers of two kinds of virus, diluting the culture before lysis begins to prevent readsorption of viral progeny to bacteria not yet lysed, and plating samples of the total yield of FiGuRE 2.—Progeny of the cross m Xr. 152 46 A. D. HERSHEY AND RAQUEL ROTMAN virus for a differential count of its component types. This procedure was first used, for another purpose, by DELBRUcK and Luria (1942). The advantage of the Xr cross is that all the genetic types of virus to which it gives rise can be recognized in a single plating on a mixture of bac- terial strains (fig. 3). This makes possible the analysis of viral yields from single bacterial cells. For this purpose the procedure already described is modified FicureE 3.—Progeny of the cross XrJ plated on mixed indicator. The acentric clearings in the ktr plaques result from secondary /: mutations. by increasing the factor of dilution to obtain only one infected bacterium to about three ml of nutrient broth. The culture is then divided up into samples of one ml, most of which will contain either no bacteria at all, or a single one. A large number of these samples are plated out after the bacteria lyse, and the elementary yields, averaging about 500 particles of virus in our experiments, are analyzed in toto. Both the mixed indicator method for differential counting of viral mixtures, and the single burst technique as here employed, were first used by DELBRUCcK (1945a, b). The remaining portion of this discussion of materials and methods is of technical interest only. Viral stocks are prepared by seeding nutrient broth cultures of E. coli strain S with material taken directly from a single plaque. Stocks of wild type and h mutant usually have titers exceeding 10'°/ml; r mutant titers are always a 153 RECOMBINATION IN BACTERIOPHAGE 47 little less. Many of the stocks, particularly of the r mutants, contain unknown substances inhibiting adsorption of the virus if the dilution into bacterial suspension is less than 1:100 or so. There is no difference in the adsorption of different stocks at high dilutions of the virus. If a mixed infection is attempted with an 7 stock which contains inhibitor, and an / or wild type stock which does not, the adsorption of both viruses in the mixture is prevented equally. No undesirable disturbance of the relative multiplicity of infection in mixtures is therefore encountered. To obtain satisfactory levels of adsorption, all r mutant stocks are sedimented in a refrigerated International centrifuge with Multispeed head, and resuspended in a solution containing 1 percent Bacto- peptone and 0.5 percent NaCl. Very little loss of virus occurs during sedimen- tation, and probably no permanent aggregation, since sedimented mixtures of r and wild type virus do not yield mixed plaques. The resuspended virus is stable for at least several weeks. To make a cross, a two hour culture of £. coli strain H in nutrient broth, containing 2X10’ bacteria per ml, is infected at 37°C in an aerated culture tube (the “adsorption tube”) with ~> volume of a mixture of diluted viral stocks containing 210° plaque forming particles of each kind per ml. After five minutes, during which equal numbers (about 50 percent) of each virus are adsorbed, a 10‘ dilution is made into broth (the “growth tube”) for further incubation, and a second diluted sample is spun for the assay of unadsorbed virus. Sixty minutes after infection, an assay from the growth tube gives the average yield of virus from about 40,000 mixedly infected bacteria. For the single burst experiments, an additional dilution from the adsorption tube into antiserum to neutralize the unadsorbed virus (DELBRUCK 1945c), is made at the end of the adsorption period. Five minutes later, a further dilution from the antiserum tube is made into broth to contain about one infected bacterium per three ml. Before the 20th minute after infection, sam- ples measuring one ml are distributed into a series of small tubes. The virus yields in these tubes are assayed by plating 0.3 ml 60 minutes or more after infection. The remainder of each sample, excepting those containing no virus or unmixed yields, is plated on two additional plates the next day. About 10 percent of each sample is mechanically lost. An important feature of these experiments is the guard against contamination of materials provided by the fact that about { of the samples contain no virus, whereas the remainder contain more than 100 particles. Viral yields from the growth tube are plated on sensitive bacteria (strain S), on the indicator strain (No. 2 B/2H, 2K), and on a mixture containing one volume S and two volumes indicator (day-old broth cultures). On the mixed indicator all four types of virus can be recognized (fig. 3), and their sum equals the count on S. Mixed indicators plates always show a few doubt- ful plaques which can be identified only by sampling and retesting, but their number is too small to be of importance, and mixtures of pure stocks can be counted with satisfactory accuracy. The counts on the single indicator, giving only the # mutants, are also satisfactory with mixtures of pure stocks. These counts tend to be low, however, for mixed yields of # and k* virus. The cause 154 48 A. D. HERSHEY AND RAQUEL ROTMAN of this “mixed indicator effect,” similar to that observed by DEeLBRUcK and BAILEY (1946), remains obscure. It does not appear to be the result of segre- gation of multiple # factors, because no intermediate genetic types can be found in crosses between / and wild type. In Xr crosses, it affects equally counts of parental and recombinant virus. The validity of the mixed indicator count itself rests on three lines of evi- dence. First, plaques sampled and retested always conform to the genetic type deduced from inspection. Second, the ratio of / to A+ virus in the yield following mixed infection, measured by the mixed indicator count, is the same as the corresponding ratio of infecting viruses, with minor exceptions to be mentioned below. Third, the yield of the two recombinants in either / Xr or hr X wild type crosses is very nearly equal, and the slight bias (actually of doubtful signifi- cance) correlates not with the #, but with the 7 pair of alleles. For these reasons, and because of its statistical efficiency, only the mixed indicator plating is used for counting single bursts. The reproducibility of the counts so obtained may be judged from the examples shown in table 1. TABLE 1 Mixed Indicator Counts of Viral Types in the First Eight Bursts Examined from the Cross hXr7. The counts shown are for 3 aliquots of 0.3 ml from each tube. The volumes are not measured very accurately, owing to the effort made to plate the entire sample. In computing results, it was assumed that totals of counts for each tube represented 90 percent of the actual virus content, 10 percent of the fluid being lost mechanically. TUBE htr* hr* htr hr TUBE htrt hyr* her hr 2 9 57 46 19 35 21 80 71 23 9 Th 56 20 24 74 65 18 11 50 47 18 14 68 39 15 10 18 17 95 13 36 9 136 30 9 10 14 1s 8 if 100 22 7 10 22 118 15 14 120 18 9 26 9 84 Sit 8 38 9 139 30 9 10 124 53 § 9 130 19 13 8 78 34 fl 4 102 31 4 34 2 20 58 17 40 1 24 54 1 6 26 78 32 3 38 83 10 6 33 55 16 3 24 66 if The nutrient broth referred to above is composed of Bacto-peptone 10 g, Bacto-beef extract 3 g, NaCl 5 g, glucose 1 g, per liter distilled water. The pH (unadjusted) is about 6.8. An occasional batch of broth prepared according to this formula proves unfavorable to the adsorption of the virus. Nutrient agar plates are poured with a minimum of 35 ml per 9 cm Petri dish of Bacto-agar 10 g, Bacto-Tryptose 10 g, NaCl 8 g, sodium citrate crys- tals 2 g, glucose 1 g, per liter distilled water. The pH of the agar is adjusted to 6.8 to 7.0. Contrary to an early experience, we have recently found that Bacto-Tryptone can be substituted for Tryptose. Poured plates are stored in 155 RECOMBINATION IN BACTERIOPHAGE 49 the refrigerator. Agar for layer plating has the same nutrient composition, but contains only 0.5 to 0.6 percent agar, the optimal concentration depending on the age of the plates and on other variables. All platings of virus are made by the agar layer method, by adding an aliquot of virus measuring ().1 to 0.5 ml and about 0.1 ml of a day old unaerated broth culture of bacteria, to 2 ml of melted soft agar at 45°C, and pouring the mix- ture over the surface of an agar plate at room temperature. The plates are in- cubated 18 to 24 hours at 37°C without inverting, during which time fluid collects on the agar surface without undesirable effects if conditions are optimal. Bacterial counts are made by spreading 0.1 ml aliquots on the surface of dried agar plates. THE LINKAGE SYSTEM The mutants m and k (minute plaque and host-range modification, respec- tively) have been crossed with wild type, with the mutants called r/, r7, and r13, and with each other. The crosses with wild type yield only the parental types of virus, confirming that the mutants are unit modifications. The crosses with ry mutants, and the intercross, extend the linkage system previously de- scribed (HERSHEY and RorMAN 1948) as shown in fig. 4. The / locus is closely linked to the locus r/3; the m locus belongs in a third linkage group C. It should be understood that the diagram of fig. 4 is only a convenient representation of linkage relations whose structural basis remains to be eluci- dated (HERSHEY and RorMan 1948). The question of linear structure will be returned to in the discussion of this paper. At this point we insert an experi- ment which supports the general interpretation in terms of linkage. rl Pri4 A {Z AT 15% as poo h P13 Cea ONO SAG.7. 5 Bh 15% 7h 15% FicurE 4.—Linkage relations among mutants of 72H. The percentages indicate yields of wild type in two factor crosses. The double mutants ir7 and hr13 were isolated by making the respective crosses hXr7 and hXr13. The three crosses r7 Xr13, hr7Xr13, and hr13Xr7 were then compared with respect to the yield of rt virus, classified without 156 50 A. D. HERSHEY AND RAQUEL ROTMAN respect to the host-range character. In each case the yield was the same with- in experimental error, seven percent of the total virus. This experiment shows that the small yield of recombinants (one percent of wild type) in the cross hXr13 cannot be attributed to suppression of a hypothetical conjugation, which would affect the interaction of h r7 with r13 as well, but indicates some kind of linkage between the genetic factors concerned. AVERAGE YIELDS OF VIRUS IN CROSSES BETWEEN HOST-RANGE AND 7 MUTANTS Six crosses have been studied by the single burst technique; namely, iXr1, hXr7, hXr13 and the corresponding reverse crosses, h rX wild type. Each cross was made three to five times for the collection of the single burst data, and each time the viral yield was examined also from a culture tube containing about 40,000 mixedly infected bacteria. The average yields from the culture tubes are summarized in table 2. They show that the / locus is very closely linked to r/3 (less than one percent of wild type), and that the linkage rela- tions to rl and r7 are approximately what would have been predicted from this fact, respectively 12 and 6 percent of wild type. According to arguments previously given (HERSHEY and RoTMAN 1948), the factors h, r7, and r13 are TABLE 2 Average Percent Distribution of Viral Types in Yields from about 40,000 Mixedly Infected Bacteria The results shown are from the same experiments for which single burst data are also re- ported, except that one growth tube was lost among the crosses h 77 by wild type. The total multi- plicity of infection is about five of each type per bacterium; the adsorption period is five minutes; the total incubation period one hour. The distribution of viral types is computed from the re- sults of mixed indicator platings. The column headed eop(h) gives the efficiency of plating of h virus on single indicator as compared with mixed indicator, and illustrates the mixed indicator effect mentioned in Methods. The column headed p(h) gives the percent of virus containing the h allele, and shows the effect of selection during growth. NO. OF nen ei 3 BURST CROSS eG htr hr htr hr a eop(h) p(h) hXrl 5 input 0 53 47 0 180 53 yield 12 42 34 12 630 0.8 54 hrIX++ 3 input 57 0 0 43 1.0 43 yield 44 14 13 29 680 0.7 43 hXr7 4 input O 49 51 0 Weal 49 yield 5.9 56 32 6.4 650 0.8 62 hr7X++ 2 input 49 0 0 51 1.0 51 yield 42 7.8 teak 43 690 0.8 Bil hXr13 3 input 0 49 51 0 hak 49 yield 0.74 59 39 0.94 510 0.8 60 hr13 K++ 4 input 52 0 0 48 0.9 48 yield 50 0.83 0.76 48 590 On7 49 157 RECOMBINATION IN BACTERIOPHAGE aL linked to each other, but r/ is probably situated on an independently ex- changing structure. The results of table 2 show further that the two recombinants appear in equal numbers in any one cross, and that pairs of reverse crosses yield equal numbers of recombinants. It is these relations, which increase the resemblance to simple types of Mendelian segregation, that we wish to examine by the single burst technique. In the remainder of the experimental part of this paper we describe the results of this examination, but limit our comments chiefly to the technical problems encountered. The general implications of the data will be considered in the discussion. VARIATIONS IN YIELDS OF PARENTAL TYPES OF VIRUS AMONG INDIVIDUAL BACTERIA Yields of virus from single bacteria show large fluctuations in size (DEL- BRUCK 1945b) and, in our experiments, variations in relative yields of the two infecting viruses. The variations in total yield are shown in fig. 5 which includes the complete data for six experiments in which the proportion of multiple bursts is small (100 bursts out of 484 tubes, or 11 probable multi- ples). The burst sizes range from 150 to several thousand, with a mean of 520, or 470 corrected for probable multiples. The distribution is the same for the mixed bursts, and for the bursts containing only one viral type. Owing to these variations, it is convenient to describe the individual bursts in terms of the fractional yield of the several viral types. So Number of Bursts oN HF DH OW 00 900 1000 >1000 Burst size FicurE 5.—Distribution of total viral yields among 100 single bursts from mixedly infected bacteria. The solidly shaded areas refer to unmixed yields. Each cross yields four types of virus, wild type, the single mutants / and r, and the double mutant / r, of which two are parental and two are recombi- nant types. In order to examine the variations in relative yields of parental types independently of variations in yields of recombinants, it is convenient to ex- press the former in terms of the proportion of virus containing a specified allele, viz: 158 a2 A. D. HERSHEY AND RAQUEL ROTMAN n(hr*) +n(hr) HUE n (total) (1) a n(htr)-+n(hr) He n (total) (2) where n indicates the number of the specified viral types, p(/#) is the propor- tion of the yield containing the / allele, and 1—p(h), p(r), and 1—p(r) are re- spectively the proportions containing the /*, r, and r* alleles. The fraction p(A) is equal to 1—p(r) if the numbers of the two recombinant types are the same, or approximately equal if these numbers are small. A fair idea of the complete distribution of alleles is therefore given by the distribution of p(k) alone. Two examples of this distribution, showing the proportions of the h allele in different single bursts for the crosses hXr1 and hXr7, are given in fig. 6. Number of Bursts 0 9) 182736 45.54 63" 72.Ueh G0 299100 per cent h virus Number of Bursts O (Sul 2036. 45) SA hesva72e. eis 90 100 per cent h virus Ficure 6.—Distribution of proportions of / virus in yields from single bacteria. 159 RECOMBINATION IN BACTERIOPHAGE 53 The variations shown in fig. 6 are evidently due in part to variations in relative numbers of the two kinds of virus adsorbed, and in part to variations in viral growth. For purposes of comparison, the variation in relative multi- plicity has been computed on the assumption of a random distribution of two types of virus over a population of bacteria receiving on the average five particles of each kind. The distribution for this case is approximately that shown in table 3. TABLE 3 Ideal Distribution of Multiplicities in Mixed Infection The proportions of bacteria falling into the specified groups classified with respect to relative multiplicity of infection with two kinds of virus have been calculated for random adsorption with average multiplicity of five each of the two kinds. NUMBERS OF FIRST KIND AS PERCENT OF TOTAL VIRUS PERCENT OF BACTERIA ADSORBED 0 or 100 0.7 (exact) 1-9 or 91-99 0.2 (approximate) 10-18 or 82-90 2.0 - 19-27 or 73-81 Sek 4 28-36 or 64-72 10.8 * 37-45, 46-54, or 55-63 18.2 total 92.2 The distributions of yields actually found (fig. 6) differ from the theoretical distribution of multiplicities in showing a considerably broader spread, and a significant excess of yields containing only one kind of virus. These effects could be due in part to an inhomogeneity of the bacteria with respect to ad- sorbing power for virus; otherwise they suggest that a bacterium infected with two viruses is somewhat less likely to liberate a given one, than a bacterium ‘nfected with that one alone. DuLBecco (1949) has shown that the latter is, infact, the case. Another possible contribution to the variations described is connected with the relatively long period (five minutes) allowed for adsorption of virus, which permits some bacteria to be infected with one or more particles of one type of virus considerably in advance of infection by the second. Owing to the slow adsorption of the virus 72H and its mutants, the adsorption time cannot be much reduced without reducing the total multiplicity of infection, or introduc- ing excessive amounts of virus. This contribution to the variation in composi- tion of viral yields has not, therefore, been assessed. COMPETITION BETWEEN VIRAL MUTANTS The competition between viral mutants expresses itself in two ways; first, by the complete suppression of one virus or the other in mixedly infected bac- teria, and second, by excessive growth of one of the two types in bacteria liber- ating both. These effects are slight among mutants of T2H so far examined, 160 54 A. D. HERSHEY AND RAQUEL ROTMAN and probably do not influence appreciably the yields of recombinants in genetic crosses, as the following discussion will show. The two distributions shown in fig. 6 illustrate the competitive relations encountered. In the cross #Xr7, most of the unmixed yields contain the h rather than the 7 parental type. Corresponding to this, there is a tendency for the mixed yields to contain an excess of # virus. The combined effect is to cause a definite increase in yield of / virus at the expense of r, as compared with the input proportions. The cross 4Xr13 also shows these characteristics, the ef- fects being evident in table 2. In the cross kXr/, on the other hand, the unmixed yields of each kind are approximately equal in number, and the mean proportion of / virus in the mixed bursts, in the total yield, and in the input mixture of viruses is the same. The crosses between wild type and /rl,hr7, and hrl3 are like hXrlin this respect, as shown in table 2. It might be supposed that the suppression of one virus by a second is favored by an excess of the second. This is true only in a special sense, as DULBECCO (1949) has shown, and we have confirmed. An excess of one virus tends to sup- press a minority type completely in some bacteria, but there is a compensating excess of this type among the mixed bursts, so that the average proportion of the minority virus in the yield averaged over many bacteria is the same as in the input mixture. This identity has been established with considerable pre- cision for proportions of r/ between 7 and 50 percent in mixed infection with wild type. The nature of this relationship, which is at first sight perplexing in the case of unequal multiplicity, has been explained by DuLBeEcco (1949) in terms of a limitation to the number of viral particles which can participate in growth in a single bacterium. If all those viral particles in excess of a certain number attached to the same bacterium fail to grow, and if the excluded ones are chosen at random, the result will be precisely the one described, provided there is no selection during the growth of the successful particles. It is apparent that with certain viral pairs, the excluding mechanism does not operate at random, or there is continuing selection during growth. Thus h mutant slightly suppresses 77 or r/3, but not rl or wild type. Wild type suppresses 7/3, but not r/ or r7 (HERSHEY and Rotman 1948). There is no selection with respect to either / or r factors when wild type is crossed with hrl,h 17, or h r13 (table 2). The competitive relations discussed above are of immediate interest only in the negative sense that they probably do not influence the yields of recom- binant virus in crosses. The evidence for the latter conclusion, drawn from data presented elsewhere in this paper, may be summarized as follows: (1) the link- age relations deduced from average yields of virus are the same as those de- duced from single bursts selected for equality of yields of the two infecting viruses; (2) in the reverse crosses hXr7 and h r7 X wild type, one gets with- in experimental error equal numbers of all four recombinants in spite of the fact that in one case the infecting pair, and in the other the recombinant pair, have unequal excluding power; (3) in all crosses, the distribution of yields of recombinants among single bursts does not show one peak at zero and another 161 RECOMBINATION IN BACTERIOPHAGE 55 above the mean, as is the case with the yields of a minority infecting type, but shows a single mode slightly less than the mean. The last two lines of evidence cited seem to show that the principle of limited participation (DuLBEcco 1949) referred to above, operates only during the initial stages of infection, or at any rate does not influence the yields of genetic recombinants arising within the mixedly infected bacteria. They suggest further that the # mutant is superior to 77 or r13 in excluding power only, not as a competititor during actual multiplication. YIELDS OF GENETIC RECOMBINANTS FROM SINGLE MIXEDLY INFECTED BACTERIA In order to study the variations in yields of recombinants intrinsic to the recombination process, one would like to exclude as many as possible of the accessory sources of variation. The most important of these are variations in burst size, and variations in the relative numbers of the two infecting viruses adsorbed to individual bacteria. It will be seen presently that effects of varia- tions in burst size can be avoided by the simple expedient of computing propor- tionate yields of recombinants, these being independent of burst size. The effect of variations in relative multiplicity could be minimized either by going to very small or very large total multiplicities. Low multiplicities are uneconomi- cal, because at multiplicities sufficiently small so that most of the mixedly infected bacteria receive only one viral particle of each type, very few of the test cultures will yield a mixed burst. High multiplicities also introduce dif- ficulties (DuLBECCO 1949). We have chosen to use total multiplicities between 10 and 20, within which range the yield of recombinants is constant. As previously described, the elementary viral yields vary considerably in the relative numbers of the two parental types of virus and, as expected, these variations influence in turn the yields of recombinants. A correction for this source of variation was devised as follows. Assuming that the genetic inter- action occurs between unlike viral pairs, and that the composition of the viral yield provides a direct measure of the composition of the intracellular viral population during growth, one computes an interaction coefficient k = p(A)[1 — p()] (3) in which p(h) is given by (1), and k expresses the influence of the composition of the population on the number of unlike viral pairs present in the cell, neg- lecting effects of genetic recombination. The coefficient k has a maximum of 0.25 when half the viral yield contains the h allele. Dividing the proportions of recombinants by 4k serves therefore as a correction for inequality of yields of the parental viruses. This correction is ambiguous only for bursts in which the yields of the two recombinants are large and unequal, and bursts from which either recombinant is absent. A summary of the single burst data is given in table 4, which includes the mixed bursts only. The bursts have been separated into the classes k20.21 and k <0.20, to show the effect of the correction described above. It will be seen that the uncorrected mean proportion of recombinants is larger for the 162 56 A. D. HERSHEY AND RAQUEL ROTMAN bursts with the larger k, and that the effect of dividing each proportion by 4k is to make the results homogenous. These facts justify the use of the correction. Its theoretical significance is clarified in the discussion. TABLE 4 Single burst data for hXr crosses k=a measure of disproportion between yields of parental types (Eq. 3). x =average yield of the 7+ recombinant as percent of total virus. y =average yield of the r recombinant as percent of total virus. n=average burst size. r(x, y) =coefficient of correlation between proportionate yields of the two recombinants. r(n, x+y) =coefficient of correlation between burst size and sum of proportions of the two recombinants. The variations of x, y and n/100 shown are standard deviations within the sample. The standard errors of the means are obtained by dividing these by the square root of the number of bursts. The correction referred to is described in the text. NO. OF — CROSS Sowers k 4k x J n/100 r(x, y) r(n, x+y) hXrl 25 <0.20 0.65 uncorr. 8.9+3.8 NZ pelictiOve Smo 2. 4 —0.25 corr. IGF OEt 52.3 19.04+9.2 0.09 52 = 0-21 0.94 uncorr. 15.5+6.9 gfe lee 7a Satan sin/4 0.15 corr. 16.6+6.3 Sl ietaribere, —0.01. hriX++ 19 <0.20 0.67 uncorr. 13), 62: 51.9) 13.2+4.4 Saliczle9 —0.26 corr. ZOR EE 20.8+8.5 —0.27 36 0221 0.94 uncorr. 16.5+5.8 171256 5.4+3.0 —0.11 corr. Ai sos G2 18.2+6.0 0.16 hXr7 13 <0.20 0.64 uncorr. Sofa seS 6.6+4.6 6.2+1.9 —0.43 corr. 8.8+4.8 92959: 0.65 35) 20.21 0.93 uncorr. 6.8+4.2 002455 Ne aes jq7} 0.16 corr. Hepat ee} 9.8+4.8 0.53 hr7X++ 17 <0.20 0.63 uncorr. 6.44+3.1 4.84+2.9 6.44+7.8 —0.45 corr. 10.04+4.3 8.0+5.0 0.12 26 20.21 0.95 uncorr. ava sie7 8.94+5.4 Ha sao sei) 0.02 corr. ed t=3).10 O53 SI6 0.47 h&r13 29 <0.20 0.61 uncorr. 0.50.5 0.8+0.8 Se 2ict2 al 0.07 0.35 29 20.21 0.94 uncorr. 0.9+0.9 1.1+0.9 §.14+2.3 —0.05 —0.30 hr13X%++ 21 <0.20 0.69 uncorr. 0.6+0.7 0.6+0.9 4.64+1.4 0.15 0.22 46 20.21 0.94 uncorr. 0.8+0.7 0.7+0.6 4 ODES. 0.21 0.04 The data of table 4 for mixed single bursts confirm fairly well the average data of table 2, except that the yields of recombinants are somewhat greater owing to the exclusion of the unmixed bursts, and that the yields corrected for unequal growth of parental viruses are higher still. The chief point of interest is the question of the correlation between yields of the two recombinants in single bursts. This has been measured in terms of the correlation coefficient r (RIDER 1939). This measure varies between — 1 and +1, a value near 0 indicating independence of variates, and values near unity indicating negative or positive correlation, respectively. The data of table 4 show clearly that there is no significant correlation between the proportions of the two recombinants in single bursts except for the crosses h Xr7 and hr7X wild type. Even for these crosses the correlation is weak and not entirely convincing, especially since the data are not completely unselected (see below). 163 RECOMBINATION IN BACTERIOPHAGE Si The correlation between the uncorrected proportions of the two recombi- nants is shown in the form of scatter diagrams in fig. 7. These data might be expected to show some degree of spurious correlation owing to the fact that bursts with disproportionate yields of the two parental types tend to contain diminished numbers of both recombinants. This tendency can be seen in the 44) Zh'r* hal hxrl 28 @e x 24 ; ’ ee 20 16 12 8 4 76 4 8 IZ iG 20) 324 28) 32ese °5 4 8 12 16 20 24 28 32 Cae i ee hx rl3 4) %hr° 4 ° pe hri3xhr 3 e Bt F6teos & ° I 0.8, *o o% e 0 bh eke . One %hrr Ce O 1 2 3 4 227 %hrr* ° 1G hxr7 14 12 10 e 8 6 ce x 1 x 4 2 4-68 10) wiz la. IG aos °6 Beas 6 8). NO. 2 ala eG: Urey Ficure 7,—Correlation between proportionate yields of the two recombinants in single bursts. The open circles indicate bursts with disproportionate yields of the two parental viruses. Crosses indicate yields omitted from the data of table 4. 164 58 A. D. HERSHEY AND RAQUEL ROTMAN diagrams in which the disproportionate yields (k $0.20) are indicated by open circles. Actually this effect is of minor importance, since the variations due to unknown causes are so much greater than those due to variations in k. Con- sequently, the uncorrected data lead to the same conclusions as the corrected data; namely, that the proportionate yields of the recombinants are uncor- related in the crosses hXr1, hXr13, and the corresponding reverse crosses, but that there is a weak positive correlation for the crosses #Xr7 and hr7X wild type. As mentioned earlier, the correlation data in table 4 are not unselected. The diagrams of fig. 7, however, show all the mixed bursts for the respective ex- periments, those omitted from the table being indicated by crosses in the dia- gram. It is plausible that some of the discrepant bursts came from bacteria infected with a spontaneous mutant present in one of the parental stocks of virus. In fact, two bursts from the cross X77 were found to contain a large proportion of mutants, m in one case and weak inhibitor (HERSHEY 1946b) in the other, which almost certainly arose in this way. The / stock used in these crosses contained about 0.1 percent of #7 virus, so that in crosses with r at least one bacterium in 200 was infected with three types of virus, and would be likely to yield an excess of one of the recombinant phenotypes. Unfortu- nately the recombinant progeny in the exceptional bursts were not checked by crossing with the parental stocks, which should be done in any further experi- ments of this type. For the present these bursts throw some doubt on the sig- nificance of the positive correlation between proportions of recombinants in the crosses hXr7 and hr7X wild type. The results of the cross hXr13 are of special interest because of the small yield of recombinants. The distribution of the bursts with respect to absence of recombinants is shown in table 5. Nine of the 125 mixed bursts fail to show either recombinant, and 31 more lack one recombinant or the other. One can test the hypothesis that the two sister recombinants arise independently as follows. About 20 percent of the bursts fail to show a specified one of the two possible recombinants. If the absence of the one were independent of the ab- sence of the other, about (0.2)? or four percent of the bursts should show neither recombinant. The number found, 9/125, is larger than this, but not significantly so. Moreover, bursts lacking one recombinant do not show less than the average proportion of the other (table 5). The data evidently fail to exclude the hypothesis of independent origin of the two recombinants, but do not, of course, rule out the hypothesis of reciprocal exchange. Another question that arises in connection with the data is concerned with the number of genetic exchanges per bacterium. For explicitness, we consider separately the hypotheses of reciprocal and non-reciprocal exchange. If ex- changes are reciprocal, the bursts lacking a single recombinant are the result of failure to recognize the few plaques of either type, of losses in the ten per- cent of each culture not examined, and of unspecified biological accidents. As previously computed, four percent or 5 of the 125 bursts fail to show either recombinant for one or another of these reasons, leaving only 4 without re- combinants possibly owing to failure of exchange. This number is too small 165 RECOMBINATION IN BACTERIOPHAGE 59 if some of the single absences result from erroneous recognition of one recom- binant, but is otherwise subject only to its sampling errors. Taken as a measure of failure of reciprocal exchange, the fraction 4/125 implies an average of 3.4 exchanges per bacterium. If, on the other hand, exchanges are not recipro- cal, the fraction (20 percent) of the bursts lacking any given recombinant cor- responds to 1.7 genetic transfers per bacterium. These estimates may be too low if conditions vary from bacterium to bacte- rium in such a way that genetic exchange is suppressed in some bacteria. TABLE 5 Distribution of Bursts with Respect to Absence of Recombinants See legend table 4. The proportions of recombinants have not been corrected for dispropor- tionate yields of parental types of virus. NO. OF = CROSS CLASS x y ii k BURSTS hXrl13 both absent 2 0.0 0.0 390 0.20 htr* absent 10 0.0 0.81 460 0.19 h r absent 2 2.4 0.0 390 0.19 total 58 0.72 0.97 520 0.20 hls Ke a both absent 7 0.0 0.0 340 0.21 hr* absent 8 0.0 0.66 360 0.21 htr absent 11 0.75 0.0 380 0.22 total 67 0.73 0.64 480 0.22 Yields with small k and small burst size must have this effect, but are evi- dently not very important in the data of table 5, since the different classes are very similar with respect to k and burst size. In short, it is necessary to con- clude that there are at least two or three genetic exchanges per bacterium, independently of the mechanism by which recombinants arise. A different kind of estimate of the number of exchanges per bacterium is obtained from the number of recombinants actually found. The data are sum- marized in table 6 in the form of distributions of numbers of the several re- combinant types. One finds on the average 3.4 recombinants of any one kind per bacterium. This evidently furnishes an upper limit to the number of ex- changes per bacterium, insofar as exchanges yield viable and countable pro- geny. This result, taken in conjunction with the preceding estimates, leads to several remarkable conclusions. First, since the two methods of estimation, one minimal and one maximal, yield about the same result, there must be in fact only two or three exchanges per bacterium in the crosses between closely linked factors. Second, the recombinants must undergo little multiplication after they arise in the cell. Third, the conditions of viral growth in different bacterial cells must be equally favorable to genetic recombination; otherwise a larger proportion of bursts would fail to show recombinants. 166 60 A. D. HERSHEY AND RAQUEL ROTMAN These conclusions are substantially confirmed by the distributions of num- bers of recombinants shown in table 6, which are essentially of the Poisson type, with variance only moderately greater than the mean. In other words, the individual particles of any one recombinant must often arise independ- ently of each other in the same bacterial cell, and with equal probability in different bacterial cells. The deviations from the Poisson distribution are nevertheless significant, and can be attributed to a moderate amount of growth of recombinants. TABLE 6 Distributions of Numbers of Recombinants in Single Bursts from the Crosses hXr13 and h r13Xwild type The Poisson distributions show the numbers of tubes in the various classes expected if there were no growth of recombinants. The distribution with mean 3.4 is appropriate to the hypothesis of reciprocal exchange, and the distribution with mean 1.7 to the hypothesis of non-reciprocal genetic interaction. NO. OF RE- NO. OF TUBES FOUND COMBINANTS —— shee PER TUBE htr* hr hrt her Me es as 0) Ah2 4 tS 18 Deol iba Ses) 1 ial ala 11 14 Uae) 19.6 2 9 8 8 6 12m? 16.7 3 6 9 11 9 13.8 9.5 4 3 7 AE 4 Ai 4.0 5 7 3 4 2 8.0 1.4 6 1 2 3 6 4.5 0.4 7 3 6 0 2 DED 0.1 8 2 2 3 3 0.9 0.0 9 1 2 2 1 0.3 0.0 10-18 3 2 3 2 0.1 0.0 19-27 0) 1 1 0 0.0 0.0 28-36 0 1 0 0 0.0 0.0 No. of tubes 58 58 67 67 63 63 Mean per tube Sell 4.5 3.3 2.8 3.4 WS7 Variance 9.2 27 16 10 3.4 de The conclusion that genetic recombination is not suppressed in some of the bacteria is also supported by the results of the other crosses, in which no bursts yielding both parental viruses and lacking both recombinants were found. Only one burst, from the cross hXr7, failed to show one of the recombinants; it contained 95 percent of the / parent. INDEPENDENCE BETWEEN PROPORTION OF RECOMBINANTS AND BURST SIZE The data of table 4 do not show any significant correlation between burst size and proportion of recombinants, which means that the number of re- combinants must be very nearly proportional to the total yield of virus in single bursts. This conclusion must be qualified in view of the following considerations. 167 RECOMBINATION IN BACTERIOPHAGE 61 Some of the tubes contain one or more mixed bursts plus one or more unmixed bursts. Such tubes would tend to show less than average values of k and less than average yields of recombinants together with greater than average burst size. The number of such tubes is about five percent of the total in our experi- ments and these tend to be concentrated in the class k $0.2. This probably explains the negative correlation between proportions of recombinants and burst size among tubes selected for small k. A larger proportion of the tubes, between 10 and 20 percent in our experi- ments, contain two or more mixed bursts. These tubes would tend to have greater than average k and greater than average burst size, but will not show exceptional proportions of recombinants. These tubes have the effect of weak- ening any correlation that may exist between burst size and proportion of re- combinants, especially among the class k20.21. The data previously consid- ered do not, therefore, exclude the possibility of a weak positive correlation between proportion of recombinants and size of bursts. Advantage was taken of the finding that high pH reduces size of bursts, to examine the relation between burst size and yield of recombinants in another TABLE 7 Small Bursts from the Cross hXr7 in Broth of pH 9.0 See legend table 4. The proportions of recombinants have not been corrected for dispropor- tionate yields of parental types of virus. The individual cultures contain an average of about 1.4 mixed bursts. NO. OF ce ie 4k x y n r(x, y) rn, x-+Fy) 20 <0.20 0.61 6.3+3.8 NOAA SEO a7 99 + 50 0.53 —0.34 34 20.21 0.93 7.443.5 10.5+4.6 M23 \sto 73 0.61 0.35 54 all 0.81 (Wsesis a ORGEte 522 114+ 66 0.56 0.12 way. Since increased pH was found also to cause some of the bacteria to fail to liberate virus, the single burst technique was chosen. Bacteria were infected in the usual way with mutants / and 77 in broth of pH 6.8. At the end of the five minute adsorption period, and without treatment with antiserum, single burst cultures were prepared after diluting in broth of pH 9.0. Preliminary experiments showed that the yield of virus was complete under these conditions within one hour, and than no inactivation occurred during two additional hours at 37°C, or overnight in the refrigerator. Entire samples were plated on single mixed indicator plates. One successful experi- ment yielded 66 bursts, of which 54 were mixed, among 104 tubes receiving on the average 1.4 bacteria per tube. Evidently most of the bacteria liberated some virus. The average burst size, after subtracting the virus carried over from the input (totalling 36 particles per tube), was only 114 per tube, or about 70 per bacterium corrected for the probable multiples. The average proportion of recombinants was nevertheless of normal size (table 7). It will be noticed also that the correlation between the numbers of the two recombinants in these bursts is exceptionally good. The correlation is, however, 168 62 A. D. HERSHEY AND RAQUEL ROTMAN slightly exaggerated owing to the fact that the 54 tubes contain on the average mixed yields of virus from about 1.4 bacteria per tube. THE EFFECT OF A SHORT PERIOD OF ADSORPTION AND LOW MULTIPLICITY OF INFECTION ON THE DISTRIBUTION OF RECOMBINANTS The following experiment shows that when the multiplicity of infection in the cross hXr7 is reduced from five to about one of each viral type per bac- terium, the distribution of recombinants among single mixed bursts is little if any altered. Six crosses were made in the usual way, except that the period allowed for adsorption was reduced to one minute, without reducing the total input of virus. The amount of virus adsorbed was too small to be measured, but the multiplicity of infection can be estimated from the data given below. The single burst cultures collected from the six crosses are sufficiently simi- lar to be considered together. The mean number of bacteria per tube for the six sets, determined by colony counts from the growth tubes immediately before adding virus, is 0.23. The mean number of infected bacteria per tube determined by plaque counts of samples taken before lysis is 0.20. The mean number of bursts per tube calculated from the proportion, 141 out of 720, of tubes containing virus is 0.22. The 141 tubes therefore contained about 157 infected bacteria. From the distribution of viral types among the tubes, namely, 69 containing h only, 22 containing r only, 50 containing both, and 579 containing neither, one finds the probable distribution with respect to bacteria to be 80 infected with / only, 28 with r only, 49 both, and 46 neither. The multiplicity of in- fection is therefore about 1.0 with respect to 4, and 0.48 with respect to r. One can estimate further that about seven of the tubes contained one or more mixed bursts plus one or more unmixed; and that about three contained both h and r bursts without any mixed bursts. Also, among the mixedly infected bacteria, 45 percent were infected with one particle only of each viral type. In making the above computations we have neglected the probability that the k mutant suppresses the growth of r in some bacteria adsorbing both types of virus. The apparent inequality of infection is probably due in some part to this effect. However, in other experiments with low multiplicity of in- fection with / and 17 designed to check this point, the split into h, r, and mixed yielders was nearly equal. It seems likely, therefore, that in the experiments reported here the two viral types were unequally adsorbed for unknown reasons. The tubes containing only one viral type may be dismissed by saying that their average content of virus did not differ significantly from that of the mixed yields, and that the / and r yields per bacterium were the same. There was one exceptional burst containing only 4 r* and hr phenotypes. The char- acteristics of the remaining 49 cultures containing / and r virus are summarized in table 8. The data show no unusual features excepting the small burst size, which is a direct effect of the low multiplicity of infection, and the somewhat 169 RECOMBINATION IN BACTERIOPHAGE 63 small proportion of recombinants, probably due to the appreciable number of superimposed unmixed bursts. TABLE 8 Single Bursts from the Cross hXr7 with Low Multiplicity of Infection See legend table 4. The proportion of recombinants have not been corrected for dispropor- tionate yields of parental types of virus. Adsorption time one minute. Multiplicity 1.0 # and 0.57 per bacterium. NO. OF — it Degur k Ak x y n/100 r(x, y) r(n, x+y) 17 <0.20 0.59 Hpolaeesa(0) ASO 2916 0.38 —0.17 32 20.21 0.95 4.4+3.1 OER 5.4 Sion 230 0.20 Osos 49 all 0.84 AS TEESic5 Se te A9 e4andlow) 0.24 —0.30 The principal point to be made here is that the variation in yields of recom- binants from tube to tube is not exceptional. In this connection it must be mentioned that 5 of the 49 tubes contained # and 7 virus without any recom- binants. Of these, one was exceptional in containing only 24 viral particles, and another for the extreme disproportion of parental types (88 percent #). The remaining three, each containing from 450 to 570 particles, are probably superimposed unmixed / and ¢ bursts. It should be noted also that the correlation between the yields of the two recombinants in this set is not significant, since the observed correlation is exaggerated by the tubes containing unmixed / and 7 bursts without recom- binants. Whether the poor correlation is accidental, or an effect of the low multiplicity of infection, remains to be determined. The negative correlation between burst size and proportion of recombinants can, however, be ascribed to the superimposed mixed and unmixed bursts, as well as to the unmixed h and r bursts, in some of the tubes. IDENTITY OF RECOMBINANTS WITH THE CORRESPONDING ANCESTRAL TYPES According to any simple hypothesis of factorial recombination, one expects the recombinant virus arising in crosses not to differ genetically from the cor- responding ancestral type. Two kinds of test indicate that this is so. In the first kind of test (HERSHEY and RotmAN 1948) stocks of the phenotypic wild type arising from the cross between two different r mutants were backcrossed to authentic wild type. No ry mutants appeared in such crosses, and it was concluded that the stocks were genetically identical. The second kind of test is the following. The double mutant / 17, itself ob- tained by crossing the two single mutants, was crossed with wild type and the recombinants / and r were re-isolated. These were then tested by making the homologous (parental i by recombinant / and parental r by recombinant r) and heterologous (parental # by recombinant 7 and parental r by recombinant h) back crosses. In both cases, the homologous cross yielded only one type of 170 64 A. D. HERSHEY AND RAQUEL ROTMAN virus, and the heterologous cross yielded recombinants in the same proportion as found with the parental stocks. These tests show not only that the recom- binants contain the same genetic markers as the corresponding progenitive types, but also that the region between the markers is unchanged. MIXED YIELDS CONTAINING ONLY ONE PARENTAL TYPE OF VIRUS Only four mixed bursts lacking one of the parental types of virus were found among the experiments reported in this paper. One, from the cross /XrJ, contained 86 percent htr and 14 percent hr. A second, from the cross hXr7 contained 99.6 percent 4 r* and 0.4 per cent hr. A third, from the same cross with low multiplicity of infection, contained 83 percent # rt, and 17 percent hr. The fourth, from the cross h r7 X wild type, contained 23 percent hr* and 77 percent ir. In this case the yield of r+, which appeared to be homogenous, formed atypical plaques and proved on isolation to differ from any known mutant of 72H. It seems reasonable to suppose that these exceptional bursts contained progeny stemming from mutants contaminating the parental stocks of virus. On the other hand, the one exceptional burst following low multi- plicity of infection, together with the failure to find similar bursts among the crosses involving # and r/3, suggest that a different interpretation should be looked for. Genetic tests which might have clarified this point are lacking. For the present we conclude, as a first approximation, that recombinants arise only in those bacteria in which both parental types of virus succeed in multi- plying. It may be added here, because the question arises in connection with these exceptional bursts, that no correlation can be seen between the proportion in mixed bursts of the total virus containing the / allele, and the proportion of the recombinant virus containing the / allele. We have therefore omitted this datum from the tables. DISCUSSION In collecting and analyzing the data just described, we have had in mind the following questions. Does genetic exchange occur in the course of matings between viral particles, or is it the expression of a mechanism of growth such as that visualized by Luria (1947), according to which the multiplying units in the cell are not phage particles, but simpler structures derived from them? Can the linkage relations represented in fig. 1 be interpreted in terms of linear chromosome-like structures? Are the genetic exchanges reciprocal, as one ex- pects for simple cases of crossing over, or must one look for an alternative mechanism more intimately connected with the mode of reproduction of the virus? It was soon apparent that the data for crosses between linked and unlinked factors tended to give different answers to these questions, and we were led to consider a model based on two distinct mechanisms of exchange. The neces- sity for this arises from the following facts. First, the linkage data indicate a limitation at about seven percent to the proportion of wild type found in crosses between linked factors, which is dif- 171 RECOMBINATION IN BACTERIOPHAGE 65 ficult to reconcile, in terms of a single mechanism, with the existence of a second class of crosses yielding about 15 percent of wild type. Second, the correlation between proportions of the two recombinants in the cross hXr7, and the lack of a corresponding correlation in the cross hXrJ, is incompatible with a single mechanism for the two crosses. It must be recalled, however, that the correlations found are too weak to be wholly convincing. Third, Lurra’s (1947) evidence for a mechanism of independent multiplica- tion and transfer of subunits of the virus, and ours for a system of linkage, re- quire dissimilar types of interpretation. The model to which these considerations seem to lead is described below, but we do not consider that we have decisive answers to any of the questions originally posed. The remainder of this discussion is of value only insofar as it clarifies the questions, and systematizes the experimental results so far ob- tained. The two linkage structures bearing the markers r/ and h, respectively, are assumed to be examples of the class of independently multiplying subunits of the virus whose minimal number Luria and DurBecco (1949) estimate at about 25. The reconstitution of virus from these units must be regulated in such a way that each particle receives one representative of each kind of unit. In the cross Xr the choice between / and kt, and between r and r*, is de- cided nearly at random to yield on the average 37 percent of recombinants and 63 percent of the parental types in bacteria yielding equal numbers of the two parents. The deficit of recombinants below 50 per cent is unexplained, but may be thought of as an effect of incomplete mixing between neighboring clones of multiplying virus in the cell. According to this hypothesis one expects from the cross Xr/ proportionate yields of the two recombinants in a single burst to be: ne n( n(ht*) )/( n(r+) -) (4) B ODUM) Ma) + n(7) n(h) ( n(r) ) hr) = 5 Bi) m( n(ht) + =) n(r+) + n(r) (5) where the expressions on the left refer to proportions of recombinant virus, the corresponding expressions on the right refer to the intra cellular yields of the respective unit linkage structures, and the coefficient m expresses the fraction of the intracellular virus which may be regarded as a random mixture of the two parental types, the remainder being considered unmixed. If the structures carrying the markers h, h+, r, and r* grow independently in the cell, their yields will fluctuate independently, and no correlation will be ex- pected between the numbers of the two recombinants in sufficiently small yields of virus. This expectation is borne out by the data for viral yields from single bacteria. The average yields of the two recombinants are equal, however, showing that the several unit structures grow at equal rates. According to equations (4) and (5), the proportionate yield of recombinants should not be 172 66 A. D. HERSHEY AND RAQUEL ROTMAN influenced by burst size unless the latter has an effect on the fraction m. According to the data it does not, and m is a parameter having the average value 37/50. This interpretation requires that 26 percent of either kind of parental virus in the cell should multiply in effective isolation from the other parent. It will be noticed that the expressions (4) and (5) reduce to mk if one makes the approximations mentioned in connection with equation (3). This provides a theoretical basis for the correction we have applied to yields of recombinants in crosses between unlinked factors. An analogous justification for its use in crosses between linked factors will appear in the discussion to follow. The predictions for the cross 4 Xr7 are different from the preceding case, be- cause here the markers are situated on homologous linkage structures, so that recombination requires something like crossing over, which in turn requires something like synapsis. The expected fractional yields of recombinants are p(ktrt) = p(hr) = 3 msc, (6) in which m has been defined previously, s is a fraction independent of m ex- pressing frequency of pairing, and c is a crossover frequency. In order to make c independent of m and s it is evidently sufficient to define the product ms as the fraction of the viral yield made up of particles in which the marked unit has descended from an unlike synapsed pair. The application of (6) leads to ambiguity if exchanges can occur between descendants of unlike synapsed pairs (HERSHEY and RotMaAN 1948). In what follows this difficulty does not appear to be very serious, but no rigorous analysis has yet been attempted. For the cross 4X77, if exchanges are reciprocal, one expects the correlation between proportions of the two recombinants to be disturbed only by fluctua- tions in relative growth of the two exchange products, in contrast to the cross hXr1, where the correlation is subject to fluctuations in the growth of four independent units. That these fluctuations are individually considerable is shown by the variations in relative and total viral yields in mixedly infected bacteria. A weak but probably significant correlation between proportions of the two recombinants is nevertheless visible in the cross hXr7. No such cor- relation can be seen in the cross 4 XrJ. If the mechanism of exchange for these two crosses were the same, the greater correlation would be expected in the cross h Xr1, which gives the larger yield of recombinants. The predictions for the cross hXr13 are the same as for hXr7 except as modified by the much smaller yield of recombinants, presumably owing to a smaller frequency of crossing over. We have shown that in this cross the re- combinants come from two or three individual exchanges per bacterium, and that there is little growth of recombinants subsequent to exchange. These cir- cumstances ought to be favorable for testing the hypothesis of reciprocal ex- change. The data are nevertheless inconclusive of this point. The experiments provide information about the sequence of events in the cell. A mechanism of exchange limited to an initial phase of multiplication is ruled out by the following consideration. If exchange occurred at a time when there were few replicas in the cell, any cross yielding a small average number of 173 RECOMBINATION IN BACTERIOPHAGE 67 recombinants would show some individual bursts containing no recombinants and others containing a very large proportion, especially at low multiplicity of infection. Instead one finds a comparatively uniform yield of recombinants, and the distribution of their proportions is not affected by the multiplicity of infection. Also if exchanges occurred freely throughout the period of multiplication of the virus, one would expect considerably greater variations in yields of recom- binants than we have found. For instance, in the cross 4 Xr13, in which there are only two or three exchanges per bacterium, the variations in yields of re- combinants are not much greater than those expected to result from a random variation in the number of exchanges alone. Moreover, most of the bacteria yield only a few recombinants, so that little growth can have occurred sub- sequent to exchange. The conclusion is unavoidable that the exchanges are limited to the terminal phase of multiplication, or at any rate that recombi- nants are prevented from multiplying appreciably in most of the bacteria. It is remarkable that the variations in proportions of recombinants are so little dependent on the degree of linkage (as between #Xr7 and 4 Xr13), or on the postulated mechanism of exchange (as between the above and #XrJ). The coefficients of variation in proportions of individual recombinants among single bursts are, for hXr1, about 40 percent; for 4X77, about 60 percent; and for hXr13, about 100 percent. This circumstance also supports the inference that the exchanges are limited to a late phase of multiplication. The hypothesis stated permits one to examine iurther the structure of the linkage units. Since crosses between r/3 and any of the mutants belonging to the group closely linked to r7 yield about the same proportion, seven percent, of wild type (HERSHEY and RoTMAN 1948), it might be supposed that one crossover between the distant markers is always accompanied by several others, so that 50 percent of the progeny of synapsed pairs of the units 77 and r13, for example, would be recombinant types. If this supposition is correct the terms of equation (6) can be evaluated by setting c=0.5 and p(htr*) =0.07. This gives 0.28 for the average fraction ms of virus descending from unlike synapsed pairs. If this fraction is assumed to be the same in other crosses in- volving the same linkage structure (the cross kh r7 Xr13 reported in this paper suggests that it is), one can write for them p (wild type) = 0.14 c, (7) where c is the appropriate crossover frequency and the proportion of wild type is experimentally measured. The data for the three point crosses involving r2, r3, and v6 (HERSHEY and Rotman 1948) are examined from this point of view in table 9. The proportions of wild type have been calculated for random crossing over between unit linear structures, using the crossover frequencies given by (7). It will be seen that the data are entirely compatible with the hypothesis tested. Additional tests of this kind are needed, however. It will have been noticed that the average yield of recombinants in crosses between distant linked factors is very nearly half that found for unlinked factors. Dr. M. DeLBRick has pointed out to us that this relationship can be 174 68 A. D. HERSHEY AND RAQUEL ROTMAN understood in terms of random pairing between homologous structures. In the simplest case one visualizes unrepeated pairing, that is, pairing limited to a phase in which there is no multiplication, and during which no structure finds more than one partner. For this case, the frequency of synapsis in equation (6) is simply the ratio of the number of unlike homologous pairs to the total number of homologous pairs. This ratio can be written 2ab 5s = (Sab) arta bial) where a and b are the respective numbers of the two unlike homologous structures. Inspection of (8) shows that this ratio is essentially 2k as given by equation (3) when a+b is large compared to unity. Instead of equation (6) we have, therefore, (8) p (wild type) = mkc, (9) from which the proportion of either recombinant expected in crosses between distant linked factors can be computed as follows. The parameter m, taken to be the average fraction of virus randomly mixed in the cells at the time of reconstitution of virus from subunits, was found to TABLE 9 Three Point Linkage Tests of Linear Structure The symbol r2,3 refers to the double mutant containing 7 alleles at the loci 72 and 73, etc. c; is the crossover frequency for the region between 72 and 73. C2 is the crossover frequency for the region between 73 and r6. The locus 73 is assumed to lie between r2 and 76. The factor 0.14 is explained in the discussion. FRACTIONAL YIELD OF WILD TYPE CROSS COMPUTATION EXPECTED FOUND r2Xr3 a 0.020 c:=0.020/0.14=0.14 13X16 ze 0.014 co=0.014/0.14=0.10 r2Xr6 0.030 0.024 0.14fer(1—ce) +e2(1 —c1)] 72,3 X16 0.012 0.008 0.14 cx(1—c) 13,0 Xr2 0.018 0.014 0.14 c1(1—ce) 12,6Xr3 0.002 0.003 0.14 cice be 37/50 in crosses between unlinked factors. In (9) we require the corre- sponding fraction at the time of pairing, and assume this to be the same. The average of k for bacteria giving mixed viral yields (table 4) is 0.21, or 0.19 if one includes the ten percent of bacteria yielding only one type of virus. If k and m vary independently, their mean product is the same as the product of means, or 0.14 averaged over all bacteria. The average yield of either recom- binant from equation (9), for factors sufficiently far apart so that c=0.5, is accordingly seven percent, computed solely from the data for crosses between unlinked factors. This is the maximum actually found in crosses 175 RECOMBINATION IN BACTERIOPHAGE 69 between linked factors (HERSHEY and Rotman 1948). Equation (9) also pre- dicts, in agreement with the data for single bursts, proportionality between yields of recombinants and k. The agreement supports the inferences previously drawn that the markers r7 and rl3 are attached to the same linkage unit, and that the frequency of crossing over between them is 0.5. It suggests that the pairing itself is complete without appreciable repetition, and occurs at random except that about 26 percent of the units of each kind are effectively segregated from their opposite numbers. The measure of this segregation, m, is on this view the same for crosses between linked and unlinked factors. On the other hand, this inter- pretation cannot be rigorously correct, because one can show by multiple factor crosses (HERSHEY and RoTMANn 1948) that repeated exchanges, or ex- changes among three viral particles, occur. An estimate of the amount of repeated pairing has not yet been attempted, except that the considerations just offered suggest either that it is small, or that random pairing is limited to a small proportion of the population. It follows from equation (9) that the interpretation in terms of orderly pairing accords with the fact, otherwise very puzzling, that the proportion of recombinants is not affected by size of burst even in crosses between linked factors. It has been seen that the linkage data support fairly well the idea of linear structure, but independent evidence for crossing over is meagre. According to any simple model of reciprocal exchange, a correlation between proportions of sister recombinants in individual bursts would be expected. This expectation has been only partially realized, and the question arises whether the linkage data themselves require the crossover hypothesis. The following model, suggested by Dr. A. H. STURTEVANT, shows that they do not, and also shows that the question of reciprocity is closely connected with the question whether the exchanges are material transfers. Suppose that the replication of linear structures occurs zipperwise along the pattern from one end to the other, but that the partners separate prematurely to yield fragmentary replicas. Additions to the fragments are subsequently possible only after pairing with the same or another homologous structure, which in mixedly infected bacteria could belong either to the same or a different parental line. Genetic recombination in a two factor cross will depend, then, on the contingency that the two marked regions of a given replica be laid down one after the other on homologous structures from the two unlike parents. With simple assumptions, all the consequences of the crossover hypothesis (equation (6)) follow from this model, except that the independent origin of the two recombinants provides an additional source of independent variation in their numbers. The complications peculiar to this model have to do principally with the evidence that exchanges occur only during the terminal phase of growth. These complications are not very serious if one assumes that during early stages of growth the probability is great that a fragment will be started and completed on patterns belonging to the same parental line; that is, that the 176 70 A. D. HERSHEY AND RAQUEL ROTMAN mixing of the cell contents is relatively incomplete, and the distance between unlike clones relatively great, for small total populations. It has to be stipu- lated further that the terminal mixing is independent of the final concentra- tion of virus in the cell, to account for the lack of dependence of proportion of recombinants on burst size. Some hypothesis of this sort may prove useful if further experiments fail to strengthen the present evidence for reciprocal exchange. It is notable that two very different lines of evidence, ours and that of Luria (1947), have led to the idea of independently multiplying subunits of the virus. Our results differ from Luria’s only in calling for a system of linkage superimposed on the set of independent units. It remains to be seen whether a combination of genetic and radiological techniques bears out the present conclusions, and perhaps leads to an identification of the radiation-sensitive units with the linkage structures. SUMMARY Genetic recombination between two viruses differing by two mutational steps has been studied by infecting bacteria with the pair, and counting the numbers of the four types of virus found in yields from single bacteria. The crosses so examined include #XrJ/ (unlinked), 4Xr7 (linked), and hXr13 (closely linked), where / refers to a mutant of altered host range, and rJ, 17, and r13 are different mutations producing the same alteration in type ol plaque. The reverse crosses, hr X wild type, were also studied. The results may be summarized as follows. Nearly all mixedly infected bacteria yield both parental types of virus and two recombinants, according to the scheme h+r=h r-+wild type. The ten percent or so of bacteria yielding only one of the parental types seldom or never yield any recombinants. The rest of the bacteria always yield two recom- binants, except for the occasional absence of one or both in the crosses between closely linked factors. The average yields of the two recombinants in any one cross are the same, and are independent of the direction of exchange, so that reverse crosses in- volving the same pair of mutant factors yield the same number of recombi- nants. The proportionate yields of recombinants from individual bacteria are independent of burst size, and of the total multiplicity of infection, but depend on the relative yields of the two parental types. The effect of the latter is not marked, however, and the variations from bacterium to bacterium must be chiefly the result of variations in the number of genetic exchanges and in the growth of recombinants subsequent to exchange. These variations may be de- scribed by saying that one finds a moderately skewed distribution, with mode less than the mean, and with mean and standard deviation dependent on the linkage relations as follows: for hXr1,15+6, for hXr7,7+4, for hXr13, 141, expressed in round numbers as percent of either recombinant in the total yield of virus. A weak but moderately convincing correlation between the proportionate 177 RECOMBINATION IN BACTERIOPHAGE i yields of the two recombinants in individual bacteria is discernible in the cross hXvr7 and its reverse, but not in the other crosses. In the cross kXr13 only two or three genetic exchanges occur during the multiplication of the virus in a single bacterial cell. These exchanges take place near the end of the period of multiplication of the virus. A hypothesis is outlined which is compatible with the genetic data and with the results of LuriIA concerning reactivation of irradiated virus in bacteria receiving two or more individually noninfective particles. The hypothesis is an extension of that of Luria, according to which one visualizes genetic interaction not between two viral particles, but between two sets of independ- ently multiplying chromosome-like structures. Genetic exchange occurs either by reassortment of these structures, or by something like crossing over between homologous pairs, depending on the structural relation between the genetic factors concerned. The interpretation made brings the linkage relations into superficial agreement with the requirements of linear structure, but there is little evidence that the genetic exchanges are reciprocal, and accordingly little evidence that they are material exchanges. LITERATURE CITED Detsrtck, M., 1945a Interference between bacterial viruses III. The mutual exclusion effect and the depressor effect. J. Bact. 50: 151-170. 1945b The burst size distribution in the growth of bacterial viruses. J. Bact. 50: 131-135. 1945c Effects of specific antisera on the growth of bacterial viruses. J. Bact. 50: 137-150. DeE.srutck, M.,and W. T. Barey, Jr., 1946 Induced mutations in bacterial viruses. Cold Spring Harbor Symp. Quant. Biol. 11: 33-37. DeEBrRUcK, M., and S. E. Luria, 1942 Interference between bacterial viruses I. Interference between two bacterial viruses acting upon the same host, and the mechanism of virus growth. Arch. Biochem. 1: 111-141. Dutsecco, R., 1949 The number of particles of bacteriophage 72 that can participate in intra- cellular growth. Genetics 34: (In press). HersHEY, A. D., 1946a Mutation of bacteriophage with respect to type of plaque. Genetics 31: 620-640. 1946b Spontaneous mutations in bacterial viruses. Cold Spring Harbor Symp. Quant. Biol. 11: 67-77. HeErsHEY, A. D., and R. Rotman, 1948 Linkage among genes controlling inhibition of lysis in a bacterial virus. Proc. nat. Acad. Sci. 34: 89-96. Luria, S. E., 1947 Reactivation of irradiated bacteriophage by transfer of self-reproducing units. Proc. nat. Acad. Sci. 33: 253-264. Luria, S. E., and R. DuLBEcco, 1949 Genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics 34: (In press). Rwer, Paur R., 1939 An introduction to modern statistical methods. ix+220 pp. New York: Wiley & Sons. 178 GENETIC RECOMBINATION AND HETEROZYGOSIS IN BACTERIOPHAGE A. D. Hershey and Martha Chase Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, New York In this paper we summarize the principal features of inheritance in the bacterio- phage T2H, and describe some new experiments. The genetic structure of this virus has been analyzed in terms of mutational patterns (Hershey, 1946) and by recombination tests (Hershey and Rotman, 1949). These two types of evidence agree in showing that mutational changes occur in localized regions of a complex genetic system. Mutations producing different effects usually occur at different loci, but one example of multiple allelism has been found (Hershey and Davidson, 1951). In this instance, the locus of the alternative mutations could be analyzed rather completely because most of the host-range mutations selected in a particular way proved to belong to a single allelic series. It was found that one pair of distinct mutants satisfied all three criteria of allelism listed below, and that another pair satisfied none of them. The criteria used were the following: (1) If the second of two successive mutations from wild type occur at the locus of the first, reversion to wild-type in a single step is possible. (2) No genetic recombination can be observed between allelic mutant pairs. (3) The map position of the locus is independent of its allelic state. BIPARENTAL RECOMBINATION The production of new genetic types of phage by intracellular interaction be- tween different bacteriophages was first observed by Delbriick and Bailey (1946), who mentioned genetic recombination as one of two possible interpretations of their result. The principle of genetic recombination was established by experi- ments with genetically defined stocks of the bacteriophage T2H (Hershey and Rotman, 1948, 1949). The main facts of genetic recombination in this bacterial virus can be illus- trated by examples of the interaction between two classes of mutant. Rapidly lysing (r) mutants are easily recognized by inspection of the plaques they produce on an agar plate seeded with sensitive bacteria. The plaques are larger, and have a sharper margin, than those of the wild-type. Host range (h) mutants are able to infect a suitable bacterial ‘indicator’ strain that is resistant to the wild-type Reprinted by permission of the authors and the Long Island Biological Association from CoLp Spring HARBOR SYMPOSIA ON QuANTITATIVE BroLoey, 16, 471-479 (1951). 179 A. D. HERSHEY AND MARTHA CHASE FIG. 1. Progeny of the cross h X r1 plated on mixed indicator. The large clear plaque is hr; small clear, h; large turbid, r; small turbid, wild-type. The eccentric clearings in the r plaque result from secondary h mutations. virus (Luria, 1945). The h mutants are normal with respect to type of plaque. By successive mutations, double (hr) mutants are readily obtained. The four kinds of virus, wild, h, r, and hr, can be recognized by plating on agar plates seed- ed with a mixture of sensitive (B) and indicator (B/2) strains of bacteria. Inde- pendently of the r character, h virus lyses both B and B/2 to produce clear plaques, and h* virus lyses only B to produce turbid plaques. Independently of the h character, r virus produces large plaques, and r* virus produces small ones. Genetic recombination is observed in a “cross” in which sensitive bacteria are infected with a few particles of h and a few particles of r per cell. The viral proge- ny coming from the mixedly infected bacteria contains both parental types, to- gether with a certain proportion of the two recombinants (Fig. 1). Analogous re- combinants are found when two different r mutants are crossed. For example, the cross r1 X r2 gives rise to the double mutant r/r2 and wild-type (Hershey and Rotman, 1948). In both types of cross, the yield of recombinants is characteristic for the 180 GENETICS OF BACTERIOPHAGE i | -_—— unit distance OS h ris reams a 3 _++—_+—. m St FIG. 2. Linkage relations among several genetic markers. mutant pair, and the numerical results can be summarized in the form of a genetic map, as shown in Figure 2. On this map we have shown only a few markers that we wish to refer to in this paper. The letter m (minute) stands for a small plaque mutant. It is well established that the three loci, r/, h,and m assort independent- ly of each other, and that the loci linked to A are arranged in linear order. Dr. N. Visconti (personal communication) has recently confirmed the earlier results on these points by a new method of three-point testing. TRIPARENTAL RECOMBINATION Important information about genetic recombination comes from experiments in which the frequency of triparental recombination is measured. If bacteria are infected with the three mutants h, m, and r1, the recombinant Amr can arise only by interactions involving all three. The results for this triple infection and its re- verse, hr X hm X mr, are shown in Table 1. About three per cent of the viral yield consists of the triparental recombinant in these crosses. This shows that in- teractions among three particles of virus occur with high frequency. A measure of this frequency can be expressed in terms of some artificial assumptions. If we suppose that multiplication precedes recombination, and that recombinants are formed during random successive pairings between phage particles, each particle would have to pair with three other particles to explain the results shown in Table 1. The genetic factors used in these experiments are unlinked. The data for tri- parental recombination in experiments with linked markers (Hershey and Rotman, 1948) lead to the same quantitative conclusion. This conclusion is a stumbling block to the further understanding of the mecha- nism of genetic recombination, since it is extremely difficult to distinguish be- tween successive interactions by pairs, and other types of interaction that might involve larger groups. The available information is insufficient, therefore, to de- cide between alternative hypotheses of viral interaction so far considered. 181 A. D. HERSHEY AND MARTHA CHASE FIG. 3. Mottled plaques from bacteria infected with r and r+ virus. EXPERIMENTS WITH CLUMPED PHAGE The following observations are pertinent to the proper interpretation of experi- ments to be described in a later part of this paper. Since they are also of general interest, we record them separately. The r mutants are unique among the known mutants of phage in that mixed colonies of r and r* phage are easily recognizable as mottled plaques. Mottled plaques are conveniently prepared by infecting bacteria with r and r* phage, and plating before lysis so that the plaques originate not from single phage particles, but from the mixed population liberated locally when the bacterium lyses (Fig. 3). We have made use of this characteristic mottling as a test for the aggregation of phage particles. Platings of mixtures of r and rt phage do not show mottled plaques (except rarely by the overlapping of plaques), even after the mixture has been packed in a centrifuge to allow every opportunity for the particles to stick together. Mottling clumps can be produced, however, by agglutinating the mixture with antiserum. To prepare mottling clumps of phage, one adds to a mixture containing half r and half r* phage, at a concentration of about 10!! particles per ml, an amount of 182 GENETICS OF BACTERIOPHAGE TABLE 1. TRIPARENTAL RECOMBINATION TESTS WITH THREE INDEPENDENTLY ASSORTING GENETIC FACTORS Cross Per cent distribution in yield wild h r m hr hm mr hmr hX 30% 20 (?) (2) The crystallographic unit contains two polynucleotide chains. The argument is crystallographic and so will only be given in outline. Structure B has a very strong 3.4 A reflexion on the meridian. As first pointed out by Astbury (1947), this can only mean that the nucleotides in it occur in groups spaced 3.4 A apart in 195 J. D: WATSOM AND FivH: C\CRICK the fiber direction. On going from Structure B to Structure A the fiber shortens by about 30 per cent. Thus in Structure A the groups must be about 2.5 per cent A apart axially. The measured density of Structure A, (Franklin and Gosling, Figure 2. X-ray fiber diagram of structure A of desoxyribonucleic acid. (H. M. F. Wilkins and H. R. Wilson, unpub.) 1953c) together with the cell dimensions, shows that there must be two nucleo- tides in each such group. Thus it is very probable that the crystallographic unit consists of two distinct polynucleotide chains. Final proof of this can only come from a complete solution of the structure. Structure A has a pseudo-hexagonal lattice, in which the lattice points are 22 A apart. This distance roughly corresponds with the diameter of fibers seen in the electron microscope, bearing in mind that the latter are quite dry. Thus it is probable that the crystallographic unit and the fiber are the one and the same. III]. DEsScRIPTION OF THE PROPOSED STRUCTURE Two conclusions might profitably be drawn from the above data. Firstly, the 196 THE STRUCTURE OF DNA structure of DNA is regular enough to form a three dimensional crystal. This is in spite of the fact that its component chains may have an irregular sequence of purine and pyrimidine nucleotides. Secondly, as the structure contains two Fiaure 3. X-ray fiber diagram of Structure B of desoxyribonucleic acid. (R. E. Franklin and R. Gosling, 1958a.) chains, these chains must be regularly arranged in relation to each other. To account for these findings, we have proposed (Watson and Crick, 1953a) a structure in which the two chains are coiled round a common axis and joined to- gether by hydrogen bonds between the nucleotide bases (see Figure 4). Both chains follow right handed helices, but the sequences of the atoms in the phos- phate-sugar backbones run in opposite directions and so are related by a dyad perpendicular to the helix axis. The phosphates and sugar groups are on the out- side of the helix whilst the bases are on the inside. The distance of a phosphorus atom from the fiber axis is 10 A. We have built our model to correspond to Struc- ture B, which the X-ray data show to have a repeat distance of 34 A in the fiber direction and a very strong reflexion of spacing 3.4 A on the meridian of the X-ray 197 J. D. WATSON AND FA. C.ACRICK FicureE 4. This figure is diagrammatic. The two ribbons symbolize the two phosphate-sugar chains and the horizontal rods the pairs of bases holding the chain together. The vertical line marks the fiber axis. pattern. To fit these observations our structure has a nucleotide on each chain every 3.4 A in the fiber direction, and makes one complete turn after 10 such intervals, i.e., after 34 A. Our structure is a well-defined one and all bond dis- tances and angles, including van der Waal distances, are stereochemically accept- able. THE STRUCTURE OF DNA ADENINE © THYMINE Figure 5. Pairing of adenine and thymine. Hydrogen bonds are shown dotted. One carbon atom of each sugar is shown. GUANINE a CYTOSINE Figure 6. Pairing of guanine and cytosine. Hydrogen bonds are shown dotted. One carbon atom of each sugar is shown. The essential element of the structure is the manner in which the two chains are held together by hydrogen bonds between the bases. The bases are perpendic- ular to the fiber axis and joined together in pairs. The pairing arrangement is very specific, and only certain pairs of bases will fit into the structure. The basic reason for this is that we have assumed that the backbone of each polynucleotide 199 J. D. WATSON AND F. H. C. CRICK chain is in the form of a regular helix. Thus, irrespective of which bases are present, the glucosidic bonds (which join sugar and base) are arranged in a regu- lar manner in space. In particular, any two glucosidic bonds (one from each chain) which are attached to a bonded pair of bases, must always occur at a fixed distance apart due to the regularity of the two backbones to which they are joined. The result is that one member of a pair of bases must always be a purine, and the other a pyrimidine, in order to bridge between the two chains. If a pair con- sisted of two purines, for example, there would not be room for it; if of two pyrimidines they would be too far apart to form hydrogen bonds. In theory a base can exist in a number of tautomeric forms, differing in the exact positions at which its hydrogen atoms are attached. However, under physi- ological conditions one particular form of each base is much more probable than any of the others. If we make the assumption that the favored forms always oc- cur, then the pairing requirements are even more restrictive. Adenine can only pair with thymine, and guanine only with cytosine (or 5-methyl-cytosine, or 5- hydroxy-methyl-cytosine). This pairing is shown in detail in Figures 5 and 6. If adenine tried to pair with cytosine it could not form hydrogen bonds, since there would be two hydrogens near one of the bonding positions, and none at the other, instead of one in each. A given pair can be either way round. Adenine, for example, can occur on either chain, but when it does its partner on the other chain must always be thymine. This is possible because the two glucoside bonds of a pair (see Figures 5 and 6) are symmetrically related to each other, and thus occur in the same po- sitions if the pair is turned over. It should be emphasized that since each base can form hydrogen bonds at a number of points one can pair up isolated nucleotides in a large variety of ways. Specific pairing of bases can only be obtained by imposing some restriction, and in our case it is in a direct consequence of the postulated regularity of the phos- phate-sugar backbone. It should further be emphasized that whatever pair of bases occurs at one par- ticular point in the DNA structure, no restriction is imposed on the neighboring pairs, and any sequence of pairs can occur. This is because all the bases are flat, and since they are stacked roughly one above another like a pile of pennies, it makes no difference which pair is neighbor to which. Though any sequence of bases can fit into our structure, the necessity for spe- cific pairing demands a definite relationship between the sequences on the two chains. That is, if we knew the actual order of the bases on one chain, we could automatically write down the order on the other. Our structure therefore consists of two chains, each of which is the complement of the other. IV. EvipENcE In Favor OF THE COMPLEMENTARY MopEL The experimental evidence available to us now offers strong support to our | model though we should emphasize that, as yet, it has not been proved correct. | The evidence in its favor is of three types: (1) The general appearance of the X-ray picture strongly suggests that the | 200 THE STRUCTURE OF DNA basic structure is helical (Wilkins et al., 1953; Franklin and Gosling, 1953a). If we postulate that a helix is present, we immediately are able to deduce from the X-ray pattern of Structure B (Figure 3), that its pitch is 34 A and its diameter ap- proximately 20A. Moreover, the pattern suggests a high concentration of atoms on the circumference of the helix, in accord with our model which places the phosphate sugar backbone on the outside. The photograph also indicates that the two polynucleotide chains are not spaced equaily along the fiber axis, but are probably displaced from each other by about three-eighths of the fiber axis period, an inference again in qualitative agreement with our model. The interpretation of the X-ray pattern of Structure A (the crystalline form) is less obvious. This form does not give a meridional reflexion at 3.4 A, but instead (Figure 2) gives a series of reflexions around 25° off the meridian at spacings be- tween 3 A and 4A. This suggests to us that in this form the bases are no longer perpendicular to the fiber axis, but are tilted about 25° from the perpendicular position in a way that allows the fiber to contract 30 per cent and reduces the longitudinal translation of each nucleotide to about 2.5 A. It should be noted that the X-ray pattern of Structure A is much more detailed than that of Struc- ture B and so if correctly interpreted, can yield more precise information about DNA. Any proposed model for DNA must be capable of forming either Struc- ture A or Structure B and so it remains imperative for our very tentative interpre- tation of Structure A to be confirmed. (2) The anomolous titration curves of undegraded DNA with acids and bases strongly suggests that hydrogen bond formation is a characteristic aspect of DNA structure. When a solution of DNA is initially treated with acids or bases, no groups are titratable at first between pH 5 and pH 11.0, but outside these limits a rapid ionization occurs (Gulland and Jordan, 1947; Jordan, 1951). On back titration, however, either with acid from pH 12 or with alkali from pH 23, a dif- ferent titration curve is obtained indicating that the titratable groups are more accessible to acids and bases than is the untreated solution. Accompanying the initial release of groups at pH 11.5 and in the range pH 3.5 to pH 4.5 is a marked fall in the viscosity and the disappearance of strong flow birefringence. While this decrease was originally thought to be caused by a reversible depolymerization (Vilbrandt and Tennent, 1943), it has been shown by Gulland, Jordan and Taylor (1947) that this is unlikely as no increase was observed in the amount of sec- ondary phosphoryl groups. Instead these authors suggested that some of the groups of the bases formed hydrogen bonds between different bases. They were unable to decide whether the hydrogen bonds linked bases in the same or in adjacent structural units. The fact that most of the ionizable groups are orig- inally inaccessible to acids and bases is more easily explained if the hydrogen bonds are between bases within the same structural unit. This point would defi- nitely be established if it were shown that the shape of the initial titration curve was the same at very low DNA concentrations, when the interaction between neighboring structural units is small. (3) The analytical data on the relative proportion of the various bases show that the amount of adenine is close to that of thymine, and the amount of guanine 201 J. D. WATSON AND F. H.-C. CRICK close to the amount of cytosine + 5-methyl cytosine, although the ratio of ade- nine to guanine can vary from one source to another (Chargaff, 1951; Wyatt, 1952). In fact as the techniques for estimation of the bases improve, the ratios of adenine to thymine, and guanine to cytosine + 5-methyl cytosine appear to grow very close to unity. This is a most striking result, especially as the sequence of bases on a given chain is likely to be irregular, and suggests a structure involving paired bases. In fact, we believe the analytical data offer the most important evidence so far available in support of our model, since they specifically support the biologically interesting feature, the presence of complementary chains. We thus believe that the present experimental evidence justifies the working hypothesis that the essential features of our model are correct and allows us to consider its genetic possibilities. V. GENETICAL IMPLICATIONS OF THE COMPLEMENTARY MODEL As a preliminary we should state that the DNA fibers from which the X-ray diffraction patterns were obtained are not artifacts arising in the method of pre- paration. In the first place, Wilkins and his co-workers (see Wilkins et al., 1953) have shown that X-ray patterns similar to those from the isolated fibers can be obtained from certain intact biological materials such as sperm head and bacterio- phage particles. Secondly, our postulated model is so extremely specific that we find it impossible to believe that it could be formed during the isolation from living cells. A genetic material must in some way fulfil two functions. It must duplicate itself, and it must exert a highly specific influence on the cell. Our model for DNA suggests a simple mechanism for the first process, but at the moment we cannot see how it carries out the second one. We believe, however, that its speci- ficity is expressed by the precise sequence of the pairs of bases. The backbone of our model is highly regular, and the sequence is the only feature which can carry the genetical information. It should not be thought that because in our structure the bases are on the ‘‘inside,”’ they would be unable to come into contact with other molecules. Owing to the open nature of our structure they are in fact fairly accessible. A MeEcHANISM FoR DNA REPLICATION The complementary nature of our structure suggests how it duplicates itself. It is difficult to imagine how like attracts like, and it has been suggested (see Pauling and Delbriick, 1940; Friedrich-Freksa, 1940; and Muller, 1947) that self duplication may involve the union of each part with an opposite or comple- mentary part. In these discussions it has generally been suggested that protein and nucleic acid are complementary to each other and that self replication involves the alternate syntheses of these two components. We should like to propose instead that the specificity of DNA self replication is accomplished without recourse to specific protein synthesis and that each of our complementary DNA chains serves as a template or mould for the formation onto itself of a new com- panion chain. 202 THE STRUCTURE OF DNA For this to occur the hydrogen bonds linking the complementary chains must break and the two chains unwind and separate. It seems likely that the single chain (or the relevant part of it) might itself assume the helical form and serve as a mould onto which free nucleotides (strictly polynucleotide precursors) can attach themselves by forming hydrogen bonds. We propose that polymerization of the precursors to form a new chain only occurs if the resulting chain forms the proposed structure. This is plausible because steric reasons would not allow monomers “‘crystallized”’ onto the first chain to approach one another in such a way that they could be joined together in a new chain, unless they were those monomers which could fit into our structure. It is not obvious to us whether a special enzyme would be required to carry out the polymerization or whether the existing single helical chain could act effectively as an enzyme. DIFFICULTIES IN THE REPLICATION SCHEME While this scheme appears intriguing, it nevertheless raises a number of difficulties, none of which, however, do we regard as insuperable. The first difficulty is that our structure does not differentiate between cytosine and 5- methyl cytosine, and therefore during replication the specificity in sequence in- volving these bases would not be perpetuated. The amount of 5-methyl cytosine varies considerably from one species to another, though it is usually rather small or absent. The present experimental results (Wyatt, 1952) suggest that each species has a characteristic amount. They also show that the sum of the two cytosines is more nearly equal to the amount of guanine than is the amount of cytosine by itself. It may well be that the difference between the two cytosines is not functionally significant. This interpretation would be considerably strength- ened if it proved possible to change the amount of 5-methyl cytosine in the DNA of an organism without altering its genetical make-up. The occurrence of 5-hydroxy-methyl-cytosine in the T even phages (Wyatt and Cohen, 1952) presents no such difficulty, since it completely replaces cytosine, and its amount in the DNA is close to that of guanine. The second main objection to our scheme is that it completely ignores the role of the basic protamines and histones, proteins known to be combined with DNA in most living organisms. This was done for two reasons. Firstly, we can for- mulate a scheme of DNA reproduction involving it alone and so from the view- point of simplicity it seems better to believe (at least at present) that the genetic specificity is never passed through a protein intermediary. Secondly, we know almost nothing about the structural features of protamines and histones. Our only clue is the finding of Astbury (1947) and of Wilkins and Randall (1953) that the X-ray pattern of nucleoprotamine is very similar to that of DNA alone. This suggests that the protein component, or at least some of it, also assumes a helical form and in view of the very open nature of our model, we suspect that protein forms a third helical chain between the pair of polynucleotide chains (see Figure 4). As yet nothing is known about the function of the protein; perhaps it controls the coiling and uncoiling and perhaps it assists in holding the single polynucleotide chains in a helical configuration. 203 J. D. WATSON AND: PAH? C.CRICK The third difficulty involves the necessity for the two complementary chains to unwind in order to serve as a template for a new chain. This is a very funda- mental difficulty when the two chains are interlaced as in our model. The two main ways in which a pair of helices can be coiled together have been called plectonemic coiling and paranemic coiling. These terms have been used by cytologists to describe the coiling of chromosomes (Huskins, 1941; for a review see Manton, 1950). The type of coiling found in our model (see Figure 4) is called plectonemic. Paranemic coiling is found when two separate helices are brought to lie side by side and then pushed together so that their axes roughly coincide. Though one may start with two regular helices the process of pushing them together necessarily distorts them. It is impossible to have paranemic coiling with two regular simple helices going round the same axis. This point can only be clearly grasped by studying models. There is of course no difficulty in “‘unwinding”’ a single chain of DNA coiled into a helix, since a polynucleotide chain has so many single bonds about which rotation is possible. The difficulty occurs when one has a pair of simple helices with a common axis. The difficulty is a topological one and cannot be sur- mounted by simple manipulation. Apart from breaking the chains there are only two sorts of ways to separate two chains coiled plectonemically. In the first, one takes hold of one end of one chain, and the other end of the other, and simply pulls in the axial direction. The two chains slip over each other, and finish up separate and end to end. It seems to us highly unlikely that this occurs in this case, and we shall not consider it further. In the second way the two chains must be directly untwisted. When this has been done they are separate and side by side. The number of turns necessary to untwist them completely is equal to the number of turns of one of the chains round the common axis. For our structure this comes to one turn every 34 A, and thus about 150 turns per million molecular weight of DNA, that is per 5000 A of our structure. The problem of uncoiling falls into two parts: (1) How many turns must be made, and how is tangling avoided? (2) What are the physical or chemical forces which produce it? For the moment we shall be mainly discussing the first of these. It is not easy to decide what is the uninterrupted length of functionally active DNA. As a lower limit we may take the molecular weight of the DNA after isolation, say fifty thousand A in length and having about 1000 turns. This is only a lower limit as there is evidence suggesting a breakage of the DNA fiber during the process of extraction. The upper limit might be the total amount of DNA ina virus or in the case of a higher organism, the total amount of DNA in a chromo- some. For T2 this upper limit is approximately 800,000 A which corresponds to 20,000 turns, while in the higher organisms this upper limit may sometimes be 1000 fold higher. The difficulty might be more simple to resolve if successive parts of a chromo- some coiled in opposite directions. The most obvious way would be to have both right and left handed DNA helices in sequence but this seems unlikely as we have only been able to build our model in the right handed sense. Another 204 THE STRUCTURE OF DNA possibility might be that the long strands of right handed DNA are joined together by compensating strands of left handed polypeptide helices. The merits of this proposition are difficult to assess, but the fact that the phage DNA does not seem to be linked to protein makes it rather unattractive. The untwisting process would be less complicated if replication started at the ends as soon as the chains began to separate. This mechanism would produce a new two-strand structure without requiring at any time a free single-strand stage. In this way the danger of tangling would be considerably decreased as the two-strand structure is much more rigid than a single strand and would resist attempts to coil around its neighbors. Once the replicating process is started the presence, at the growing end of the pair, of double-stranded structures might facilitate the breaking of hydrogen bonds in the original unduplicated section and allow replication to proceed in a zipper-like fashion. It is also possible that one chain of a pair occasionally breaks under the strain of twisting. The polynucleotide chain remaining intact could then release the accumulated twist by rotation about single bonds and following this, the broken ends, being still in close proximity, might rejoin. It is clear that, in spite of the tentative suggestions we have just made, the difficulty of untwisting is a formidable one, and it is therefore worthwhile re- examining why we postulate plectonemic coiling, and not paranemic coiling in which the two helical threads are not intertwined, but merely in close apposition to each other. Our answer is that with paranemic coiling, the specific pairing of bases would not allow the successive residues of each helix to be in equivalent orientation with regard to the helical axis. This is a possibility we strongly oppose as it implies that a large number of stereochemical alternatives for the sugar-phosphate backbone are possible, an inference at variance to our finding, with stereochemical models (Crick and Watson, 1953) that the position of the sugar-phosphate group is rather restrictive and cannot be subject to the large variability necessary for paranemic coiling. Moreover, such a model would not lead to specific pairing of the bases, since this only follows if the glucosidic links are arranged regularly in space. We therefore believe that if a helical structure is present, the relationship between the helices will be plectonemic. We should ask, however, whether there might not be another complementary structure which maintains the necessary regularity but which is not helical. One such structure can, in fact, be imagined. It would consist of a ribbon-like arrangement in which again the two chains are joined together by specific pairs of bases, located 3.4 A above each other, but in which the sugar-phosphate backbone instead of forming a helix, runs in a straight line at an angle approx- imately 30° off the line formed by the pair of bases. While this ribbon-like structure would give many of the features of the X-ray diagram of Structure B, we are unable to define precisely how it should pack in a macroscopic fiber, and why in particular it should give a strong equatorial reflexion at 20-24 A. We are thus not enthusiastic about this model though we should emphasize that it has not yet been disproved. Independent of the details of our model, there are two geometrical problems 205 J. D. WATSON AND F. H. C. CRICK ADENINE THY MINE ADENINE CYTOSINE Ficure 7. Pairing arrangements of adenine before (above) and after (below) it has undergone a tautomeric shift. which any model for DNA must face. Both involve the necessity for some form of super folding process and can be illustrated with bacteriophage. Firstly, the total length of the DNA within T2 is about 8 X 10°A. As its DNA is thought (Siegal and Singer, 1953) to have the same very large M. W. as that from other sources, it must bend back and forth many times in order to fit into the phage head of diameter 800 A. Secondly, the DNA must replicate itself without getting tangled. Approximately 500 phage particles can be synthesized within a single bacterium of average dimensions 104 & 104 K 2 X 104 A. The total length of the newly produced DNA is some 4 X 108 A, all of which we believe was at some interval in contact with its parental template. Whatever the precise mechanism of replication we suspect the most reasonable way to avoid tangling is to have the DNA fold up into a compact bundle as it is formed. A PossiBLE MECHANISM FOR NATURAL MuTATION In our duplication scheme, the specificity of replication is achieved by means of specific pairing between purine and pyrimidine bases; adenine with thymine, 206 THE STRUCTURE OF DNA and guanine with one of the cytosines. This specificity results from our assump- tion that each of the bases possesses one tautomeric form which is very much more stable than any of the other possibilities. The fact that a compound is tautomeric, however, means that the hydrogen atoms can occasionally change their locations. It seems plausible to us that a spontaneous mutation, which as implied earlier we imagine to be a change in the sequence of bases, is due to a base occurring very occasionally in one of the less likely tautomeric forms, at the moment when the complementary chain is being formed. For example, while adenine will normally pair with thymine, if there is a tautomeric shift of one of its hydrogen atoms it can pair with cytosine (Figure 7). The next time pairing occurs, the adenine (having resumed its more usual tautomeric form) will pair with thymine, but the cytosine will pair with guanine, and so a change in the sequence of bases will have occurred. It would be of interest to know the precise difference in free energy between the various tautomeric forms under physiological conditions. GENERAL CONCLUSION The proof or disproof of our structure will have to come from further crystal- lographic analysis, a task we hope will be accomplished soon. It would be surprising to us, however, if the idea of complementary chains turns out to be wrong. This feature was initially postulated by us to account for the crystal- lographic regularity and it seems to us unlikely that its obvious connection with self replication is a matter of chance. On the other hand the plectonemic coiling is, superficially at least, biologically unattractive and so demands precise crystallographic proof. In any case the evidence for both the model and the suggested replication scheme will be strengthened if it can be shown unambig- uously that the genetic specificity is carried by DNA alone, and, on the molecular side, how the structure could exert a specific influence on the cell. REFERENCES Astpury, W. T., 1947, X-Ray Studies of nucleic acids in tissues. Sym. Soc. Exp. Biol. 1: 66-76. CuaraaFr, E., 1951, Structure and function of nucleic acids as cell constituents. Fed. Proc. 10: 654-659. Crick, F. H. C., and Watson, J. D., 1953, Manuscript in preparation. FRANKLIN, R. E., and Gosuine, R., 1953a, Molecular configuration in sodium thymo- nucleate. Nature, Lond. 171: 740-741. 1953b, Fiber diagrams of sodium thymonucleate. I. The influence of water content. Acta Cryst., Camb. (in press). 1953c, The structure of sodium thymonucleate fibers. Il. The cylindrically symmetri- cal Patterson Function. Acta Cryst., Camb. (in press). Fraser, M. 8., and Fraser, R. D. B., 1951, Evidence on the structure of desoxyribo- nucleic acid from measurements with polarized infra-red radiation. Nature, Lond. 167: 760-761. Friepricu-Frexksa, H., 1940, Bei der Chromosomen Konjugation wirksame Krafte und ihre Bedeutung fiir die identische Verdoppling von Nucleoproteinen. Naturwissen- shaften 28: 376-379. 207 J. D. WATSON AND Fae C..CRICK GULLAND, J. M., and Jorpan, D. O., 1946, The macromolecular behavior of nucleic acids. Sym. Soc. Exp. Biol. 1: 56-65. GuLuanp, J. M., Jorpan, D. O., and Taytor, H. F. W., 1947, Electrometric titration of the acidic and basic groups of the desoxypentose nucleic acid of calf thymus. J. Chem. Soc. 1131-1141. Huskins, C. L., 1941, The coiling of chromonemata. Cold Spr. Harb. Symp. Quant. Biol. 9: 13-18. JorDAN, D. O., 1951, Physiochemical properties of the nucleic acids. Prog. Biophys. 2: 51-89. Kanter, H., and Luoyp, B. J., 1953, The electron microscopy of sodium desoxyribo- nucleate. Biochim. Biophys. Acta 10: 355-359. Manton, I., 1950, The spiral structure of chromosomes. Biol. Rev. 25: 486-508. MuLteER, H. J., 1947, The Gene. Proc. Roy. Soc. Lond. Ser. B. 134: 1-37. Pav.ine, L., and DeLBrivcx, M., 1940, The nature of the intermolecular forces operative in biological processes. Science 92: 77-79 Srecat, A., and Sincer, 8. J., 1953, The preparation and properties of desoxypen- tosenucleic acid. Biochim. Biophys. Acta 10: 311-319. VILBRANDT, C. F., and Tennent, H. G., 1943, The effect of pH changes upon some properties of sodium thymonucleate solutions. J. Amer. Chem. Soc. 63: 1806-1809. Watson, J. D., and Crick, F. H. C., 1953a, A structure for desoxyribose nucleic acids. Nature, Lond. 171: 737-738. 1953b, Genetical implications of the structure of desoxyribose nucleic acid. Nature, Lond. (in press). Wiuxins, M. H. F., Gostine, R. G., and SEeps, W. E., 1951, Physical studies of nucleic acids —- nucleic acid: an extensible molecule. Nature, Lond. 167: 759-760. Witxins, M. H. F., and Ranpatu, J. T., 1953, Crystallinity in sperm-heads: molecular structure of nucleoprotein in vivo. Biochim. Biophys. Acta 10: 192 (1953). Witkins, M. H. F., Sroxss, A. R., and Witson, H. R., 1953, Molecular structure of desoxypentose nucleic acids. Nature, Lond. 171: 738-740. WituiaMs, R. C., 1952, Electron microscopy of sodium desoxyribonucleate by use of a new freeze-drying method. Biochim. Biophys. Acta 9: 237-239. Wyatt, G. R., 1952, Specificity in the composition of nucleic acids. In “The Chemistry and Physiology of the Nucleus,” pp. 201-213, N. Y. Academic Press. Wyatt, G. R., and Conen, 8. 8., 1952, A new pyrimidine base from bacteriophage nucleic acid. Nature, Lond. 170: 1072. 208 FINE STRUCTURE OF A GENETIC REGION IN BACTERIOPHAGE By SEYMOUR BENZER BIOPHYSICAL LABORATORY, DEPARTMENTS OF BIOLOGICAL SCIENCES AND PHYSICS, PURDUE UNIVERSITY, LAFAYETTE, INDIANA Communicated by M. Delbriick, April 6, 1955 This paper describes a functionally related region in the genetic material of a bacteriophage that is finely subdivisible by mutation and by genetic recombination. The group of mutants resembles similar cases which have been observed in many organisms, usually designated as ‘‘pseudo-alleles.”” (See reviews by Lewis! and Reprinted by permission of the author and the National Academy of Sciences from the PROCEEDINGS OF THE NATIONAL ACADEMY OF ScrENcES, 41 (6), 344-354, June, 1955. 209 Vou. 41, 1955 GENETICS: S. BENZER 345 Pontecorvo.”) Such cases are of special interest for their bearing on the structure and function of genetic determinants. The phenomenon of genetic recombination provides a powerful tool for separating mutations and discerning their positions along a chromosome. When it comes to very closely neighboring mutations, a difficulty arises, since the closer two mutations lie to one another, the smaller is the probability that recombination between them will occur. Therefore, failure to observe recombinant types among a finite number of progeny ordinarily does not justify the conclusion that the two mutations are inseparable but can only place an upper limit on the linkage distance between them. A high degree of resolution requires the examination of very many prog- eny. This can best be achieved if there is available a selective feature for the de- tection of small proportions of recombinants. Such a feature is offered by the case of the rII mutants of T4 bacteriophage described in this paper. The wild-type phage produces plaques on either of two bacterial hosts, B or K, while a mutant of the rII group produces plaques only on B. Therefore, if a cross is made between two different rII mutants, any wild-type recombinants which arise, even in proportions as low as 107%, can be detected by plating on K. This great sensitivity prompts the question of how closely the attainable resolu- tion approaches the molecular limits of the genetic material. From the experi- ments of Hershey and Chase,’ it appears practically certain that the genetic infor- mation of phage is carried in its DNA. The amount of DNA in a particle of phage T2 has been determined by Hershey, Dixon, and Chase* to be 4 X 10° nucleotides. The amount for T4 is similar.’ If we accept the model of DNA structure proposed by Watson and Crick,® consisting of two paired nucleotide chains, this corresponds to a total length of DNA per T4 particle of 2 & 10° nucleotide pairs. We wish to translate linkage distances, as derived from genetic recombination experiments, into molecular units. This cannot be done very precisely at present. It is not known whether all the DNA in a phage particle is indispensable genetic material. Nor is it known whether a phage ‘“‘chromosome’”’ (i.e., the physical counterpart of a linkage group identified by genetic means) is composed of a single (duplex) DNA fiber or whether genetic recombination is equally probable in all chromosomal regions. For the purpose of a rough calculation, however, these notions will be assumed to be true. Thus we place the total linkage map of T4 in correspondence with 2 & 10° nucleotide pairs of DNA. The total known length of the three linkage groups’ in phage T4 amounts to some 100 units (one unit = 1 per cent recombination in a standard cross). In addition, there is evidence® for roughly another 100 units of length connecting two of the groups. Therefore, if we assume 200 recombination units to correspond to 2 X 10° nucleotide pairs, the recombination per nucleotide pair is 10~% per cent. That is to say, given two phage mutants whose mutations are localized in their chromosomes at sites only one nucleotide pair apart, a cross between these mutants should give rise to a progeny population in which one par- ticle in 10° results from recombination between the mutations (provided, of course, that recombination is possible between adjacent nucleotide pairs). This compu- tation is an exceedingly rough one and is only intended to indicate the order of mag- nitude of the scale factor. Some preliminary results are here presented of a pro- gram designed to extend genetic studies to the molecular (nucleotide) level. 210 346 GENETICS: S. BENZER Proc. N. A.S. r Mutants.—The wild-type phages T2, T4, and T6 produce small plaques with rough edges when plated on strain B of Escherichia coli. From sectors of clearing in these plaques, mutants can be readily isolated which produce large, sharp-edged plaques (Hershey). These mutants have been designated ‘‘r’’ for rapid lysis; they differ from the wild type by a failure to cause ‘‘lysis inhibition” on strain B (Doermann”). The wild type has a selective advantage over r mutants when the two types grow together on B. The genetics of r mutants was studied by Hershey and Rotman,!! who found three regions in the linkage map of T2 in which various mutations causing the r phenotype were located, including one large ‘‘cluster’”’ of mutants which were shown to be genetically distinct from one another. The genetic study of T4 by Doermann and Hill’ showed r regions corresponding to two of those in T2. T6 also has at least two such r regions. The rll Group.—For all three phages, T2, T4, and T6, the r mutants can be sep- arated into groups on the basis of their behavior on strains other than B. This paper will be concerned only with one group, which will be called the ‘“rII group.” Mutants of the rII group are distinguished from those of other groups, and from wild type, by a failure to produce plaques on certain lysogenic strains!? of E. coli which carry phage A. As shown in Table 1, a mutant of the rII group produces TABLE 1 PHENOTYPES (PLAQUE MorpHo.ocy) or T4 WILD AND rII Mutant PLATED ON Various Hosts Host STRAIN E. coli E. colt E. coli B K128 K128 (A) T4 wild type Wild Wild Wild T4rlII mutant r Type Wild r-type plaques on strain B, wild-type plaques on strain K12S (nonlysogenic strain sensitive to X), and no plaques on K12S (A) (derived from K12S by lysogenization with A). The wild-type phage produces similar plaques on all three strains. In the case of T4, with which we shall be concerned in this paper, the efficiencies of plating are approximately equal on the three strains, except, of course, for rII on K128 (A). The three bacterial strains will be here designated as “B,” “S,” and SKE 72 . Approximately two-thirds of the independently arising r mutants isolated on B are of the rII type. This group includes the ‘cluster’? of r mutants of T2 de- scribed by Hershey and Rotman and the r47 and r51 mutants described by Doer- mann and Hill in the corresponding map region of T4 but does not include r mu- tants located outside that region. Similarly, all newly isolated mutants showing the rII character have turned out to fall within the same region, as indicated in Figure 1. The properties of the rII group are especially favorable for detailed genetic study. An rII mutant has three different phenotypes on the three host strains (Table 1): (1) altered plaque morphology on B, (2) indistinguishable from wild type on §, and (3) unable to produce plaques on K. These properties are all useful. By virtue of their altered plaque type on B, r mutants are readily isolated, and those of the rII group are identified by testing on K. Where it is desired to avoid a selective disadvantage compared with wild type, e.g., in measuring mutation 211 Vou. 41, 1955 GENETICS: S. BENZER 347 rates, S can be used as a nondiscriminating host. The failure of rll mutants to plate on K enables one to detect very small proportions of wild-type particles due to reversion or due to recombination between different riI mutants. ? “10% rit region Fic. 1.—Partial linkage map of T4 (Doermann), indicating the location of the rII region. m and tu designate ‘‘minute plaque” and “‘turbid plaque’’ mutations. The circular inset shows, diagrammatically, the corresponding dimensions of the DNA chain magnified 1,000 diameters. Fate of rll Mutants in K.—Wild-type and rII mutants adsorb equally well to strains S and K. Whereas the wild type provokes lysis and liberation of a burst of progeny on both strains, the rII mutant grows normally only on 8. Infection of K with an rII mutant provokes very little (and/or very late) lysis, although all in- fected cells are killed. The block in growth of rII mutant is associated with the presence of the carried phage }. The reason for this association is unknown. Quantitative Differences in Phenotype-——While all rII mutants show the same phenotypic effect of poor multiplication on K, they differ in the degree of this effect. A certain proportion of K infected with rII actually liberates some prog- eny, which can be detected by plating the infected cells on B. The fraction of infected cells yielding progeny defines a ‘‘transmission coefficient’’ characteristic of the mutant. The transmission coefficient is insensitive to the multiplicity of in- fection but depends strongly upon the physiological state of the bacteria (K) and upon temperature. Under given conditions, however, the coefficient can be used as a comparative index of degree of phenotypic effect, a “leaky”’ mutant having a high coefficient. As can be seen in Table 2, a wide range of values is found. TABLE 2 PROPERTIES OF T4 MUTANTS OF THE rII Group* Mutant Map Transmission Reversion Number Position Coefficient Index (units of 10) r47 0 0.03 <0.01 r104 is .91 "| Hi no reversion detected Cc Fic. 2.—Genetic maps showing the locations of spontaneous and 5-bromouracil-induced mutational alterations in the rII region of phage T4 divided into cistron A and B. Each mutant is represented by a box, placed in the proper segment of the map and shaded to indicate its re- version index. The relative order (but not the lengths) of segments 1, 2, 3, 4, 5, 6, 8, and 10 have been established. Segments 7, 9, and 11, are lumped together and considered as one segment. The order and position of different mutations within one segment have not been determined. they were leaky. The results of mapping the remaining 67 mutants are shown in Figure 2. This set of mutants reveals ‘5-bromouracil hot spots” that are located at different positions from the spontaneous ones. It must be emphasized that the vertical scales in Figure 2 are very different for the two sets of mutants. The over-all induced mutation rate was several hundred- 224 Vou. 44, 1958 BIOCHEMISTRY: BENZER AND FREESE Wil7s fold higher, and only half as many induced mutants are mapped; therefore, the occurrence of an induced mutant once represented in the induced set corresponds to roughly 10° times higher mutability than the occurrence of a spontaneous mutant once represented in the spontaneous set. Hence, for those “hot spots” in the induced set for which no spontaneous mutation has been observed, the mutability with bromouracil is at least 104 times larger than is the spontaneous mutability. Reverse Mutations.—Among the spontaneous rII mutants, not only are forward mutation frequencies different for different sites, but the reversion indexes as well cover an enormous range. Among the 132 spontaneous rII mutants, reversion in- dexes range as high as 5 X 10~? down to less than 10-°. For 19 of them, reversion has not been detected. The induced mutants, on the other hand, are more homogeneous with respect to reversion index. There is only one mutant of very high reversion index (N 76) and two non-reverting mutants (N 101, N 32). Most of the remainder have reversion indexes of the order of 10-*. Notably rare are mutants containing larger aberra- tions (i.e., ones that fail to give standard-type recombinants with two or more other mutants that do themselves show recombination). Some preliminary experiments have been performed on the induction of reversion of various mutants with 5-bromouracil. For some mutants, in particular, chosen from some of the ‘5-bromouracil hot spots,” increases over the spontaneous rate of reversion by factors as high as 104 have been observed. Some spontaneous mutants show no or only much smaller effects. These experiments are still in progress. DISCUSSION Clearly, the mutagenic effect of 5-bromouracil is not merely a general enhance- ment of spontaneously occurring mutations. Rather, it is a specific effect. In de- noting the action of a mutagen as specific, one might be comparing the effects on different phenotypic characteristics in the whole organism, different cistrons, or different locations within a single cistron. Were it not for the high resolution of our genetic techniques, it might have been erroneously concluded that 5-bromouracil acts aspecifically, since, among all the induced mutants of the r phenotype, the proportion of rII mutants and even the ratio of A cistron to B cistron mutants are quite comparable to the spontaneous values. It is only at finer resolution that the different effects are revealed, and it is seen that mutations are, for the most part, induced at specific places in the genetic structure. Since the proportion of large aberrations and non-reverting mutants is notably smaller among the 5-bromouracil-induced mutations, the induced changes in the genetic structure appear to involve small molecular substitutions rather than large changes of the genome. Further, the more homogeneous properties of the induced mutants with respect to reversion rates indicate that a certain class of molecular transitions may be involved here. Under the conditions used, the total frequency of occurrence of all rII mutants in d-bromouracil was raised several hundred fold above the spontaneous rate. The increase in rate at specific locations in the genetic structure was much larger. For example, 11 occurrences of mutation at one site were observed out of 67 induced rII mutants, while none occurred at that site among twice as many spontaneous rII 225 118 BIOCHEMISTRY: BENZER AND FREESE Proc. N2 Aisi mutants. Thus the probability of mutation at that point, per replication of the phage, was raised by a factor greater than twice eleven times several hundred, or of the order of 10%. Conversely, note the site at which there were 19 occurrences among 132 spon- taneous rll mutants, while among the 67 induced mutants none were observed. Therefore, 5-bromouracil had little, if any, positive mutagenic effect at that point. The disappearance of the spontaneous “hot spot’? does not, of course, mean that spontaneous mutations at that point were suppressed but simply that they were not increased in proportion to other mutations. Only three sites (indicated by dotted lines in the figure) are represented in both sets of mutants. In each of those cases, there was only a single occurrence in one of the sets, so that the ratio of induced to spontaneous frequencies cannot be re- liably computed. The mutation rate seems to have been truly increased by 5- bromouracil in 2 of the cases (2 and 7 occurrences in the induced set) because one occurrence among the induced series represents a thousandfold greater probability of mutation. In the third case, there is, in the induced set, a single occurrence of a highly revertable mutant that appearaed nine times in the spontaneous set. This single event probably belongs to the small background of spontaneous mutations among the induced ones (ca | or 2 in 100). The methods used in the isolation of the two sets of mutants were quite different, and one cannot be sure whether factors other than the addition of 5-bromouracil could have affected the results. Although sulfanilamide alone does not appreciably increase the total mutation rate, the possibility that the distribution of mutations might change has not been excluded. Sulfanilamide, as an analogue of p-amino benzoic acid, inhibits the formation of folic acid, thereby inhibiting nearly all the methylation and hydroxymethylation steps (Cohen and Barner)."* In our sul- fanilamide medium, nearly all the major chemicals containing the methyl or hy- droxymethyl group are added except the deoxyribonucleotides of thymine and 5- hydroxymethyleytosine, which thus are expected to be deficient. The deficiency in thymine facilitates the incorporation of 5-bromouracil into DNA. Whether the deficiency in 5-hydroxymethyleytosine enhances the probability of the false incorporation of 5-bromouracil into a hydroxymethyleytosine site of the phage DNA remains to be seen. One is not in a position, from these experiments alone, to reach a clear conclusion as to the molecular mechanism of mutagenesis; thus, at this stage, it cannot be de- cided whether or not the tautomeric shift of the DNA bases from the keto- to the enol- form is mainly responsible for the production of point mutations, as suggested by Watson and Crick.! If it is assumed that only two kinds of nucleotide pairs are present in DNA (i.e., adenine—thymine, and guanine—5-hydroxymethylcytosine), the existance of hot spots of spontaneous mutation and the appearance of different hot spots with 5-bromouracil would suggest that not every nucleotide pair of a given type mutates with the same probability. Rather, the mutability of a nu- cleotide pair would have to depend upon its position. In any case, the striking results of this preliminary investigation indicate that it would be fruitful to pursue this line of investigation, using a range of mutagenic substances in systems where the chemical events are under proper control. 226 Vou. 44, 1958 BIOCHEMISTRY: BENZER AND FREESE 119 SUMMARY A set of spontaneously arising rII mutants of phage T4 is compared with a set of mutants induced by the action of 5-bromouracil. When analyzed by genetic mapping techniques of high resolution, the two sets of mutants are found to be quite different. The mutagen does not merely enhance the over-all mutation rate but acts at specific locations in the hereditary structure. The induced mutants are mostly of the nature of small, revertible alterations rather than gross defects. The reversion rates of induced mutants are less varied than those of spontaneous mutants, indicating that a certain class of molecular transition is involved. These preliminary results encourage the hope that this sort of genetic analysis can lead toward an understanding of the mechanism of mutation and the identifica- tion of the specific chemical configurations composing the genetic structure. Note: The occurrence at a given location of one mutation in the induced set corresponds to roughly 103 times higher mutability of this spot in 5-bromouracil than is indicated by the occurrence of one mutation in the spontaneous set. * Aided by grants from the National Science Foundation and the American Cancer Society + Present address: Medical Research Council for Molecular Biology, Cavendish Laboratory, Cambridge University, England. t Research Fellow of the Damon Runyon Memorial Fund for Cancer Research. Present address: Biological Laboratories, Harvard University, Cambridge, Massachusetts. 1 J. D. Watson, and F. H. C. Crick, Cold Spring Harbor Symposia Quant. Biol., 18, 123, 1953. 2S. Benzer, in The Chemical Basis of Heredity, ed. McElroy and Glass (Baltimore: Johns Hop- kins Press, 1957). 3H. J. Muller, Sczence, 66, 84, 1927. 41. Stadler, Science, 68, 1928. 5 C. Auerbach and J. M. Robson, Nature, 157, 302, 1946. 6 F. Oehlkers, Z. Ind. Abst. u. Vererbungsl., 81, 313, 1943. 7M. Demerec, Caryologia, Vol. suppl. 201, 1954. 8M. Westergaard, Experientia, 13, 224, 1957. 9R. Latarjet, Compt. rend., 228, 1345, 1948. 10 J. J. Weigle, these PROCBEDINGs, 39, 628, 1953. 1J,, Silvestri, Bull. 7.S.M., 28, 193, 1949. 12B, Fernandez, F. L. Haas, and O. Wyss, these PRocEDDINGs, 39, 1052, 1953. 13R. de Mars, Nature, 172, 964, 1953. 147). B. Dunn and J. D. Smith, Nature, 174, 304, 1954. 18 R, M. Litman, and A. B. Pardee, Nature, 178, 529, 1956. 1 R. M. Herriott, and J. L. Barlow, J. Gen. Physiol., 36, 17, 1952. 118. Benzer, these PROCEEDINGS, 41, 344, 1955. 18 Tn order to minimize the background of r mutants present in the stock of standard type, which is usually around 2 X 10~4, several stocks of T4 were grown, and one with a small proportion of r mutants was selected. In the stock used, this proportion was less than 1074. 19§.S. Cohen and H. D. Barner, J. Bacteriol., 71, 588, 1956. 227 EXPERIMENTS ON PHOTOREACTIVATION OF BACTERIOPHAGES INACTIVATED WITH ULTRAVIOLET RADIATION! R. DULBECCO Department of Bacteriology, Indiana University, Bloomington, Indiana? Received for publication October 24, 1949 Kelner (1949), working with conidia of Streptomyces griseus, discovered that light belonging to the visible range is capable of reactivating biological ma- terial that has been rendered inactive by ultraviolet radiation (UV). Shortly after Kelner’s discovery was known, a similar phenomenon in bacteriophages (bacterial viruses) was observed by accident. Plates of nutrient agar containing UV-inactivated phage and sensitive bacteria had been left for several hours on a table illuminated by a fluorescent lamp. After incubation it was noticed that the number of plaques was higher on these plates than on similar plates incu- bated in darkness. A short report of this phenomenon of “‘photoreactivation” (PHTR) has already been published (Dulbecco, 1949). The present paper con- tains the results of a first group of experiments concerning PHTR of seven bac- teriophages of the T group active on Escherichia colz, strain B. MATERIALS AND METHODS Stocks of each phage were prepared by inoculating material from a single plaque into a culture of H. coli B in a synthetic medium M9,? except for phage T5, of which a stock in Difco nutrient broth was used. In some experiments the phage was purified by two or three steps of differential centrifugation ; the phage was resuspended in M/15 phosphate buffer pH 7, with MgSO, added to a con- centration 10-3 m. Unless otherwise specified, the experiments described in this paper were performed with phage T2. Escherichia colt, strain B, was used through- out. In some experiments bacteria were grown in nutrient broth with aeration and the culture was infected with phage when it was in the logarithmic phase of growth (about 108 cells per ml); these bacteria will be referred to as ‘‘bac- teria in broth.” In other experiments bacteria were grown in broth up to a con- centration of about 2 X 108 cells per ml, then washed with saline (0.85 per cent NaCl) and resuspended in saline, kept at 37 C for 30 minutes, and then infected ; these bacteria will be referred to as ‘resting bacteria.” 1'This work was done under an American Cancer Society grant, recommended by the Committee on Growth of the National Research Council, under the direction of Dr. S. E. Luria. The author wishes to express his appreciation to Dr. Luria for facilitating this work materially and for numerous discussions during its progress. The manuscript was completed at the California Institute of Technology. The author also wishes to acknowledge his in- debtedness to Dr. M. Delbriick for helpful discussions on the interpretation of the data. 2 Present address: Kerckhoff Laboratories of Biology, California Institute of Technology, Pasadena 4, California. 3 NH,Cl,1.0 g; KH2POu,, 3.0 g; Na2HPO,,6.0 g; NaCl,0.5 g; MgS0O,,0.1 g; distilled water, 1,000 ml; 4 g per liter glucose added after separate sterilization. Reprinted by permission of the author and the Williams and Wilkins Co., from the JouRNAL OF BacTERIOLOGY, 59 (3), 329-347, March, 1950. 228 330 R. DULBECCO [vou. 59 Inactivation of the phages was accomplished with a low-pressure mercury discharge lamp (General Electric ‘‘germicidal” lamp, 15 watts), giving most of the UV energy in the line 2,537 A. The output of the lamp was kept constant by alimenting it through a “sola” stabilizer and by using it only after it had been burning for at least 20 minutes. The stocks to be irradiated were diluted in phosphate buffer plus MgSO, and exposed to the lamp at a 20-inch distance either in an open petri dish with con- tinuous shaking (3 ml of phage in a 10-cm petri dish) or in a quartz cell 2 mm thick with parallel faces. Relative measurements of the incident UV doses were made in some experiments by timing the exposure; in other experiments rela- adsorbed UVP. Light Condenser Figure 1. Diagram of the apparatus employed for illumination in liquid. tive and absolute measurements were conducted with a calibrated Westinghouse SM-200 meter with tantalum photocell WL-775. A dose of UV will be expressed as seconds of exposure to the germicidal lamp. The reactivating light was used in two different ways: Illumination on the plate. The plates, prepared by the agar layer method (Gratia, 1936; Hershey eé al., 1943), were exposed right side up to the light of two parallel fluorescent discharge lamps, 40 watts each, at a distance of 12 inches at room temperature. Illumination in liquid. The apparatus used is illustrated in figure 1. A mercury discharge lamp, medium pressure (General Electric H-5 lamp, 250 watts) was used as the light source. The light was condensed through a spherical pyrex flask 229 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 331 filled with distilled water and passed through suitable filters (see later section) ; for white light experiments infrared rays were absorbed by a filter of CuSO,- 5H.O (5 per cent in water, 1 inch thick) and ultraviolet rays shorter than 330 mu by a Corning glass filter no. 738. A mixture of phage and bacteria was ex- posed to light in a small beaker (5 ml of mixture in a beaker 4 cm in diameter) kept in a thermostatically regulated water bath and shaken by a reciprocating motion in a horizontal plane to ensure uniform distribution of the material and uniform illumination. Some experiments were done with a 100-watt General Electric H-4 lamp without a condenser. In the experiments with illumination in liquid the ratio “phage particles: bac- teria’? was kept very low (about 10-*) to decrease the probability of multiple infection of bacteria and the occurrence of reactivation by multiplicity (Luria, 1947). EXPERIMENTAL RESULTS Role of the Bacteria in the PHTR of Inactive Phage Phage particles inactivated by UV (UVP) can be reactivated by light only if the particles are mixed with sensitive bacteria during illumination. []lumination of UVP alone is without effect, as is shown by the following experiment: Phage T2 was irradiated with the germicidal lamp for 30 seconds (dark survival = 2X 10-5) and divided into two equal samples. The first sample was immediately plated and incubated in darkness; the second one was exposed to the light of a fluorescent lamp (80 watts at a 12-inch distance) for 1 hour at room temper- ature and then divided into two parts, one of which was plated and incubated in darkness, the other under the same light. The sum of plaque counts of two plates for each sample are given in table 1 (1). In another similar experiment the UVP was first spread on the surface of a nutrient agar plate and then exposed to the light; after illumination sensitive bacteria were spread on the same plate in darkness. In this condition also PHTR was not produced. These experiments clearly indicate that illumination of UVP in the absence of bacteria has no reactivating effect; they do not show, however, whether PHTR occurs only for adsorbed phage or also for nonadsorbed phage in the presence of bacteria. This point was investigated by mixing UVP with bacteria in nutrient broth without added NaCl (under these conditions the adsorption is slight), illuminating the mixture, and testing for reactivation of the nonadsorbed phage particles. A sample of phage irradiated with the germicidal lamp for 30 seconds was mixed with a culture of bacteria in broth without added NaCl, containing 10° cells per ml. The mixture was exposed to the light of an H-4 lamp at a 6- inch distance for 10 minutes at 28 C, then centrifuged; samples from the super- natant were plated and incubated both in darkness and in the light. The plaque counts (two plates for each sample) are given in table 1 (II), together with an assay of the irradiated phage diluted in broth by a factor equal to the one used in the experiment. The result of this experiment clearly indicates that the un- adsorbed phage particles are not reactivated by light. 230 332 R. DULBECCO [voL. 59 Illumination of bacteria alone followed by the addition of UVP does not pro- duce any PHTR. Bacteria spread on the surface of several nutrient-agar plates were exposed to the light of a fluorescent lamp (80 watts, 12-inch distance) for 4 hours at room temperature; then UVP were spread on the same plates in dark- ness, and the plates were incubated in darkness. Control plates, spread with bacteria at the same time, were kept in darkness and received UVP at the same time as the illuminated plates. Equal numbers of plaques were found in all plates whether the bacteria had been preilluminated or not, showing that preillumina- tion of bacteria does not cause PHTR of UVP added later. In another experi- ment a suspension of resting bacteria was illuminated with a light of 365-mp wave length at 37 C for a period long enough to give a very high PHTR in ad- TABLE 1 Effect of light on inactivated phage T2 alone and on unadsorbed inactivated phage T2r mixed with sensitive bacteria PLAQUE EXPERIMENT TREATMENT COUNT 0.1 ML I. Illumination of UVP alone 1. UVP not illuminated and plated with 17 B. Plates incubated in darkness. 2. UVP illuminated alone and plated 6 with B. Plates incubated in darkness. 3. UVP illuminated alone and plated 609 with B. Plates incubated under light. II. Effect of light on unadsorbed | 1. UVP alone. 72 phage in presence of bacteria 2. UVP mixed with B in saltless broth; 86 illuminated 10 minutes; centrifuged; supernatant plated with B; plates in- cubated in darkness. 3. Same as II, 2, but with plates in- | >1,000 cubated under light. sorbed UVP, and the UVP was added at the very moment at which the light was turned off; no measurable PHTR was observed. If bacteria killed by heating to 60 C for 20 minutes are substituted for living bacteria, no PHTR takes place. Actually the plaque count decreases, probably because of an irreversible adsorption of phage by the dead bacteria without the release of new phage. Illumination of bacteria prior to infection does not diminish the photoreac- tivability of UVP added later, as shown by the following experiment: Bacteria were spread on the surface of nutrient agar plates and exposed to the light of a fluorescent lamp (80 watts, 12-inch distance) for 4 hours at room temperature; then UVP was spread on the plates, which were afterwards incubated under the same light. After incubation the plates showed the same number of plaques as control plates containing nonpreilluminated bacteria and UVP, incubated under the same light. 231 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 333 From these experiments with phage T2 one may conclude that PHTR occurs only for UVP adsorbed on sensitive bacteria and that illumination either of UVP or of bacteria before infection has no detectable effect. To test how soon after phage adsorption PHTR can occur, UVP and bacteria were mixed on several plates, and the plates were immediately exposed to the light of an H-4 lamp at an 8-inch distance at 28 C. The exposure was continued for 10, 20, 30, or 50 seconds. The plaque count was found to increase even after 10 seconds, showing that no measurable delay exists between adsorption and the beginning of PHTR and that PHTR has no measurable latent period. Action of Bacterial Extracts on PHTR Some attempts were made to obtain PHTR by illuminating mixtures of UVP with cell-free bacterial extracts. Bacteria were grown in nutrient broth to a concentration of about 5 X 108 cells per ml and harvested in a Sharples centri- fuge. Two extraction procedures were used: (a) the thick bacterial suspension was frozen at —30 C, the frozen paste was then ground with carborundum pow- der and extracted with phosphate buffer (pH 7.5) for about 10 minutes, and the extract was clarified by centrifugation; (b) the bacteria were broken in a sonic vibrator after the bacterial paste was diluted with an equal volume of phosphate buffer, and the extract was clarified in the centrifuge. In both cases the super- natant was a thick, yellowish liquid, which showed a high degree of enzymatic activity (methylene blue reduction, tryptophanase). Both extracts still con- tained a few living cells, which could be eliminated either by filtration or by repeated freezing at —30 C. UVP was mixed into various dilutions of the extracts, and the mixtures were kept either in light or darkness and assayed for active phages at different times. Only extracts still containing living cells gave some PHTR. Removal of almost all living cells eliminated PHTR. PHTR as a Function of the Dose of the Inactivating UV Light For several phages of the T group (T2, T4, T5, T6) the curve obtained by plotting the logarithm of the active fraction against the UV dose approaches a straight line (Latarjet and Wahl, 1945), at least for high values of the dose, whereas an inflection with downward concavity, of dubious origin, may appear for low doses. Three other phages (T1, T3, T7) show, on the contrary, an inflec- tion with upward concavity of unknown origin. If the inactivated phages are adsorbed on bacteria and exposed to light of high intensity for a sufficient length of time, the active fraction increases and reaches a maximum (see later section). After this maximum is reached, the curve showing the logarithm of the active fraction against the UV dose has for each phage the same shape as the curve obtained in darkness, but for a given UV dose the slope of the curve obtained after PHTR is lower than the slope of the curve in darkness. The fact that both curves in the light and in darkness tend to be straight lines with different slopes for high UV doses is an indication that absorption of 232 334 R. DULBECCO [voL. 59 UV light in the phage has a probability, a, of producing a photoreactivable in- activation and a probability, b, of producing a nonphotoreactivable inactivation; a + b, the probability of producing any inactivating damage, is proportional to the cross section of the phage for UV. Assuming a + 6b = 1, a is the photo- reactivable sector of the cross section, b the nonphotoreactivable sector; b is measured by the ratio of the slope of the curve after maximum PHTR to the slope of the curve in darkness, both measured in the straight parts. The photoreactivable sector, a, varies between 1 (complete photoreactivability) and 0 (no photoreactivability) and can therefore be used as an index of the photoreactivability. Values of a for different phages are given in table 2. Influence on PHTR of the Interval of Time between Infection and Exposure to Light In the experiments reported in the present and following sections the influence of various experimental conditions on PHTR was analyzed. A quantitative de- termination of PHTR was made by measuring either the ‘active fraction” or the ‘amount of PHTR” in an UVP sample after a given exposure to light. The active fraction is the ratio of the number of active particles after PHTR to the total number of adsorbed particles and is equal to the sum of the fraction active TABLE 2 Photoreactivability of the phages of the T group PHAGE Tl T2 T3 T4 TS T6 T7 Photoreactivable sector of cross section 0.68 | 0.56 | 0.39 | 0.20 | 0.20 | 0.44 | 0.35 (a) in the darkness plus the fraction reactivated by light; the amount of PHTR is the reactivated fraction. The influence on PHTR of the interval of time between infection and exposure to light was determined for UVP adsorbed on bacteria in broth and on resting bacteria (see ‘‘Material and Methods”’). Bacteria and UVP were mixed in dark- ness; samples of the mixture were kept in darkness for various intervals of time and then exposed to light for a period long enough to produce maximum PHTR. After illumination, samples were plated and incubated in darkness, and the ac- tive fraction was determined. In this procedure the bacteria infected with ir- radiated phage particles had to be exposed to light much longer than the latent period between infection and liberation of phage adsorbed on bacteria in broth. When bacteria in broth were used, therefore, the mixtures were plated before the end of the latent period and illumination was continued by exposing the plates; when resting bacteria were used, illumination could be continued indefinitely in liquid, since no phage liberation takes place under these conditions. Experiments with bacteria in broth. The experiments were performed with phage T2 at 28 C. The amount of PHTR decreased rapidly as the time interval between infection and the beginning of exposure to light increased; after about 20 minutes only a small amount of PHTR was produced, as is shown in table 3. 233 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 335 This decrease in PHTR might be caused by a gradual decrease in the amount of PHTR per time unit as the time interval between infection and illumination increases, by a limitation of the time interval after infection in which PHTR can occur, or by both. The amount of PHTR per time unit was determined in experiments in which exposure to light was started at various times after in- fection. The results, shown in figure 2, indicate that the amount of PHTR per time unit remained practically constant for about 15 minutes. The decline in maximum PHTR must be due, therefore, to a limitation of the time within which PHTR can occur after infection, the useful time interval ending between 20 and 30 minutes after infection under the experimental conditions; after this time very little or no PHTR can take place. Experiments with resting bacteria. As is shown in table 4, the maximum amount of PHTR obtainable in phage T2r irradiated with the germicidal lamp for 18 seconds remains fairly constant for at least 70 minutes after infection at 37 C; TABLE 3 The effect of the time interval between infection and exposure to light (bacteria in broth) Phage T2r, irradiated for 20 seconds with the germicidal lamp, was mixed with bacteria and adsorption was allowed to continued for 2 minutes, after which it was interrupted by serum anti-I'2. Exposure to light (H-4 lamp, 12-inch distance) was begun at various times and was continued for 100 minutes at 28 C. Amount of PHTR is lower than in experiment reported in table 4, because in the present experiment a lower light intensity was used, and the time in which the light could be utilized for reactivation was limited, since bacteria in broth were used. TIME INTERVAL BETWEEN INFECTION AND EXPOSURE TO LIGHT ACTIVE FRACTION min 0 Dron Ome 10 Me NO 20 Sie >< MO 30 NW << NOT Active fraction in darkness 3.0) <0 longer intervals have not been tested. The amount of PHTR per time unit is not influenced by the time interval between infection and illumination. he differences between experiments with bacteria in broth and with resting bacteria indicate that under the experimental conditions the system ‘‘UVP- metabolizing bacteria” undergoes a gradual change that in its late phases pre- vents PHTR, a change absent in the system ‘‘UVP-testing bacteria.” Kinetics of PHTR PHTR as a function of the time of exposure to the reactivating light. The following experiments employed inactive phage T2r and resting bacteria, with illumina- tion in liquid. Inactive phage diluted in phosphate buffer was mixed with bac- teria at time 0 at 37 C in darkness, and 10 minutes were allowed for complete adsorption. At the eleventh minute a sample was plated in darkness; at the twelfth minute the mixture was exposed to light, and samples were taken at 234 336 R. DULBECCO [voL. 59 (6) 4 S 4 [= 8 4 re) S | Xu I 4 te O v 3 6) I 4 153 = eS ee eee ! af! 4) oO 5 10 15 20 Ty, minut ieee Pe ( ee es) Figure 2. The fraction of active particles as a function of the time of illumination (in minutes) and of the interval between infection and exposure to light. Each curve gives the active fraction as a function of the time of illumination (in minutes) for a different interval between infection and exposure to light; the interval is indicated (in minutes) at the right end of each curve. Phage T2r was irradiated for 20 seconds with the germicidal lamp, ad- sorbed on bacteria in broth, and exposed to light in broth at 28 C. TABLE 4 The effect of the time interval between infection and exposure to light (resting bacteria) Phage T2r, irradiated for 18 seconds with the germicidal lamp, was adsorbed onto resting bacteria suspended in saline. Exposure to light (H-5 lamp with condenser, wave length 365 mu) began at various times. Illumination was carried out in liquid. TIME INTERVAL BETWEEN INFECTION AND EXPOSURE TO LIGHT ACTIVE FRACTION min 0 5 X 107? 10 5 xX 10- 30 ODOR 50 5.4 X 107? 70 6 X 10-2 Active fraction in darkness 10-3 various time intervals thereafter and plated in darkness; all plates were incu- bated in darkness. The experiments lasted 140 minutes at most; control experi 235 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 337 ments showed that resting bacteria that have adsorbed active phage do not liberate any phage in this time interval. The active fraction always increased with the time of illumination, the increase becoming less and less with increasing time, so that a maximum was reached as is shown in figure 3. The time at which the maximum was reached depended on the light intensity, a longer time being required when the intensity was lower; when the light intensity was varied in such a way that the maximum was reached in a period between 20 and 140 minutes, approximately the same maximum was reached in all cases, as is shown in figure 3. 6 x 10% Active Fraction O /0 20 30 40 50 60 70 80 90 Exposure to the Reactivating Light (minutes) Figure 3. The fraction of active particles as a function of the time of illumination and of the light intensity. The active fraction is plotted against the time of illumination (in minutes). Phage T2r was irradiated for 20 seconds with the germicidal lamp, adsorbed on resting bacteria, and illuminated in liquid at 37 C. Curve 1 was obtained with a light of intensity 10 (in arbitrary units), curve 2 with a light of intensity 2.9, and curve 3 with a light of intensity 0.6. The amount of PHTR (defined in previous section) observed in a sample of UVP after a given time of illumination (p(t)), divided by the amount of PHTR obtained in the same sample when PHTR has reached the maximum value (p (2 )), will be indicated at the ‘‘relative amount of PHTR”’; it can vary between zero and one. By subtracting the relative amount of PHTR from unity, one obtains the fraction of photoreactivable particles that are still inactive after a time, ¢, of p (t) p (2) the time of illumination always gave a straight line for different intensities of illumination (- 1-— iF The logarithm of this quantity plotted against 236 338 R. DULBECCO [voL. 59 the reactivating light and for different doses of the inactivating UV. A curve of this type is reproduced in figure 4. The linearity of the experimental curves was found to be statistically significant by comparing, with the x? test, the experi- mental data for the active fractions with data calculated on this assumption, as is shown in table 5. This result shows that PHTR is a one-hit phenomenon; a Leg (1-8) 10 (6) 20 40 60 80 100 120 140 Time of Illumination (minutes) Figure 4. The logarithm of the fraction of photoreactivable particles that has not been P(t) p() ination (in minutes). Phage T2r was irradiated for 20 seconds with the germicidal lamp, adsorbed on resting bacteria, and illuminated in liquid at 37 C. reactivated after a given time of illumination (: - plotted against the time of illum- TABLE 5 A comparison between observed and calculated active fractions after different times of illumination EXPERIMENT NO. UV DOSE IN SECONDS DEGREES OF FREEDOM x? uy 312 10 10 12.1 >0.20 313 30 11 10.6 >0.40 315 20 12 14.3 >0.20 319 20 11 19.8 0.05 photoreactivable particle is reactivated by one quantum only, independently of the UV dose. The dependence of the amount of PHTR on the time of illumination is ex- pressed by the equation pit) = (1 — e 4) FW) in which ¢ is the time of illumination, F(D) the photoreactivable fraction, which 1s a function of the dose, D, of UV. Value f is the probability per time unit that 237 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 339 a particle is photoreactivated and can be called the PHTR rate; it is proportional p (t) p(#) f may depend on several variables, such as dose of UV, intensity of the reactivat- ing light, temperature, and metabolic condition of the bacteria during PHTR. This dependence will be examined in the next sections. Dependence of PHTR rate on dose of UV and intensity of the reactivating light. PHTR rate (f) was determined for UVP inactivated with different UV doses, adsorbed on resting bacteria, and illuminated in liquid at 37 C with light of constant intensity; it was found to be approximately constant for doses of UV between 10 and 30 seconds. The results, however, are not yet definite on this point, and a decrease of f by a factor 1.2 when the UV dose increases from 10 to 30 seconds cannot be excluded. This result, shows that the probability for an adsorbed quantum to reactivate a photoreactivable phage particle is practically independent of the inactivating UV dose. Value f was also determined for different intensities of the reactivating light on UVP inactivated with the same UV dose, adsorbed on resting bacteria, and illuminated in liquid at constant temperature. The intensity was varied either by changing the distance of the sample from an H-4 lamp—assuming the in- tensity to be inversely proportional to the square of the distance—or by lowering the intensity of a monochromatic light by filters and measuring with a thermo- pile the relative intensities. Value f was found to increase almost linearly with light intensity for low intensities but for high intensities to tend to a maximum as is shown in figure 5. The highest value of the PHTR rate observed in these experiments was about 1.4 X 10-3 sec“! and corresponds to a half-time of about 8 minutes. For low light intensities, f being a linear function of the intensity, the proba- bility of PHTR occurring in a bacterium-phage complex is a linear function of the dose of the reactivating light (equal to intensity < time), whereas for high intensities the same dose has less effect. For low intensities and relatively short exposures the dependence of amount of PHTR on light dose is also approxi- mately linear. to the slope of the line giving log (1 — ) versus time of illumination. Value Action Spectrum of PHTR Seven wave lengths were tested for photoreactivating activity. The corre- sponding monochromatic lights were obtained in the following ways (see Bowen, 1946): (1) Group of lines near 313 my of the mercury are (with a small amount of 334 my). Light: mercury lamp H-4 without glass envelope. Filter: 3 em NiSOu- 7H.0, 350 g + CoSQ:-7H.0, 10 g made up to a liter with water; 1 cm potas- sium hydrogen phthalate, 5 g in 1,000 ml water. (2) Group of lines 365 my of the mercury arc. Mercury lamp H-5 (General Electric); Corning glass filter combination nos. 738, 5860. (3) Group of lines 404 mp of the mercury arc. Lamp H-5. Filter: 2 em Cu(NO;)2:6H2O, 200 g in 100 ml water. Iodine 0.75 g in 100 ml carbon tet- rachlo ride. 238 340 R. DULBECCO [voL. 59 (4) Group of lines 434 my of the mercury arc. Lamp H-5. Filter: 2 em CuSQ,- 5H20, 25 g + 300 ml ammonium hydroxide (d = 0.88), made up to 1 liter with water; 1 em NaNO, 75 g in 100 ml water. (5) Band around 500 my (between 480 and 520 mu, center 500 my). Pro- jection lamp with ribbon filament. Filter: Wratten no. 47, Wratten no. 58, 2 cm CuSO,, 5 per cent. (6) Line 546 my of the mercury arc. Mercury discharge lamp H-5. Corning glass filter combination, nos. 3484, 4303, 5120. 14x10? 12x107 6x 104 4x 107 oO 1 Fd 3} 4 SS 6 te 8 i) 10 Relative Light Intensity (arbitrary units) Figure §. The PHTR rate as a function of the intensity of the reactivating light. The PHTR rate is expressed in sec”! and the light intensity in arbitrary units. Phage T2r was irradiated for 20 seconds with the germicidal lamp, adsorbed on resting bacteria, and il- luminated in liquid at 37 C. (7) Group 576-579 my of the mercury are. Lamp H-5. Filter: 1 em mixture of CuCl.-2H,O, 10 g in 10 ml water and CaCl, 3 m, 90 ml; 2 em K.Cr,07, 15 g in 200 ml water. The efficiency of the different lights was determined in the following way: For each light the range of intensity was first determined, in which the PHTR rate is approximately proportional to the light intensity; the intensity of the most effective wave lengths was reduced by filters until it fell into this range. The rate of PHTR was then determined for each wave length, and the relative intensities were measured with a thermopile. The time of illumination was short, so that the amount of PHTR was very nearly proportional to the dose of reac- tivating light (see previous section). The dose of light of each wave length necessary to give a standard amount of 239 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 341 PHTR in a given time was calculated from these data, and the reciprocal of this dose (given in arbitrary units) was taken as a measure of the activity of that wave length. In figure 6 the activity of the wave lengths tested is plotted against the wave length. The activity of a given light may be underestimated, since it is known that light of the wave lengths used in PHTR may damage the bacteria (Hollaender, 1943) or the phages (Wahl and Latarjet, 1947). The killing action of the seven wave lengths on active phage adsorbed on bacteria was therefore determined, and it was found that with the light intensity and the time of illumination used in the PHTR experiments an appreciable killing activity was only evident for wave length 313 mu. To correct for this killing activity, the amount of PHTR 6 N x) Achiv ity (arbitrary units) w | a a aaa (Fee ae eal Soa0 4000 5000 6000 °o Wave Length in A Figure 6. The action spectrum of PHTR. The activity of each wave length, given in arbitrary units, is plotted against the wave length. Phage T2r was irradiated for 20 seconds with the germicidal lamp, adsorbed on resting bacteria, and illuminated in liquid at 37 C. obtained after a given exposure to this light was increased by a factor equal to the decrease in titer of active phage adsorbed on B exposed to the same light for the same length of time in equal experimental conditions. The curve of PHTR as a function of the time of exposure to 313 my light, obtained in this way, was almost linear and was used in calculating the activity of the light. The activity of the seven wave lengths tested gives only the general shape of the action spectrum. It consists of a band covering the range from about 300 mu on the side of the short wave lengths to about 500 my on the side of the long ones, with a maximum around 365 mu. The greatest photoreactivating activity occurs therefore in the near ultraviolet. The action spectrum of PHTR is related to the absorption spectrum of the 240 342 R. DULBECCO [voL. 59 pigment that absorbs the reactivating light (see Loofbourow, 1948, for dis- cussion of this relation); we tried, therefore, to obtain on this basis some infor- mation about the photosensitive pigment. The action spectrum is not detailed enough to give a specific indication; it shows, however, that the pigment is not contained in the unmodified phage, since the absorption spectrum of purified -500 w 9 is) Optical Density -200 100 240 260 280 300 320 340 360 380 400 420 40 Wave Length, mye Figure 7. Absorption spectra of purified phage T2. The optical density is plotted against the wave length. The spectra were obtained with a Beckman quartz spectrophotometer. The phage was suspended in phosphate buffer (m/15, pH 7) plus MgSO, 107% m. A. Active phage, concentration 5.5 X 10” infecting units per ml. B. Upper curve: absorption spectrum of active phage for wave lengths longer than 320 my, concentration 2.3 X 10! infecting units per ml; lower curve: absorption spectrum of the same phage after 2 hours’ irradiation with the germicidal lamp at a distance of 12 inches. phage has no band comparable to the action spectrum of PHTR. This is shown by figure 7A, which reproduces the absorption spectrum of purified phage T2. For wave lengths longer than 320 my the absorption closely follows Rayleigh’s law of scattering and is, therefore, due to scattering of the light. This is shown more convincingly by plotting the logarithm of the optical density at different wave lengths against the logarithm of the wave length; according to Rayleigh’s 241 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 343 law one should obtain a straight line with slope 4 (see Oster, 1948, 323, formula 6). As shown in figure 8A, the curve, obtained from the same data used for figure 7A, is a straight line in the range of wave lengths 320 to 450 muy; the slope of the curve is 3.7, instead of 4, owing to the size of the phage particles, which is larger than required for the strict application of Rayleigh’s law (La Mer, 1948). The pigment might be formed in the phage after UV irradiation. To check this point a suspension of purified phage T2 containing 2.3 X 10" particles per ml was irradiated in an open shallow container with the germicidal lamp at a dis- tance of 12 inches for variable lengths of time up to 4 hours, and the absorption 2.500 2.550 2600 2650 log Xx Figure 8. The logarithm of the optical density, D, of purified phage T2 versus the lo- garithm of the wave length for the range 320 to 450 mu. A. Active phage (the same data as in figure 7A). B. Phage after long UV irradiation (same data as in figure 7B). Dashed line shows the curve expected for pure scattering. spectrum was determined at regular intervals. The absorption spectrum was found to undergo complex changes as the irradiation proceeded; we shall limit our attention to the modifications occurring in the range of wave lengths longer than 320 mu. A general decrease in absorption in this region was observed, and after about one hour of irradiation a faint band was noticed, which became more evident during the next hour (figure 7B). This band has maximum absorption around 330 my and extends to about 380 my on the side of longer wave lengths (figure 88); its limit on the side of shorter wave lengths cannot be determined because of overlapping with the general phage absorption. 242 344 R. DULBECCO [voL. 59 The maximum absorption of this band is located at a shorter wave length than the maximum of the action spectrum of PHTR; this difference, however, is not such as to exclude the band from belonging to the photosensitive pigment, because the location of the band may be shifted toward longer wave lengths if the pigment is bound with some bacterial constituent after adsorption of the inactive phage on bacteria. Influence of the Metabolic Conditions of Bacteria in PHTR of Phages PHTR is not appreciably different whether the phage is adsorbed by bacteria in broth or in a synthetic medium; the rate of PHTR is somewhat lower with resting bacteria than with bacteria suspended in nutrient media. The influence of oxygen on PHTR was determined. Resting bacteria and UVP were placed in separate compartments of a Thunberg tube, nitrogen was bubbled for about 20 minutes through the bacterial suspension, the tube was then evacu- ated by a pump, and air was replaced with nitrogen, the operation being re- TABLE 6 PHTR rate and Qo at different temperatures The Qio was determined from the ratio of Qa of the rates at two successive temperatures, using the formula: Qio = (Qa)!°/4, where d is the temperature interval between two ob- servations. Phage T2r irradiated for 20 seconds with the germicidal lamp, illuminated in liquid. TEMPERATURE (C) PHTR RATE (SEC!) Qio 3 ior 105? 11 Of2) sale? 7.5 16 13) x 10s 4.2 24 253) OX 10Re 21 30 Sno OS LO 2.4 37 O00) oS Om Med peated 4 times. Phage and bacteria were mixed and the tube was exposed to light. The control consisted of an open tube from which oxygen had not been removed. The same amount of PHTR was observed in both tubes. Oxygen is therefore not necessary for PHTR, at least not for the initial photochemical re- action. We also found that cyanide in 10-2 mM concentration does not affect PHTR. The Effect of Temperature on PHTR PHTR can occur at temperatures too low to allow growth of active phage (+1 C). To obtain a measurable amount of PHTR at this temperature, one must mix UVP and bacteria in the dark at 37 C to allow enough adsorption, then chill the mixture to 1 C and expose it to light. Determinations of PHTR rate with constant illumination were made at 3, 11, 16, 24, and 37 C, using phage T2 irradiated for 20 minutes with the germicidal lamp. The Qi was determined for each interval, and the results are reproduced in table 6. 243 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 345 The behavior of the Qyo is similar to that found for complex bacterial activities and for enzymatic reactions (Rahn, 1932) and is an indication that the physio- logical state of the bacterium conditions the probability of the photoreactivating event. PHTR of Phage Inactivated with X-Rays It was previously reported (1949) that no PHTR had been detected for phage inactivated by X-rays. This statement is valid only if X-ray irradiation is per- formed on phage in synthetic medium. With phage T2 inactivated by X-rays in nutrient broth a slight amount of PHTR can be observed. This amount is proba- bly reduced by the poor adsorption of phage inactivated by X-rays, as discovered by Watson (1948). After correction for the limited adsorption (data kindly sup- plied by Watson), the PHTR of X-ray-inactivated phage is still considerably less than that for phage inactivated to the same extent by UV. SUMMARY AND DISCUSSION In the following brief discussion we shall try to arrange the results of our ex- periments in an order that will bring out their theoretical implications, and we shall present a working hypothesis for the mechanism of PHTR. (1) The damage caused by UV in bacteriophage consists of two kinds: photo- reactivable and nonphotoreactivable damage. These two kinds of damage occur with comparable cross sections; they may reflect the presence of two kinds of UV-absorbing constituents in each phage particle. Further information should be obtainable by determining the action spectrum for the two types of inac- tivation. (2) Only phage particles adsorbed on bacteria undergo PHTR; PHTR can occur within a few seconds after adsorption, indicating that PHTR is due to reactions occurring in the early phase of the interaction between inactive phage and bacterium; perhaps surface reactions are involved, and this may account for the failure to reproduce PHTR with bacterial extracts. PHTR does not re- quire the presence of external metabolic substrates or of oxygen and is not in- hibited by cyanide, but is influenced by the physiological condition of the bac- teria after infection. (3) The photosensitive pigment has an action spectrum with a maximum near 365 my. Normal phage does not have an absorption band corresponding to this action spectrum. UV-treated phage shows an absorption band with a maximum near 330 mu. Perhaps this is the photosensitive pigment created by UV irradia- tion in the phage. The shift from 330 to 365 my could be due to the binding of the pigment with bacterial constituents upon adsorption of a phage particle on the bacterium. The alternative possibility is that the photosensitive pigment exists in the bacterium prior to infection. Further studies of the absorption band of UV-irradiated phage and of the action spectrum of PHTR are needed. (4) At low intensities of illumination the probability of PHTR occurring in a bacterium-phage complex is proportional to the dose of light. From this we might conclude that the individual light quanta absorbed by the bacterium- 244 346 R. DULBECCO [voL. 59 phage complex do not co-operate to produce PHTR but that each quantum in- dividually has a chance to accomplish PHTR and that this chance is independent of any other quanta absorbed by the complex. We would thus be led to conjec- ture that PHTR is due to a primary photochemical reaction. (5) The picture is complicated by the finding that at high intensities the rate of PHTR ceases to be proportional to the intensity of illumination, reaching a maximum value, and by the finding of a complex temperature dependence. These findings require the participation of dark reactions in the mechanism of PHTR. (6) The probability of PHTR per time unit (PHTR rate) is practically in- dependent of the dose of UV used for inactivation. This finding shows that photoreactivation is an all-or-none phenomenon, and it may indicate that photo- reactivable inactivation is always due to one injury, elimination of which resti- tutes activity. To explain all the known features of PHTR, we propose the following eae hypothesis: The photoreactivable inactivation is due to formation of molecules of an inhibitor in the phage; although many of these molecules may be formed in one phage particle, just one molecule is the inactivating one in each case, for example, by blocking a reaction necessary for phage growth in a small area of the contact surface between phage and bacterium. Restoration of phage ac- tivity requires the permanent removal of the inactivating inhibitor molecule. Dissociation does not occur by thermal activation; but absorption of a light quantum of a given wave length produces a transient and reversible dissociation. During the time the inhibitor is dissociated it can be captured by a receptor and destroyed with dark reaction. This makes the removal permanent and consti- tutes reactivation. PHTR therefore requires a system made by phage, inhibitor, pigment, and receptor. The inhibitor belongs to the phage, the receptor to the bacterium being perhaps of enzymatic nature; the pigment may belong to either one and may be identified either with the inhibitor or with the receptor. The system is com- pleted after adsorption of the inactive phage on bacteria. The probability of PHTR (PHTR rate) for low light intensity is proportional to the time integral in which the inhibitor is dissociated and therefore to the number of quanta absorbed (dose of the reactivating light). For high intensities the activation times due to absorption of different quanta overlap somewhat, so that equal doses become less efficient. When the light intensity is so high that the inhibitor is dissociated without interruption during illumination, the proba- bility of PHTR reaches a maximum value. The probability of PHTR is also pro- portional to the probability that the dissociated inhibitor will be captured and destroyed by the receptor in the time unit and therefore depends on temper- ature, which influences the dark reactions, and on some physiological conditions of the bacteria, which may affect the efficiency or the amount of the receptor. REFERENCES Bowen, E. J. 1946 The chemical aspects of light. Clarendon Press, Oxford. Durpecco, R. 1949 Reactivation of ultraviolet-inactivated bacteriophage by visible light. Nature, 163, 949-950. 245 1950] PHOTOREACTIVATION OF INACTIVATED BACTERIOPHAGES 347 GratiA, A. 1936 Des rélations numériques entre bactéries lysogénes et particules de bactériophage. Ann. inst. Pasteur, 57, 652-676. Hersuey, A. D., KALMANSON, G., AND BRONFENBRENNER, J. 1943 Quantitative methods in the study of the phage-antiphage reaction. J. Immunol., 46, 267-280. HouLuaENpDER, A. 1943 Effect of long ultraviolet and short visible radiation (3500 to 4900A) on Escherichia coli. J. Bact., 46, 531-541. Kewner, A. 1949 Effect of visible light on the recovery of Streptomyces griseus conidia from ultra-violet irradiation injury. Proc. Natl. Acad. Sci. U.S., 35, 73-79. La Mer, V. K. 1948 Monodisperse colloids and higher-order Tyndall spectra. J. Phys. Colloid Chem., 52, 65. LaTARJET, R., AND WAHL, R. 1945 Précisions sur l’inactivation des bactériophages par les rayons ultra-violets. Ann. inst. Pasteur, 71, 336-339. Loorsourow, J. R. 1948 Effects of ultraviolet radiation on cells. Growth, 12, Suppl., 75-149. Luria, 8. E. 1947 Reactivation of irradiated bacteriophage by transfer of self-reproduc- ing units. Proc. Natl. Acad. Sci. U.S., 33, 253-264. Luria, S. E., anp De.Briick, M. 1942 Interference between bacterial viruses. II. Interference between inactivated bacterial virus and active virus of the same strain and of different strain. Arch. Biochem., 1, 207-218. OstER, G. 1948 The scattering of light and its applications to chemistry. Chem. Rev., 43, 319-365. Raun, O. 1932 Physiology of bacteria. P. Blakiston’s Son and Co., Philadelphia. Refer to p. 123, 221. Waut, R., anp LaTarsET, R. 1947 Inactivation de bactériophages par des radiations de grande longeur d’onde (3400-6000 A). Ann. inst. Pasteur, 73, 957-971. Watson, J. D. 1948 Inactivating mutations produced by X-rays in bacteriophages. Genetics, 33, 633. 246 GENETIC RECOMBINATIONS LEADING TO PRODUCTION OF ACTIVE BACTERIOPHAGE FROM ULTRAVIOLET INACTIVATED BACTERIOPHAGE PARTICLES! S. E. LURIA anp R. DULBECCO Department of Bacteriology, Indiana University, Bloomington, Indiana WITH AN APPENDIX BY R. DULBECCO Received June 28, 1948 HE potentialities of bacteriophage genetics have been revealed by the discovery of genetic recombinations among related phage particles in- fecting the same bacterial cell (DELBRUCcK and Bartry 1946). The complexities of these genetic systems have been further illustrated by the work of HERSHEY and RotMAN (1948) on a number of different genetic determinants involved in the determination of the alternative phenotypes r+ and 7 in phage 72H. A different approach to the genetic mechanisms of bacteriophages originated from the chance observation by DELBRUcK and BaILey that, after exposure to ultraviolet light, some phages gave variable plaque counts, depending on the relative concentrations of phage lysates and host cells at the time of assay. In investigating this phenomenon, one of us (LuRIA 1947) discovered a mecha- nism of phage reactivation by interaction among inactive particles in the course of intracellular growth. The detailed investigation of this phenomenon has indicated new possibilities for a quantitative analysis of the genetic struc- ture of these viruses and has suggested a possible mechanism for their repro- duction. A preliminary discussion of some of the results reported in this paper has appeared (Luria 1947); their implications for a number of problems have been discussed in a forthcoming publication (Lurt1a 1948). The present article is intended to present the results in detail, and, by describing techniques and methods of analysis, to serve as a background for future publications on this topic. The analysis of the results presented in the following pages is based on the hypothesis that inactivation of bacteriophage particles by ultraviolet light is due to production of discrete alterations in individual portions of genetic ma- terial. Although the internal evidence in support of this hypothesis, as pre- sented in this paper, is quite satisfactory, it must be said that satisfactory external evidence from other lines of attack is not yet available. The conclu- sions reached in this article must be considered for the time being as working hypotheses for further investigation. MATERIAL AND GENERAL METHODS The system of phages T1-77, their r mutants, and their common host Escherichia coli strain B have repeatedly been described, as well as the use of 1 This work was done under an AMERICAN CANCER SOCIETY grant recommended by the COMMITTEE ON GROWTH OF THE NATIONAL RESEARCH CouNCcIL. We wish to acknowledge the able assistance of Mrs. J. P. Heappy. Reprinted by permission of the authors and Genetics, Inc., from GENETICS, 34, 93-125, March, 1949. 247 94 S. E. LURIA AND R. DULBECCO bacterial mutants resistant to one or more phages as indicators for one phage in the presence of another (see DELBRUCK 1946). Plate counts for viable bac- teria, and plaque counts in agar layer for active phage were used throughout, employing 1.1 percent agar in “Difco” nutrient broth plus 0.5 percent NaCl. All plates were incubated at 37°C. Experimental bacterial cultures in the logarithmic phase of growth were grown with aeration at 37°C from standard inocula. The phage stocks were lysates in glucose+-ammonia (or lactate+-ammonia) medium. These media give negligible absorption of the ultraviolet light used in this work. High titer phage lysates (over 1X10! particles per ml) might give some ultraviolet screening effect because of bacterial debris and of phage itself. Whenever possible, therefore, phage was irradiated after a dilution 1:5 or higher in the same medium. The source of ultraviolet was a General Electric Company germicidal bulb, 15 watts, alimented through a stabilizer. At a distance of 50 cm from the center of this bulb, the flux—measured with a West- inghouse SM-200 meter with tantalum phototube WL-775—is about 7 erg Xmm-~? sec~!. The beam contains mainly radiation of wavelength 2537 A. Samples were irradiated in a thin layer (not over 0.4 mm) in open Petri dishes rocked during exposure. The technique of “one-step growth” experiment in its various forms has been described in detail previously (DELBRUcK and Luria 1942). EXPERIMENTAL Inactivation and reactivation of bacteriophages Plaque counts on phage suspensions exposed to ultraviolet for various lengths of time generally give survival ratios whose logarithms are proportional to the dose, that is, to the time of exposure (see LATARJET and Want 1945, and figure 1). The logarithmic rate indicates a one-hit mechanism of inactivation (LEA 1947), and we can assume that the hit consists of the successful absorption of one quantum. The probability that one quantum produces inactivation is, however, very small: for phage 72, for example, one inactivating hit is pro- duced by a dose corresponding to almost 10* quanta absorbed per particle (M. ZELLE, personal communication). Only one absorption in 104 on the aver- age is, therefore, effective, the others probably producing excitations that do not lead to the inactivating effect. When the average number of effective hits per particle is r, the proportion of active to total phage will be e~’. For r=1, e-”=0.37; the corresponding dose is the “inactivation dose” in LEa’s terminology (1947). If doses are expressed in multiples of the inactivation dose, their values give directly the average number of hits per particle. . Phage particles inactivated by ultraviolet light are adsorbed by bacteria (Luria and DELBRUCcK 1942). This is detected because adsorption of one par- ticle by a bacterium causes death of the latter. One can, therefore, measure the rate of adsorption of inactive particles from the survival of bacteria in mixtures containing bacteria and irradiated phage in known proportions. If, on the average, x particles are adsorbed per bacterium, a fraction e~? of the 248 ACTIVE FROM INACTIVATED BACTERIOPHAGE 95 10 20 30 40 =10 20 30 40 50 60 70 10 20 30 SECONDS Ficure 1.—Inactivation of various phages by ultraviolet light. P/Po=proportion of active phage particles after irradiation. y=In Po/P=average number of lethal hits per phage particle. The doses are expressed in seconds of exposure. The deviations for high doses in the curves for T2, T4, and T6 are due to reactivation occurring on the assay plates (see text). The broken lines represent extrapolations from the logarithmic portions of the curves. bacteria should receive no particle and survive. By this means, we could establish that for irradiated phages T2, T4, T5, and T6, even for high doses, the rate of adsorption is the same as for unirradiated phage. An experiment of this type is shown in table 1. Only for very high doses a slight reduction occurs in the ability of phage to kill bacteria. This reduction is never such as to require important corrections in the analysis presented in later sections. With phages T2, T4, T5, and T6 (the “large particle” phages) the plaque counts on irradiated samples are not independent of the mode of assay. They depend on the concentration of the samples when first mixed with bacteria, in a way illustrated in table 2. In these experiments, bacteria were mixed with various concentrations of irradiated phage. Before lysis and phage liberation TABLE 1 Killing of bacteria by irradiated phage 0.9 ml of a bacterial culture was mixed with 0.1 ml of each of five suspensions of phage T2r that had received various doses of radiation. After 10 minutes, samples were diluted and plated for viable bacterial count. <7 DOSE OF RADIATION PHAGE EXPERI- aban BACTEREY (eee SURVIVING SURVIVAL, Nansorpen INPUT, INPUT, ea MENT HITS BACTERIA INPUT PER BAC- PARTICLES CELLS NO. SECONDS PER PER ML es TERIUM PER ML PER ML PARTICLE x 0 0 2.3108 0.20 1.60 60 18 2.4108 0.21 1.56 129 2109 WIS 108 70 21 2.2108 0.19 1.66 80 24 1.6108 0.14 1.96 100 30 2.7X108 O23 1.47 249 96 S. E. LURIA AND R. DULBECCO took place, dilutions were made to bring the total dilution of the irradiated sample to a constant value, and an aliquot plated for plaque count. The plaque counts represent infected bacteria that liberate active phage. Although the total dilution of the irradiated phage on all plates is the same, it is seen that the plaque counts are higher when bacteria have first been placed in contact with a more concentrated phage lysate. This means that bacteria may produce active phage if they pick up the ir- TABLE 2 Dependence of plaque counts on irradiated phage T6r on the concentration of the phage sample that is mixed with bacteria A sample of phage T6r containing 1.510!° particles/ml was irradiated for 20 seconds. The bacterial suspension (B) contained 2X 10° cells/ml. Each plate received 0.05 ml of phage dilution and 0.2 ml of suspension (B). TOTAL DILU- DILUTION TION FROM PLAQUE OF PHAGE THE ORIGINAL EXPERI- MIX- COUNT WHEN PHAGE TO THE MENT TURE PROCEDURE (SUM OF FIRST SUSPENSION NO. NO. TWO MIXED FROM WHICH PLATES) WITH (B) SAMPLES ARE PLATED 1 0.1 ml T6r—0.9 ml (B); kept 10 min. at 37°C; diluted 1:10%, 0.05 cc plated 1:10 1:10! 1318 2 0.1 ml (T6r 1:10)—0.9 ml (B); kept 10 min. at 37°C; diluted 1:10, 0.05 ml plated 1:10? 1:10 474 5 3 0.1 ml (T6r 1:103)—0.9 ml (B); kept 10 min. at 37°C; diluted 1:10, 0.05 ml plated 1:104 1:108 250 4 0.05 ml (T6r 1:10) plated Less than 1:10 1:104 57 (on plate) radiated particles from a concentrated phage suspension, but not from a dilute one. The immediate explanation is that from a concentrated lysate the bac- teria receive some other “factor,” which, inside the bacterium, somehow reactivates an “inactive” particle and which is not present in dilute lysates. An “inactive” particle can be defined as one that has lost the ability to initiate production of active phage unless adsorbed by a bacterium together with the unknown “factor.” Reactivation still occurs after storage of irradiated phage for weeks in an ice-box. Reactivation gives rise to fully active phage particles. This can be proved either by sampling phage from the plaques or by letting the bacteria, in which reactivation occurs, lyse in liquid and then testing the lysate for active particles. 250 ACTIVE FROM INACTIVATED BACTERIOPHAGE 97 The occurrence of reactivation for certain phages accounts for deviations from the logarithmic inactivation rate found for these same phages (see figure 1). As the dose of radiation increases beyond a certain point, the survival, as determined by plaque count, appears to diminish less rapidly. This is due to an unavoidable partial reactivation. In a phage titration, we mix approximately 5X10’ bacteria with an amount of phage suspension such as to give approximately 100 plaques, and pour the mixture on an agar plate. On the one hand, if we are dealing with a fully active sample, the bacteria only come in contact with 100 active particles. On the other hand, in assaying an irradiated suspension containing, for example, one active particle in 10°, we expose the bacteria to 10* inactive particles plus a corre- spondingly large amount of other lysate constituents, besides the residual 100 active particles. Conditions permitting reactivation, therefore, obtain in these plating mixtures, and reactivation disturbs and often completely ob- scures the count of the residual active phage. For this reason, the survival of fully active phage for high doses must be obtained by extrapolation from the logarithmic part of the curve, a necessarily inefficient procedure. If deviations in the survival rate for high doses occurred, this extrapolation would not be justified. One possible cause of error—screening of some phage particles from radiation by components of the lysate itself—was excluded by irradiating concentrated phage T6 mixed with phage T/, and testing for the inactivation rate of the latter, which is not disturbed by reactivation phenomena. The inactivation rate of T7 remains the same as in the absence of T6 up to doses that correspond to 100 hits per particle of phage 76. Identification of the reactivating factor What is the “factor” present in irradiated stocks of phages T2, T4, T5, or T6, which, if acting on bacteria that have adsorbed inactive phage particles, allows production of active phage? Since phage stocks are lysates produced by lysis of the common host E£. coli B, the factor might be either of phage or of bacterial origin. The factor was identified as being phage itself, inactive or active, in that reactivation occurs in bacteria that adsorb either more than one inactive particle of a given phage, or one inactive particle of one phage plus some active or inactive particles of a related phage. The evidence for this conclusion, which is illustrated in part by the data in table 3, can be summarized as follows. (a) Addition of an excess of supernatant from a heavy bacterial culture to a mixture of dilute irradiated phage plus bacteria gives no increase in plaque count. The factor in the lysates is not a normal bacterial secretion. (b) Concentrated lysates of phages T/, T5, or T7 added to a mixture of bacteria and dilute irradiated 72 (or T4, or T6) do not cause reactivation. No heterologous lysate causes reactivation of 75. The test for cross-reactivation is done by plating the mixtures, before lysis, with bacterial indicator strains sensitive to the phage whose reactivation is tested, but not to the others. Since all phage stocks are lysates of common host cells, the factor is not an unspecific bacterial product liberated upon lysis. 251 98 S. E. LURIA AND R. DULBECCO TABLE 3 Reactivation of phage T2 under various conditions Phage 72, containing 210" particles per ml, was irradiated for 35 seconds. Phages T6 and T4, containing 4X10" particles per ml, were irradiated for 30 seconds. Phages T/ and T5 con- tained 310" particles per ml. The bacterial suspensions (B) and (B/6) contained 10° cells per ml. PLAQUE COUNT eae OFS CONTENTS AFTER 10 MINUTES (SUM OF ae NO. TION TWO PLATES) — 2 Se 1 0.1 ml Phage 72 dil. 1:500+1.9 ml (B) 0.05 ml plated with B 22 = 2 0.1 ml Phage T2 dil. 1:500+-1.9 ml (B/6) 0.05 ml. plated with B/6 20 _ 3 0.1 ml Phage T2 pure +1.9 ml (B) dil. 1:500, 0.05 ml plated with B 4000 -- 4 0.1 ml Phage 72 pure +1.9 ml (B/6) dil. 1:500, 0.05 ml plated with B/6 3000 + 5 0.1 ml Phage 72. dil 1:500+0.1 ml irrad. 0.05 ml plated with B/6 670 + T6+1.8 ml (B) 6 0.1 ml Phage 72 dil. 1:500+0.1 ml unirrad. | 0.05 ml plated with B/6 190 a T6+1.8 ml (B) 7 0.1 ml Phage 72 dil. 1:500+0.1 ml irrad. 0.05 ml plated with B/4 661 + T4+1.8 ml (B) 8 0.1 ml Phage 72 dil. 1:500+0.1 ml irrad. 0.05 ml plated with B/6 10 = T6+1.8 ml (B/6) 9 0.1 ml Phage 72 dil. 1:500+0.1 ml unirrad. | 0.05 ml plated with B/6 12 = T6+1.8 ml (B/6) 10 0.1 ml Phage 72 dil. 1:500+0.1 ml irrad. 0.05 ml plated with B/4 883 + T4+1.8 ml (B/6) 11 0.1 ml Phage 72 dil. 1:500+0.1 ml unirrad. | 0.05 ml plated with B/1, 5 18 = T1+1.8 ml (B) 12 0.1 ml Phage 72 dil. 1:500+0.1 ml unirrad. | 0.05 ml plated with B/1, 5 17 - T5+1.8 ml (B) De EE a (c) Concentrated lysates of phages T2 (or T4, or TO), whether fully active or irradiated, can reactivate irradiated particles of any other T-even phage. These phages are morphologically, serologically, and probably genetically related, whereas T5 belongs to a fully separate group (DELBRUcK 1946). The “factor” in the lysates appears to carry the same pattern of relatedness. It is not produced by irradiation, since its presence can be proved in unirradiated lysates by the technique of cross-reactivation. (d) Reactivation is independent of contact between phages prior to infection of the host: a mixture of bacteria and phages gives the same amount of re- activation independently of how long the phages have been together before adding the bacteria. (e) Phage T4r purified by fast centrifugation (kindly supplied by Dr. T. F. ANDERSON) shows both self-reactivation as a function of concentration in the mixtures, and ability to reactivate phages T2 or T6. The factor, therefore, is present in such a purified phage suspension. (f) Reactivation of phage T2 by lysates of T6, for example, only occurs in presence of bacteria capable of adsorbing both phages. Inactive phage 72 in presence of bacteria B/6, by which it is adsorbed, is not reactivated by T6, which is not adsorbed, but is reactivated by 74, which is adsorbed. This proves 252 ACTIVE FROM INACTIVATED BACTERIOPHAGE 99 that the “factor” has the same host specificity as the phage in whose lysate it is found. The last point, particularly, was considered crucial in showing that produc- tion of active phage from an inactive particle was actually due to infection of the same bacterial cell with other particles, either of the same or of a different but genetically related phage, active or inactive. Further confirmation came from the experiments discussed below, which showed that in a mixture of inactive phage and bacteria the number of bacteria yielding active phage is never greater than the number of bacteria that adsorb two or more inactive particles, and in some cases actually equals it. The same holds true for re- activation of an inactive phage, for instance T2, by a related one, for in- stance 74; the number of bacteria liberating active TZ is lower than—or equal to—the number of bacteria receiving at least one particle of each phage. The analysis of cross-reactivation between these different wild-type phages will not be discussed further in this paper, but will form the subject of a future publication.? (g) All bacteria which, after infection with inactive particles, do not liberate active phage also fail to lyse. This was proved by mixing bacteria and inactive phage under conditions in which some reactivation occurs, plating a sample of the mixture for plaque count, and another sample for direct microscopic ob- servation of lysis on agar. The results of such experiments proved that the fraction of bacteria that are lysed is the same as the fraction of bacteria that liberate active phage. The other infected bacteria fail to grow and divide, and can be seen still apparently unchanged 24 hours later. Reactivation and genetic transfer The limitation of cross-reactivation to the T-even phages immediately brought out a similarity between this phenomenon and that of genetic transfer described by DeLBRUcK and BatLey (1946). In the latter case, bacteria simultaneously infected with the phages T2r*+ and T4r—the r character being the result of mutation from the wild-type, which can be designated as r+—liber- ate a mixture of particles of the four types, T2r+, T2r, T4r+, and T4r, among which the second and third represent new types. These must owe their origin to some sort of recombination involving the genetic determinants for the alternative r* and r phenotypes. Evidence for the discrete nature of these determinants has since been reported by HErsHEY and RotMAN (1948). We assumed then, as a working hypothesis for the analysis of the reacti- vation phenomenon, that inactivation by ultraviolet light resulted from “lethal mutations” in a number of discrete genetic determinants among in- active particles in the same bacterium to reconstitute fully active particles. This hypothesis can be formulated quantitatively in terms of measurable * Cross-reactivation between T-even phages has the limitation that the individual phages are distinguishable only by test of differential properties such as ability to grow on different hosts and rate of inactivation by different antisera. Cross-reactivation can only be defined as the produc- tion, upon mixed infection, of active particles having the distinctive properties of an inactive parent particle. 253 100 S. E. LURIA AND R. DULBECCO quantities by making a number of simple assumptions. We shall first develop this simple theory and then describe the experiments by means of which it was tested. Theory We shall assume that in each particle of a given phage there exist 2 “units” (or “loci”) each capable of undergoing a lethal mutation when exposed to ultraviolet light. Since one effective hit is sufficient to inactivate a phage par- ticle, a lethal mutation can be defined as an effective hit, that is, as an altera- tion of one unit which makes the phage particle unable to initiate by itself the production of active pnage in a bacterium. A particle may undergo more than one lethal mutation, and mutations will be distributed at random and independ- ently among the different units, the distribution depending only on the sensitivity of each unit. We shall now make the assumption that the sensitivity of all units is the same, and show later that this assumption, if incorrect, only requires a nu- merical correction which does not invalidate the applicability of the theory. Our next assumption is that active phage cannot be produced in a bacterium unless the infecting particle or particles, taken as a group, contain at least one copy of each unit in non-lethal form. This assumption is an essential feature of the theory, and corresponds to treating each unit as a discrete, material, in- dependent hereditary unit endowed with genetic continuity and individuality. An inactive unit cannot be replaced by copies of different units. This assump- tion implies that production of active units cannot result from the cooperation of two or more lethal units, but only from actual reproduction of active units. When a population consisting of M phage particles is irradiated with a given dose, there will be produced in each particle, on the average, r lethal mutations, or a total of M Xr mutations in the whole population. Since M particles contain M Xn units, each unit will receive on the average Mr/Mn=r/n lethal muta- tions. A given unit, taken at random, will have a probability e~’” of not having a lethal mutation, and a probability (1—e~"”) of having at least one. We ask next: what is the probability that each of the x units is present in at least one non-lethal copy in a group of & particles that enter a bacterium? According to our assumptions, this probability should represent an upper limit for the probability that a bacterium produces active phage. If a bacterium is infected by & particles, the probability that a given unit is lethal in all of them is: (1—e~"”)*, and the probability that it is non-lethal in at least one of them is: 1—(1—e~7)*. The probability that at least one non-lethal copy of each of the m units is present in the k particles is the product of the probabilities referred to the individual units. Since we have assumed equal sensitivity for all units—that is, r/n constant for all units for each value of r—the product will be [PP eat) ei (1) The expression (1) represents the probability that a bacterium infected by k particles receives at least one full non-lethal complement of the ” units. 254 ACTIVE FROM INACTIVATED BACTERIOPHAGE 101 In a mixture of phage with bacteria, however, there is a distribution of the number of phage particles infecting individual bacteria. With relatively good approximation—see Appendix—this distribution can be considered as a Poisson distribution. If « is the average number of particles adsorbed per bacterium, the fraction of bacteria with k particles is: «*e-*/k! The fraction of bacteria receiving & particles which carry a full complement of non lethal units is then: xe = aalnar CHG LE (2) Finally, the fraction of the total bacterial population which receives all units in a non-lethal form is the sum of the expression (2) for all possible values of k: = k=0 This expression embodies the following consequences of our hypothesis: (a) No full complement of active units can be present in uninfected bacteria (k=0). (b) Of the bacteria with one phage particle (k=1), only those with an active particle fulfill the requirement for active phage production (Z=xe~“*e~”). (c) Any bacterium that receives at least one active particle fulfills the requirement for active phage production, whether it also receives inactive particles or not. For each unit of that particle, 1—e~’"=0; hence, (ext) = 0 and (t—(1 er") Fe — 1. (d) For any given value of r>0, Z increases with increasing x, that is, the probability of having a full complement of active units increases as the number of particles adsorbed per bacterium increases. (e) For any given value of x, Z diminishes with increasing 7, that is, the probability of having all active units diminishes as the dose of radiation increases. For the purpose of comparison with data from different experiments, it is more convenient to eliminate from the computation those bacteria that receive either zero or one phage particle, since they are not expected to contribute to reactivation. This is done by using instead of Z the expression xe k} [1 — (1 — emp (3) eke jae 2 oR [Mere (4) z represents the fraction of bacteria in the total population that have two or more phage particles, which together contain a full complement of active units. Since the fraction m of bacteria with two or more phage particles (“multiple- infected bacteria”) is m=1-—-— («+ 1)e* (5) the multiple-infected bacteria receiving a full complement of active units represent a fraction 255 102 S. E. LURIA AND R. DULBECCO Sy [1 an Gl a eqriaye n Re LR i eo = xe ! The expression y thus obtained is a function of « (average number of phage particles adsorbed per bacterium), of y (average number of lethal hits per par- ticle), and of z (number of units per particle). We shall call w the ratio between the number of bacteria that actually liber- ate active phage (plaque count) and the number of multiple-infected bacteria. For each mixture of bacteria and irradiated phage, we can determine experi- mentally 7, x, and the plaque count, and obtain from these the values of m and w. We can then compare the experimental values of w with the calculated values of y for several different values of .° The function y=F (7, x, 7) was tabulated numerically for a range of values of r (between 3 and 50), of x (between 0.05 and 20), and of m (between 10 and 60). We used for k those ranges of values for which the contributions of the corresponding classes were relevant. The corresponding curves were drawn for y=F (r) (x and m constant), and for y= F (x) (r and m constant). Before comparing the experimental results with the curves, it is useful to discuss briefly what we may expect from the comparison. If reactivation only occurs in bacteria with more than one inactive particle, w should never be greater than unity. If among the requirements for reactivation there are those stated in the assumptions of our theory, the ratio w/y should never be greater than unity. Finally, if the requirements stated in our assumptions are necessary and sufficient for reactivation, the ratio w/y should be unity, that is, active phage should be produced in all those bacteria that receive a full complement of the hypothetical units in non-lethal form. Should this obtain, it would then be possible to calculate the value of m for each phage from the experimental values of w. It is important to keep in mind that the assumptions of our theory, up to this point, do not contain any implication as to the nature, properties, or mechanism of transfer of the postulated units. They only assert that each unjt has genetic individuality and can be made lethal by radiation as a result of one photochemical reaction, which is of the “all or none” type and independent of other reactions of the same type in other units of either the same or other phage particles. Production of active phage is conditioned by the presence in one bacterium of one active copy of each unit, this copy not being replaceable by any number of inactive copies. The theory does not imply that all phage particles receive the same number of lethal hits, but that the lethal hits are distributed at random among the units of all phage particles. 3 In preliminary reports (LuRIA 1947, 1948) we used the symbol y both for the theoretical and experimental probabilities of reactivation. We also gave values of 1/y instead of y(or w). The present notation, while consistent with the previous one, makes the presentation mere logical. 256 ACTIVE FROM INACTIVATED BACTERIOPHAGE 103 Comparison of theory with experiment Quantitative experiments consisted of testing mixtures of bacteria and ir- radiated bacteriophage for the number of bacteria that liberate active phage. Only phages T2, T4, and TO were studied in detail. In a typical experiment, such as the one described in detail in table 4, a standard culture of bacteria, grown to a titer of either 10° cells per ml or 10° cells per ml, was chilled by immersion in a water bath at 5—6°C. This treatment interrupts multiplication without changing the ability to resume immediate multiplication and to support normal growth of phage upon return to 37°C. In experiments with bacteria grown to a titer of 10° (latest part of logarithmic growth phase), immersion in ice-water is not necessary, since multiplication stops almost immediately upon interruption of aeration and transfer to room temperature. Ten minutes after interrupting multiplication, a sample of the culture is diluted and assayed for viable count. If several mixtures of bacteria and phage are to be prepared, the culture may have to be used for one or two hours, in which case at least one other similar assay is made at the end of the experiment to make sure that no proliferation has occurred. At intervals, undiluted samples of the culture are placed into test tubes, and a constant volume of irradiated (or control) phage variously diluted is added. In this way, we know for each mixture the input of bacteria and of phage per ml. For irradiated samples, the input of active phage is determined from one or more assays done at very high dilution. When this is impossible—for high doses of radiation—the amount of active phage is determined by extrapolation from the first part of the inacti- vation curve. Adsorption is interrupted by heavy dilution after a short time (generally five or ten minutes). The amount of adsorption is determined either by determi- nation of free phage in the supernatant of a centrifuged sample from a mixture containing active phage, assuming similar adsorption in all other mixtures (see table 1), or by determining the bacterial survival in samples from various mixtures, With the T-even phages, 80 to 95 percent of the phage is adsorbed in ten minutes, The value for the multiplicity of infection, x, for each mixture is used in calculating the fraction m of bacteria with two or more phage par- ticles: m=1—(«+1)e7?. Before lysis begins, a suitably diluted sample of the mixture is plated with an excess of sensitive bacteria for plaque count. The plaque count—corrected, when necessary, for the active phage by subtracting the value corresponding to the latter—is divided by m to obtain the experimental value w for that mixture (see table 4). A large number of experiments, yielding a total of over 1000 values of w, for various doses of radiation and for different multiplicities, were done with the T-even phages. Phage T5 was only partially investigated, because of diffi- culties in obtaining reproducible results in view of the low and irregular adsorp- tion rate for this phage. The data for phages T-even include those for several of their r mutants, 257 A fi en ‘oanSy [eulIdep (19ySrYy 10) PuOdas 9y} Oa" ATUO P[NOM UWOTID91109 SITY} BOUTS ‘qndut 94} ul oSeyd sAtjoe 10} OPV ST UOTZDIIIOD ONT xx 01 X V7 = (Tequourtiedxa) yunod onbey[g L0T X07 =o8vYd vo1J-+ BII9}9"q pazd2jUT 1.01 XS8'T = (parefnoyes) vliazoeq peyoojuT 6070 = UoODeFUT Jo AjOTAHIN AL 801 X T=0qn} Jed eriajoeg $60 = poeqiospe uorjoe1 101 XS0'7=901 XS'T—1201 X77 = 93"Yd paqiospy ICT (ON ainyxtpl ‘Mord sospp fo uoypunusgjaq , eS ee SS ee ANS FiGuRE 3.—Same as figure 2, for phages 74 and T4r. The theoretical curves for y correspond to n=15. vation, while steadily increasing, does not keep pace with the theoretical function y. The cooperation within groups consisting of more than two par- ticles is not as successful in bringing about reactivation as required by the simple theory, whereas pairs of particles apparently collaborate with an efficiency of one hundred percent. The deviation for higher multiplicities is reflected in the curves of figures 5-7. The curves for w as a function of the dose are very close to the theoretical 262 ACTIVE FROM INACTIVATED BACTERIOPHAGE 109 curves for low multiplicities, up to «=0.5; for higher multiplicities they fall below the corresponding curves for y calculated for the same number of units. We can actually find, for each value of x, a curve for y—for the same m but corresponding to a lower «—which fits the experimental curve. The correspond- ing theoretical curves are drawn in figures 5—7. This indicates that groups of more than two particles collaborate in reactivation as if they consisted of a lower but definite number of particles. It is interesting to notice that the systematic deviation from theory for Foure 4.—Same as figure 2, for phages 76 and Tor. The theoretical curves for y correspond to n=30. 263 110 S. E. LURIA AND R. DULBECCO 5 10 IS 20 Vago) Ni FicureE 5.—The probability w of reactivation for phages T2 and 72r as a function of the dose of irradiation r (in hits per particle) for several multiplicities. Abscissae: values of r. Ordinates: values of w. @ values of w for x=0.1-0.2. O values of w for x=0.8-1.5. + values of w for x=2.5-4.0. Broken line: theoretical curve for y as a function of r for n=25, x=0.15. Solid line: theoretical curve for y as a function of r for n=25, x=0.6. Broken and dotted line: theoretical curve for y as a function of r for n=25, x=1.3. 264 ACTIVE FROM INACTIVATED BACTERIOPHAGE 111 5 lO I5 20 25 it FiGurE 6.—Same as figure 5, for phages T4 and T4r. @ values of w for x=0.1-0.2. O. values of w for x=0.8-1.5. + values of w for x=4-7. Broken line: curve of y for n=15, «=0.15. Solid line: curve of y for n=15, x=1.1. Broken and dotted line: curve of y for n=15, x=4.0. 265 142 S. E. LURIA AND R. DULBECCO 5 10 IS 20 25) ren FicurE 7.—Same as figure 5, for phages 76 and Tor. @ values of w for x=0.1-0.2. ©. values of w for x=0.8-1.5. + values of w for x=2.5—-4.0. Broken line: curve of y for n=30, «=0.15. Solid line: curve of y for »=30, «=0.5. Broken and dotted line: curve of y for n=30, «=0.9. 266 ACTIVE FROM INACTIVATED BACTERIOPHAGE 113 increasing multiplicities is less evident for phages 72 than for T6, and still less for 74, diminishing in the same order as the calculated number of units. To summarize, the results show that the probability of production of active phage from inactive particles depends on the dose of radiation and on the multiplicity of infection in a way similar to the one predicted by the simple theory, which assumes lethal mutations in discrete transferable units of equal radiation sensitivity and a hundred percent efficient recombination of active units to reconstitute active particles. All deviations can be accounted for by a limitation in the efficiency of recombination when the active units derive from more than two inactive particles. Several explanations may be offered for this limitation; some of them have been tested experimentally. Lysis from without—failure to liberate phage due to excessive multiplicity of infection (DELBRicK 1940)—was found not to take place for multiplicities of the order of those for which the deviations from theory occur. Limitations in the number of particles of a given phage that can participate in phage growth were looked for and found (see DuLBEcco 1949a), but their magnitude cannot account for the differences between w and y. The process of reactivation must involve complex mechanisms of transfer of genetic material among phage particles. Whatever these mechanisms, it is reasonable to expect that they will work less efficiently as the number of phage particles increases. Limitations may conceivably be caused by steric reasons—shape of the particles, position in the bacterium—or by physiological reasons—limited number of units of some catalyst, competition for substrates. Estimation of the number of units We have compared our results with the theoretical curves for y calculated for different values of m, the unknown number of transferable units per particle. The curves that best fit the results for phages T2 and T2r are those for n= 25; for 76 and Tor, n=30; for T4 and T4r, n=15. For phage T5, no accurate estimate of was obtained, but ” appears to be lower than for 74. If the interpretation of the results based on the simple theory is justified, we must consider the values thus obtained for ~ as minimum estimates of the number of radiation-sensitive, transferable units per phage particle. The estimates are minima because of the assumption of equal sensitivity of all units. Should there be units more sensitive than others, they would be hit more often, and in order to obtain the correct probability of reactivation we should assume more units of the less sensitive type. For example, if one unit were twice as sensitive as the average of the others, one locus of the average sensi- tivity should be added to our estimate in order to distribute the probability of inactivation over all units in such a way that the reactivation probability remains the same. In our preliminary report (LURIA 1947) we calculated 7 in a different man- ner, by assuming that for low multiplicities and low doses, where the probabil- ity of reactivation appeared to be approximately constant as a function of «, we could consider all multiple-infected bacteria as double-infected. This corre- 267 114 S. E. LURIA AND R. DULBECCO sponded to putting = 2 in formula (6) and to comparing the experimental val- ues of w with the values of y for x=0. Upon closer analysis, this method proved incorrect, because the contribution of higher multiple infection cannot be neglected, even for low multiplicities. Analysis of more data showed that the probability of reactivation is in fact not constant for low values of «, but ap- pears to be so for low doses, because the differences are small and of the order of the experimental errors. The use of the wrong approximation made our previous estimates of too high. Yield of active phage from bacteria in which reactivation occurs The yield of active phage following reactivation was studied systematically for phages T2r and T4. After infection, bacteria were diluted and allowed to lyse in liquid, as in a typical “one-step growth” experiment. The latent period before lysis is somewhat longer than for active phage (about 26 minutes instead of 21 for T2, 30 minutes instead of 25 for T4), and the rise in phage titer upon liberation somewhat slower. All yields were calculated from plaque counts after the titer had reached a steady level. The results, shown in table 6, indicate that the yields are generally somewhat lower than those from bacteria infected with active phage particles. No clear relation of yield to dose of radiation or to probability of reactivation was detected. For 727 irradiated with high doses, there is a certain tendency toward higher yields for higher multiplicities. Mixed infection with active and inactive phage Transfer of genetic material involved in reactivation must occur between active and inactive phage particles, since, as we saw before, an active particle of a T-even phage can reactivate an inactive particle of another 7-even phage. If transfer occurred by reciprocal exchanges of genetic material, we should expect that upon mixed infection with active and inactive particles of the same phage some of the active particles would receive inactive units and, therefore, be inactivated. This possibility was tested for phages T2 and T4 by experiments of the following type. Bacteria are added to mixtures containing various proportions of active phage and of phage of the same strain irradiated with different doses. For each mixture, the average numbers of active and of inactive particles adsorbed per bacterium are calculated and, hence, the number of bacteria receiving both active and inactive phage. A plaque count before lysis gives the number of bacteria that liberate phage, while a plaque count after lysis gives the yield of phage per bacterium. In this manner, we can determine whether inactive phage suppresses production of active phage from bacteria that also adsorb an active particle, or possibly affects the yield. The results of these tests can be listed as follows: (a) a bacterium receiving an active particle plus one inactive particle of the same phage—no matter how many hits the latter has received—never fails to liberate active phage; (b) part of the bacteria that adsorb one active particle plus several inactive 268 ACTIVE FROM INACTIVATED BACTERIOPHAGE 115 ones (the latter carrying enough lethal hits, so that reactivation does not occur among them) fail to liberate active phage. This suppression of active phage production is evident for multiplicities 5 or higher of inactive phage T4, and for multiplicities 8 or higher of inactive phage T2. The suppression is in- dependent of the time allowed for adsorption of the phages. It may in part be TABLE 6 The yield of active phage from bacteria in which reactivation takes place DOSE Bae aera OF INFECTION ae 0 6 12 18 0.2-0.25 114 51 122 0.5-0.6 50 42 22 2EO 89 100 29 T2r Well 59 60 9 100 70 94 10-11 73 120 59 130 HITS 0 d5 15 18 0.65 260 120 87 200 ita 200 87 162 T4 1.6 DIS 140 140 et ZAD 197 7-8 135 200 14-15 250 85 accounted for by the limitation phenomenon described by the junior author (DuLBEcco 1949a) ; (c) for those bacteria that liberate active phage, the yield per bacterium is not affected by the presence of inactive particles, but remains the same as in controls without the inactive phage; (d) when bacteria are infected with several heavily irradiated particles that do not give reactivation, and a few minutes later with one active particle, suppression of phage production occurs in a proportion of bacteria that increases with the interval between infections. Suppression is evident with an interval of 2.5 to 4 minutes and practically complete after 10 minutes. These time intervals between infections are, of course, averages, since infection may occur earlier or later for individual bacteria in the same mixture. The yield from those bacteria that liberate phage still remains normal. The suppression of active phage reproduction by inactive phage in excess indicates the existence of some type of “mutual exclusion” between particles of the same phage. Such exclusion is also indicated by the experiments of DuLBecco (1949a). More detailed analysis of the interaction between active and inactive phage, using genetic markers, will be reported in future papers. Our results 269 116 5S. E. LURIA AND R. DULBECCO discussed above are in agreement with earlier observations (Luria and DeELBRUCK 1942) on interference by a large excess of irradiated phage T2 with the growth of active phage T2 when the inactive phage was mixed with bacteria one minute and a half before the active one. Cross-reactivation between phage particles differing by one character Only one group of experiments will be discussed here, because of its bearing on the analysis of the data presented in this article. Bacteria were infected with TABLE 7 Cross-reactivation between one inactive particle of phage T2 and one inactive particle of phage T2r Compare column (3) with column (1) (1) (2)* (3) FRACTION OF BACTERIA RE- OTHER BAC- CEIVING one TERIA THAT INACTIVE PAR- COULD GIVE MULTIPLIC- MULTIPLIC- EXPERI- DOSE OF TICLE 72 AND MOTTLED FRACTION ITY OF ITY OF MENT RADIATION, one INACTIVE PLAQUES (AS. OF MOTTLED INFECTION INFECTION NO. HITS PARTICLE 72r FRACTION OF PLAQUES, FOR T2 FOR 72r AMONG THE THE BACTERIA FOUND BACTERIA THAT THAT LIBERATE LIBERATE AC- ACTIVE PHAGE), TIVE PHAGE, CALCULATED CALCULATED 1A 2.9 0.055 0.055 0.19 0.045 0.14 2A 4.6 0.055 0.049 0.335 0.053 0.16 3A OF 0.055 0.055 0.42 0.074 0.155 4A 3.4 0.053 0.053 0.24 0.046 0.14 SA 5.0 0.053 0.053 0.355 0.068 0.20 7A 3.9 0.05 0.05 0,28 0.046 0.14 8A 6.5 0.05 0.05 0.19 0.087 0.21 * The values in this column include all bacteria with two or more particles of one type and one or more of the other type, plus all bacteria with an active particle of one type and an in- active particle of the other. The values are upper limits, since only a fraction of these bacteria will actually give mottled plaques. phages T2 and T2r, both irradiated (r=5 or 6). Low multiplicities were used, so that a large proportion of the infected bacteria only received one inactive particle, and, of those that received two, a great proportion received one particle of each type. The infected bacteria were plated before lysis, and the plaques examined for the proportion of “mottled plaques,” that is, of plaques containing both 72 and T2r active phages. Such plaques can only arise from bacteria infected with both phages. It is seen from the data shown in table 7 that more than half the bacteria infected with one inactive particle of each of the two phages actually liberate a mixture of active particles of both types. This proves that recombination cannot result from reciprocal exchanges of 270 ACTIVE FROM INACTIVATED BACTERIOPHAGE iv) units between the infecting particles bringing together into one particle all the active units before multiplication begins. Evidently, this particle should be either 72 or T2r, and could not give rise to active particles of both types. This conclusion will be analyzed further in the discussion. Quantitative analysis of the number and contents of mixed yields from mixed infection with T2 and T2r shall be the subject of future publications. Phages inactivated by X-rays or nitrogen mustard In a preliminary article (LuriA 1947) it was stated that no reactivation had been detected for T-even phages inactivated by hard X-rays, using the same technique employed for ultraviolet. The same was found in our laboratory by Miss M. E. WI tis for phages T2 and T6 inactivated by a nitrogen mustard (methyl bis (6-chloroethyl) amine hydrochloride). Experiments by Mr. J. Watson, still in progress in our laboratory, have recently shown, however, that phage T2 inactivated by hard X-rays can take part in reactivation, but this reactivation occurs with such a low probability that special techniques are required for its detection. In part, the low probabil- ity of reactivation of X-ray inactivated phage is due to reduced rate of adsorp- tion. This work will be reported by Mr. Watson in a future publication. It seems possible that phages T/ and 77, for which no reactivation was detected after ultraviolet inactivation, may also be found by similar techniques to give some reactivation. It is clear that the probability of reactivation will be low if the number of transferable units is small, or if each lethal mutation in- volves several units. Its detection will be difficult whenever the number of bac- teria in which reactivation occurs is small in comparison with the number of bacteria that receive residual active phage.‘ DISCUSSION The experiments described above have given results consistent with the hypothesis that inactivation of several and possibly all bacteriophages by ul- traviolet light is to be attributed to lethal mutations in discrete units of genetic material. A genetic basis for inactivation of viruses and bacteria by radiation has often been postulated either on statistical grounds or by analogy (see RAHN 1929; Lea 1947). Our results bring new support to this view, and suggest that most, if not all, the inactivating effect of ultraviolet light (2537 A) on certain phages is due to the production of localized lethal mutations. The hypothesis of inactivation by lethal mutations and reactivation by transfer of genetic material following multiple infection, as developed in this paper, has been useful in suggesting a quantitative analysis of the reactivation phenomena and has led to fairly accurate predictions of the experimental re- sults. The following discussion assumes the correctness of this working hy- pothesis. 4 While this paper was in press, the senior author found that some reactivation by multiple infection takes place with phage TJ inactivated by ultraviolet light. For equal multiplicity of in- fection and equal number of hits, the frequency of reactivation is much lower with T/ than with any of the 7-even phages and with 75. Assuming that the type of analysis presented in this paper applies to the results with 7/, a value of 7 smaller than 5 would be obtained. 271 118 S. E. LURIA AND R. DULBECCO According to our analysis, active phage is reconstituted from inactive by re- incorporation of active units derived, directly or indirectly, from the inactive particles in a kind of hybridization. As in hybridization, the possibility of recombinations between particles of different wild-type phages suggests the existence of common genetic determinants and the absence of complete incom- patibility. It is possible that various interference phenomena among different phages may result from such an incompatibility. Reassembly of material from inactive particles into active ones is a remark- ably efficient process. Genetic material from several inactive particles may be brought together, although the relative efficiency of cooperation diminishes as the number of particles involved in this pluriparental reproduction increases. We have given estimates for the minimum number of transferable units per particle for several phages. It is interesting to notice that phage 74, more resist- ant than the related phages 72 and 76 to ultraviolet light (figure 1), appears to have fewer units. This may indicate absence of a portion of genetic material present in the other 7-even phages. Lea and SALAMAN (1946), analyzing the dependence of the rate of inacti- vation of phages by X-rays as a function of the density of ionization, and assuming that the radiosensitive material consisted of spherical units, arrived at the conclusion that a large phage contained 14 such units, whereas a small phage contained one only. Although the hypotheses involved were probably oversimplifications, the conclusion receives qualitative support from our re- sults. We must consider next the possible mechanisms of genetic transfer. We may divide the mechanisms into two groups, those in which the reproducing element is supposed to be at all times the phage particle as a whole, and those in which the reproducing elements are assumed to be component parts of the particle. The simplest hypothesis of the first group would be that reactivation results from pairing (or grouping) or the initial infecting particles, followed by reciprocal exchanges such as occur in chromosomal crossing-over; if these exchanges lead to formation of an active particle, the latter proceeds to multi- ply. This simple hypothesis can easily be disproved. The high efficiency of reactivation would require very large numbers of successive reciprocal ex- changes to bring together all active genetic material before multiplication takes place. Mixed infection with one active and one inactive particle should also lead to the occasional loss of active phage, since we know that genetic recombina- tions occur between active and inactive particles. Finally, the fact that in- fection with one particle each of inactive T2 and inactive T2r yields a mixture of active T2 and active T2r disproves this hypothesis, since any number of reciprocal exchanges between the original particles before multiplication could never lead to formation of active particles of both types. Another interpretation based on reciprocal exchanges would be that inactive particles reproduce, and that exchanges occur at various stages of the repro- duction among the original particles or their inactive offspring. These ex- changes should be numerous enough to make the probability of incorpo- ration of all active units into an active particle close to unity, and an ac- 272 ACTIVE FROM INACTIVATED BACTERIOPHAGE 119 tive particle, once formed, should be favored in multiplication. Without completely disproving it, our results make this explanation very unlikely. The fact that the probability of reactivation increases greatly by increas- ing (for example, from 8 to 20) the multiplicity of infection with heavily irradiated particles would require a very high number of exchanges. To obtain high yields of active phage in these cases we should assume, moreover, that the exchanges take place early, and that the active particles, once formed, multiply with little or no interference from the inactive particles in large excess. This seems contradicted by the fact already mentioned that active particles can actually undergo interchanges with inactive ones. Altogether, there is strong evidence that inactive units have less chance than active units of enter- ing the final particles, even when the active units derive from inactive particles. In search for a mechanism that could selectively bring together the active units, the senior author (LuriA 1947) suggested the hypothesis of independent reproduction of individual units to form a “gene pool,” from which the new active particles could be derived. Inactive units were considered to be those that cannot reproduce and that have, therefore, little chance of incorporation into the final particles. The tendency to reduction in yield may be due to oc- casional incorporation of some of the original inactive units. No hypothesis is made as to how the units reproduce or reassemble. The last step is the most difficult to visualize, and we incline to the belief that the original particles may play a role in it, possibly by supplying a framework for reassembly. This is suggested by the limited efficiency of collaboration among large groups of particles, which indicates a certain tendency of the units to remain together with their original companions. One may ask whether, inside bacteria in which reactivation does not take place, the active units present in the infecting particles reproduce or not. CoHEN (1948) states that no desoxyribose nucleic acid is synthesized in bac- teria infected with particles of T2 exposed to doses of ultraviolet light much higher than those employed in our study. Preliminary cytological evidence, collected with the collaboration of Dr. C. F. Rosrnow in our laboratory, indicates that in infected bacteria, in which reactivation does not take place, there is no accumulation of stainable material supposedly representing des- oxyribose nucleotides. If this evidence is confirmed and found to apply to the conditions of our experiments, it might then suggest, either that there is no reproduction of active units when they are not all present (which might al- together invalidate the hypothesis of independent reproduction), or that at least part of the reproduction of the active units may take place without increase in desoxyribose nucleotides. The hypothesis of a “gene pool,” although by no means the only possible one,® fits all results of reactivation. Its validity may soon be amenable to 5 Another possibility, suggested by Dr. A. H. SturTEVANT, would be a process of zipperwise replication of the various units of a phage particle. When in this process an inactive unit was reached, replication could only continue if the partial replica came in contact with another phage particle in which that unit was active. The process would then continue by addition of replicas of the active units of the second phage particle. If repeated several times, such a mechanism would provide for selective recombination of all active units. 273 120 S. E. LURIA AND R. DULBECCO critical test. In postulating a phase in phage growth in which particles, as we know them in the extracellular phase, are not present, the hypothesis accounts for the repeated failures to obtain active phage by premature artificial dis- ruption of infected bacteria. It agrees with the observation made by FosTER (1948) in our laboratory that in the presence of proflavine the reactions leading to production of active phage proceed normally for a part of the latent period, but, upon lysis, no active particle is liberated. According to FosTER (1948) active phage particles are present in the infected bacterium after the proflavine sensitive stage is passed—beginning 12 to 14 minutes after infection for phages T2 or T6. A similar conclusion was reached by A. H. DoERMANN on the basis of experiments on the effect of other inhibitors on the growth of T3 and T4r (DoERMANN 1948). That viruses multiplying inside the host cell may not have the same organization as in the extracellular form has been suggested before for certain animal viruses (see BLAND and Rosinow 1939). Our theory does not assume any degree of linkage among units, although linkage may be compatible with the theory. Each group of strongly linked units would behave as one unit, possibly as a particularly sensitive one. Weakly linked units would probably reduce the probability of reactivation for high doses, when lethal units present in one group would hinder the utilization of the linked active units for reactivation. In their work on the y and mutants of phage 72 H, HERSHEY and ROTMAN (1948, 1949) found evidence for a series of determinants exhibiting various degrees of linkage, from those apparently unlinked (high frequency of inde- pendent transfer, no correlation between the frequencies of complementary re- combinant types in the yield) to others rather strongly linked. For the latter ones, the authors considered that their results suggested the possibility of re- ciprocal exchanges. As a working hypothesis we may assume, together with HERSHEY and Rot- MAN (1949) that unlinked determinants may be located in different reactivation units, possibly transferred by a gene pool mechanism, while linked determinants may be located in the same reactivation unit. If reciprocal exchanges were found to occur, then homologous units or groups of units should be supposed to pair or group together at some stage in the growth process. Techniques recently developed in our laboratory should soon permit a study of the inactivation of individual genetic determinants and a solution of some of these problems. It appears, therefore, advisable to refrain from further discussion at the present time. The formation of active phage from inactive by transfer of discrete units requires some revision of the interpretation of experiments on irradiation of phage inside infected bacteria with ultraviolet light (LuRta and LaTaRJET 1947). In case of multiple infection, the survival curves for phage-producing ability immediately after infection indicated suppression by damage of a num- ber of centers, with the sensitivity of individual centers lower than that of extracellular phage particles. It now seems clear that what was measured was the rate of inactivation of individual units rather than of whole particles. The curves for suppression of the phage-producing ability of multiple-infected 274 ACTIVE FROM INACTIVATED BACTERIOPHAGE 121 bacteria immediately after infection are similar to the curves for the probabil- ity of reactivation for a comparable group of irradiated particles. For example, the suppression curve given by Luria and Lartarjet (1947) for bacteria infected by five particles of phage 72 is very similar to the curve for the prob- ability of reactivation w as a function of r for x=5. It is clear that in interpret- ing experiments on inactivation of intracellular phage it will be necessary to take into account the occurrence of genetic transfers.® SUMMARY Coli-bacteriophages T2, T4, T5, and T6 inactivated by ultraviolet light are still adsorbed by sensitive bacteria. Bacteria infected by only one inactive phage particle are not lysed and do not yield active phage. Infection of bacteria with more than one inactive particle leads to lysis and production of active phage in a fraction of the bacteria. This fraction diminishes with increasing doses of radiation and increases with increasing numbers of particles adsorbed per bacterium. The assumption is made that inactivation is due to lethal mutations in a number of genetic “units” of the phage particle, and that pro- duction of active phage from inactive is due to recombination of non-lethal units to form active particles. The values of the probability of active phage production calculated from these assumptions agree with the experimental results with certain limitations. In order to explain the very high frequency of recombination, the hypothesis is proposed that phage growth occurs by independent reproduction of each unit followed by reassembly of the units into complete phage particles. The minimum number of units per particle is esti- mated for various phages. LITERATURE CITED Buanp, J. O. W., and C. F. Ropinow, 1939 The inclusion bodies of vaccinia and their relation- ship to the elementary bodies studied in cultures of the rabbit’s cornea. J. Path. Bact. 48: 381-403. CouEN, S.S., 1948 The synthesis of bacterial viruses. I. The synthesis of nucleic acid and protein in Escherichia coli B infected with T2rt bacteriophage. J. Biol. Chem. 174: 281-293. Detsriick, M., 1940 The growth of bacteriophage and lysis of the host. J. Gen. Physiol. 23: 643-660. 1946 Bacterial viruses or bacteriophages. Biol. Rev. Cambridge Phil. Soc. 21: 30-40. De.sricx, M., and W. T. Bairey, Jr., 1946 Induced mutations in bacterial viruses. Cold Spring Harbor Symp. Quant. Biol. 11: 33-37. Decpriick, M., and S. E. Lurta, 1942 Interference between bacterial viruses. I. Interference between two bacterial viruses acting upon the same host, and the mechanism of virus growth. Arch. Biochem. 1: 111-141. DoerMANN, A. H., 1948 Intracellular growth of bacteriophage. Carnegie Instn. Wash. Yearb. (in press). 6 While this paper was in press, one of us (DULBECcO 1949b) discovered that ultraviolet ir- radiated phages can be reactivated by exposure to visible light of short wave length in presence of bacterial cells. This “photoreactivation” differs from reactivation by multiple infection in many of its features. Photoreactivation does not take place to any appreciable extent under the condi- tions in which the experiments reported in this paper were performed. A series of experiments of the type exemplified in table 4, but carried out in dim yellow light—under conditions that com- pletely avoid photoreactivation—gave results undistinguishable from those of the earlier experi- ments, in which no precaution had been taken to control illumination. 275 122 S. E. LURIA AND R. DULBECCO Dutsecco, R., 1949a The number of particles of bacteriophage 72 that can participate in intra- cellular growth. Genetics 34: (in press). 1949b Reactivation of ultraviolet inactivated bacteriophage by visible light. Nature (in press). Foster, R. A. C., 1948 An analysis of the action of proflavine on bacteriophage growth. J. Bact. 56: 795-809. Hersuey, A. D., and R. Rotman, 1948 Linkage among genes controlling inhibition of lysis in a bacterial virus. Proc. nat. Acad. Sci. 34: 89-96. 1949 Genetic recombination between host-range and plaque-type mutants of bacteriophage in single bacterial cells. Genetics 34: 44-71. Latarjet, R., and R. Want, 1945 Précisions sur l’inactivation des bactériophages par les rayons ultraviolets. Ann. Inst. Pasteur 71: 336-339. Lea, D. E., 1947 Actions of radiations on living cells. xii+402 pp. Cambridge: University Press. Lea, D. E., and M. H. Sataman, 1946 Experiments on the inactivation of bacteriophage by radiations, and their bearing on the nature of bacteriophage. Proc. roy. Soc., B, 133: 434-444. Luria, S. E., 1947 Reactivation of irradiated bacteriophage by transfer of self-reproducing units. Proc. nat. Acad. Sci. 33: 253-264. 1948 Bacteriophage mutations and genetic interactions among bacteriophage particles in- side the host cell. A.A.A.S. Symposium on Genetics of Microorganisms (in press). Luria, S. E., and M. DELBRUck, 1942 Interference between bacterial viruses. II. Interference between inactivated bacterial virus and active virus of the same strain and of a different strain. Arch. Biochem. 1: 207-218. Luria, S. E., and R. Latarjet, 1947 Ultraviolet irradiation of bacteriophage during intracel- lular growth. J. Bact. 53: 149-163. Rann, O., 1929 The size of bacteria as the cause of the logarithmic order of death. J. gen. Physiol. 13: 179-205, APPENDIX ON THE RELIABILITY OF THE POISSON DISTRIBUTION AS A DISTRIBUTION OF THE NUMBER OF PHAGE PARTICLES INFECTING INDIVIDUAL BACTERIA IN A POPULATION R. DuLBEcco In calculating the average number x of phage particles adsorbed per bacteri- um (multiplicity of infection) from the number of uninfected bacteria, and, from x, the proportion of bacteria with any given number & of particles, the assumption is made that the distribution of particles per bacterium is a Poisson distribution. One limitation to this assumption may arise from differ- ences in the surface area of individual bacteria. This limitation was analyzed as follows. In a mixture of P particles and B bacteria, each phage particle has a prob- ability p=c(1/B) to be adsorbed by a given bacterial cell. In actual cases where P and B are large and ¢ is between 0.4 and 0.9, we have : x=cP/B. If c is constant for all bacteria, the distribution of & is a Poisson distribution; if not, the distribution of k will be different. We assume, in first approximation, that the adsorption capacity of a bacterium is proportional to its surface, and that the surface is proportional to the length—considering bacteria as cylinders with uniform diameter and negligible end surfaces. The distribution of bacterial lengths was obtained experimentally on stand- ard cultures of E. coli B containing 108 cells per ml. Negative stains with 276 ACTIVE FROM INACTIVATED BACTERIOPHAGE 123 nigrosin were made without fixation, and, after the slides were dried, 764 cells were measured with an ocular micrometer (Filar). The distribution of lengths is given in figure 8, and will be referred to as the “B distribution.” Measurements on five cultures gave similar distributions. For the purpose of obtaining the distribution of phage particles adsorbed per bacterial cell, or “P distribution,” we shall split the bacterial population in- 300 250 MEAN=L=25.7I7 MICROMETER UNITS VARIANCE = 109.36 (MICROMETER UNITS)? 200 = 0.1653 L* 150 NUMBER OF CELLS 50 100 150 200 MICROMETER UNITS FicurE 8.—The distribution of bacterial lengths in standard cultures of Escherichia coli strain B containing 108 cells per ml. to a number of subpopulations, each comprehending one of the length classes in figure 8, and assume that the adsorption capacity of all cells within each subpopulation is constant. By the calculation given at the end of this paper, the following properties of the P distribution are derived: a) its arithmetic mean is equal to x, that is, to the multiplicity of infection, as for a Poisson distribution. b) its variance is equal to the variance x of the Poisson distribution, plus a term equal to the variance of the B distribution: var(P) =x-+var(B). If we express the variance in terms of the mean «x taken as the unit of meas- ure, we obtain for our experimental cultures: var(P) =«+0.1653x*. This equa- tion expresses the relation between the variance of the P distribution for an ideal population with uniform capacity of adsorption and the variance for a real population with a capacity of adsorption distributed with a given variance. The P distributions for several values of x, calculated from the actual B distribution, are given in table 8, together with the Poisson distributions 277 124 S. E. LURIA AND R. DULBECCO for the same values of x. It is clear that for low multiplicities of infection there is little difference between actual and Poisson distributions. For higher multiplicities, the discrepancies increase; still, for x=1.5, they only affect relevantly the proportions of bacteria with 5 or more particles. TABLE 8 The P distribution for various values of x “P” columns give the frequencies in the P distribution, ‘‘Poisson” columns give the fre- quencies in the Poisson distribution. All frequencies are multiplied by 10°. x=0.2 x=1.5 x=2.5 x=5 x=7.5 x=10 K el 12) POISSON 1D POISSON| P POISSON| P POISSON P POISSON| P POISSON 0 82115 | 81873 | 24686 | 22313 | 10400 8208 1409 674 228 55 32 5 1 15977 | 16374 | 32918 | 33468 218 45 2 1735 1637 | 23197 | 25101 765 227 3 155 109 | 11658 | 12551 1908 757 4 14 6 4751 4707 3739 1892 5 2 1721 1412 6065 3784 6 611 353 8411 6303 7 238 76 10218 9009 8 109 14 11081 | 11261 9 62 2 10924 | 12512 10 38 9942 | 12512 11 29 8489 | 11375 12 19 6902 9479 13 12 5383 7292 14 8 4089 5208 15 5 3036 3472 16 3 2219 2170 17 1 1600 1276 18 1 ! 1140 709 19 810 373 20 579 187 21-23 931 147 24-26 464 11 27-30 293 31-33 172 34-36 110 37-39 67 >39 305 The calculation of «x from the bacterial survival—assuming (B unin- fected) /B=e-*—only gives reliable results for values of x up to about 2. For higher multiplicities, it leads to an underestimation of x, THEORY The bacterial population is considered as a mixture of an infinite number of subpopulations homogeneous in adsorption capacity. Within each subpopu- lation there is a Poisson distribution of phage particles per bacterium, whose frequency function is: e-tig,* fi(k) = To (1) where x; is the multiplicity of infection in the jth subpopulation. x; is obtained from the B distribution of bacterial lengths: if we call /; the length of the bac- teria in the jth class, we have: 278 ACTIVE FROM INACTIVATED BACTERIOPHAGE 125 l; xj =x = (2) where x= multiplicity of infection in the total population, L=arithmetic mean of the B distribution (average bacterial length). The frequency function of the distribution of phage particles per bacterium in the fotal population (P distribution) is i) co je 7ix;* fk) = 2D, ngi(k) = 2) ——— (3) j=0 j=0 k! where ; is the frequency of the jth subpopulation in the B distribution mj =1 j=0 The Poisson distribution of phage particles per bacterium within each homo- geneous subpopulation of bacteria has the following first two moments: First moment about the origin = (4y’) ;=%; Second moment about the mean = (2) ;= 4; For the P distribution in the total population, remembering formula (3), we have a mean io) foo) (u1')p = Do Ds nifi(k) = >) mi 2 hfj(k) = Do n(m'); = a 3X3; j=0 j=0 from formula (2) we obtain: (ui')p = &. (4) To find the variance (u2)p of the P distribution, let us remember that, in general, He = we’ — (m’)? (S) where 2’ is the second moment about the origin. We have then: (usp = DLR? DY mifi(k) = Doms DD RR) = Do niu’); pay eB 7=0 » k=0 j=0 where (u2’); is the second moment about the origin of the distribution of phage particles per bacterium in the jth subpopulation, But (u2’);=axj;+(x;)? (since, in general, pe’=mu2+(m’)?), and, for each homogeneous subpopulation, (ue); = (u1’);=%;. We obtain, therefore: (ua!)p = Do mi(ue!)s = DE many + DD nj(as)?. (6) j=0 j=0 j= Finally, introducing the values obtained from (4) and (6) in formula (5): (u2)p = Dy miei + Do mj(x;)? — 22. (7) j=0 j=0 The first term on the right side of equation (7) equals x; the other two terms equal the variance of the B distribution, expressed as function of x. We obtain therefore, (u2)p = x + var (B). 279 INACTIVATION OF BACTERIOPHAGES BY DECAY OF INCORPORATED RADIOACTIVE PHOSPHORUS* By GUNTHER S. STENT anp CLARENCE R. FUERSTt (From the Virus Laboratory, University of California, Berkeley) (Received for publication, September 29, 1954) It was observed by Hershey, Kamen, Kennedy, and Gest (1951) that bacteri- ophages are unstable if they contain radiophosphorus P*” of high specific ac- tivity. From day to day, progressively decreasing fractions of such popu- lations of radioactive phage are still able to form plaques when plated on a sensitive bacterial strain, and the rate of loss of infective titer depends on the specific activity of the P® assimilated. It is the purpose of this communi- cation to present experiments in which these observations of Hershey et al. have been extended to the study of the lethal effects of P* decay in various strains of bacteriophage at various temperatures and to the examination of some of the biological properties of the inactivated bacteriophage particles. Some of these experiments have already been reported in preliminary form (Stent, 1953 a). Materials and Methods Bacteriophages T1, T2, T3, T5, T7, and their host, E. coli B/r, and phage \ and its host, E. coli strain K12S, were used in this study. Strain B/r, a radiation-resistant mutant derived from strain B, was kindly supplied to us by Dr. Aaron Novick. Glycerol-casamino acid medium refers to a medium devised by Fraser and Jerrel (1953). H medium is a glycerol-lactate medium of the following composition per liter of distilled water: 1.5 gm. KCl, 5 gm. NaCl, 1 gm. NH,Cl, 0.25 gm. MgSO,-7H,0, 10-4 N CaCl, 0.07 m sodium lactate, 2 gm. glycerol, 0.5 gm. bacto-peptone Difco and 0.5 gm. bacto-casamino acids Difco. H medium contains 6 mg./liter total phosphorus, of which 5 mg./liter are supplied by the casamino acids and 1 mg./liter by the peptone. Control experiments show that this phosphorus is assimilated by cultures of E. coli neither more nor less readily than inorganic phosphate. The techniques described by Adams (1950) were employed for the general pro- cedures of bacteriophagy. Radiophosphorus was obtained as carrier-free H3P*°°O, from the Isotope Division of the Atomic Energy Research Establishment, Harwell, England. Measurements * This investigation was supported by grants from the National Cancer Institute of the National Institutes of Health, Public Health Service and The Rockefeller Foundation. t Holder of a National Research Council of Canada Special Scholarship. Reprinted by permission of The Rockefeller Institute from THe JOURNAL OF GENERAL PuysioLocy, 38 (4), 441-458, March 20, 1955. 280 442 INACTIVATION OF BACTERIOPHAGES of radioactivity were made on dry samples by means of an end-window GM tube, whose counting efficiency for P* had been established by reference to a standard solu- tion of radiophosphorus supplied by the National Bureau of Standards, United States Department of Commerce. The specific radioactivity of the growth media was de- termined by radioactive counting and chemical analysis of total phosphorus in the case of a number of T2 lysates in order to establish the specific inactivation rate aN for that phage and to confirm the value obtained by Hershey et al. To conserve the supply of isotope, the specific activity of the growth medium in the case of the other phages was usually estimated only by reference to the rate of inactivation of a stock of T2 grown in an aliquot of the same medium. Bacteriophages of high specific activity were grown in the following way: A volume of the radioactive stock solution containing the desired amount of P* was evaporated to dryness in a boiling water bath and resuspended in 0.1 ml. of H medium. The radio- active growth medium was then adjusted to neutral pH and inoculated with 0.01 ml. of a culture of 2 X 10’ cells/ml. of B/r already in its exponential phase of growth in non-radioactive H medium. The growth of the radioactive culture at 37°C. was followed by microscopic counts in a Petroff-Hausser bacterial counting chamber. When the bacterial density reached 5 X 10’ cells/ml., the culture was infected with 0.01 ml. of a stock containing 10’ phages/ml. and incubated until microscopic counts indicated satisfactory lysis. At this point, the remainder of the 0.1 ml. culture was diluted into cold glycerol-casamino acid medium and assayed for its titer of infective phage particles. Experimental Results Rate of Inactivation.— Hershey ef al. observed that if a stock of T2 or T4 containing P® at high specific activity was assayed daily, the logarithm of the number of surviving phages fell linearly with the number of P® atoms that had decayed up to the time of assay. The slope of this survival curve was found to be proportional to the specific activity of the medium in which the phages had been grown, pro- vided that the stock was stored in sufficiently great dilution under conditions in which control lysates containing an equal amount of non-incorporated P® were stable. This indicated that the inactivation of one phage particle was not due to the radiation emitted by the radioactivity contained in other phages but was the consequence of the disintegration of one of its own atoms of P*. The rate of change in the fraction s of surviving phage particles with the time tin days may, therefore, be expressed as ds/dt = —aN*s (1) in which a is the fraction of the P® disintegrations which are lethal (hereafter referred to as the ‘‘efficiency of killing’), V* the number of radioactive phos- phorus atoms per phage particle, and X the fractional decay of P® per day. Integration of (1) and substitution of more practical parameters lead to logios = —1.48 X 10-6wAoN(1 — e) (2) 281 G. S. STENT AND C. R. FUERST 443 in which Ao is the specific radioactivity (in millicuries per milligram of phos- phorus) of the growth medium and NV the total number of phosphorus atoms per phage particle. Hence, a plot of logios vs. (1 — e™), the fraction of all P® atoms decayed by the ¢* day, should be a straight line with slope proportional to Ao, the relation actually observed experimentally. We have studied the inactivation by P*® decay of five virulent coliphages T1, T2, T3, T5, T7, and of the temperate coliphage i. All these strains, except the pair T3-T7, are serologically unrelated, differ in their chemical constitution, morphology, genetic structure, and manner of interaction with bacterial host cells. Radioactive stocks of each strain were grown by the procedure indicated above in media ranging in specific radioactivity from 100 to 300 mc./mg. At these specific activities, approximately 0.03 to 0.1 per cent of all phosphorus atoms are present as the P® isotope. The lysates, whose titer usually represented at least a thousandfold increase over the inoculum, were stored at 4°C. in casamino acid-glycerol medium and the number of infective centers assayed from day to day. The results are presented in Fig. 1 in which the logarithm of the fraction of the survivors in the different phage stocks is plotted against (1 — e*). It is seen that inagreement with equation (2) a straight line survival curve is obtained in every case. The specific death rates aN, having the di- mension lethal atoms per phage and obtained by dividing the observed slopes of the lines of Fig. 1 by —1.48 X 10~® Ao, are listed in Table I. Control ex- periments, not shown in Fig. 1, indicated that non-radioactive stocks of all six strains were stable in casamino acid-glycerol medium at 4°C. and that the radioactive lysates had been diluted sufficiently far to avoid inactivation by any external P*. The six phages evidently fall into two classes of sensitivity to P® inactivation. One class, composed of T2 and TS, is characterized by 4.5 X 10* lethal atoms per phage, the value already observed by Hershey et al. for T2 and T4. The sensitivity of the other group, comprising T1, T3, T7, and X, corresponds to 1.5 X 104 lethal atoms per phage. Hence the strains of the second group are only one-third as sensitive to inactivation by decay of P*® as those of the first. Phosphorus Content and Efficiency of Killing.— The efficiency of killing per disintegration, a, may be calculated from the specific death rate, aN, if the number of phosphorus atoms per infective unit is known. The phosphorus content of each phage strain was, therefore, deter- mined by means of the following procedure, the results of which are listed in Table I. A stock of each phage was grown in H medium containing P® at a low but ac- curately determined specific activity. The lysate was clarified and freed of bacterial debris by two low speed centrifugations (10 minutes at 5,000 g) and the phage sedi- 282 444 INACTIVATION OF BACTERIOPHAGES mented and washed three times in nutrient broth by high speed centrifugations (60 minutes at 10,000 r.p.m. for T2, T5; 90 minutes at 15,500 R.p.m. for T1, T3, T7). The number of plaque-forming units and the P® content of the purified suspension 0) 4 Sia lia O 4 Saali2 O 4 8 12 DAYS a ° oO < x Ww (>) = Ze > a =) wn f°) 02 04 re) 02 04 ) 02 64 |-eAt fe) AS iN (Suil2 ie YO 4 8 2 DAYS 10° me fe) oe 2 10 [a cg re (oo) PS Se S10 = @ = w [o7> 0), 02 04 | 0. Oe, Joa et Fic. 1. P® inactivation of T1, T2, T3, T5, T7, and \ at +4°C. Ao = specific ac- tivity of growth medium. were then assayed and the phosphorus content per infective unit calculated on the basis of the specific activity of the growth medium. In each case, more than 90 per cent of the P* of the purified suspension could be adsorbed specifically to sensitive bacterial cells, indicating that practically all the radioactivity resided in morpho- logically intact bacteriophage particles. The results of this analysis agree well with the phosphorus content of T2 determined by Hershey, Kamen, Kennedy, and Gest 283 G. S. STENT AND C. R. FUERST 445 (1951) and by Hershey and Chase (1952). The agreement is poor, however, with the estimations of the phosphorus contents of T1, T2, T3, T5, and T7 by Labaw (1951) whose values are about twice as great as those found here. No values are listed in Table I for the phosphorus content of A, since it was not possible to prepare a puri- fied suspension of P*-labelled \ in which the bulk of the radioactivity could be ad- sorbed specifically to sensitive bacteria. Neither the reason for this behavior of A nor the nature of the non-adsorbed material has yet been discovered. The last column of Table I lists the efficiency of killing, a, of P® decay in each of the five strains of T phage. It is seen that in all the strains studied here, a is near the value 0.09 originally observed by Hershey e¢ al.; i.e., on the TABLE I Evaluation of the Parameters of the Equation logis = —1.48 X 10-ApaN (1 — e*) at 4°C. Phage Slope of L ae P per infective N strain Ao death curve annie. unit prey me S me./mg. mg. Ale2 160* —10.5 4.5 & 10% | 2.3 X 10-4 4 Sax<. 10* | 1.8 <<10—4 3nop el 08 0.12 Atal 270t —7.0 Livi lOeeinOnd X< 10514 1.4 XK 105 0.12 T3 160* —3.1 t.3) <7 10% | 0.9 < 10} ? ? ? * Determined radiochemically. t Determined by comparison with control T2 stock. average one of about every ten P*® disintegrations inactivates any phage particle in which it occurs. Effect of Temperature on the Efficiency a.— The rate of inactivation by decay of P* was also measured at two lower temperatures in the frozen state. For this purpose, aliquots of diluted radio- active lysates of all six phage strains were stored either at +4°C., or in the frozen state at —20°C. or —196°C. (the temperature of boiling liquid nitro- gen). Samples were then thawed from day to day and assayed for the fraction of surviving infective centers. Frozen controls with corresponding non-radio- active lysates showed that, depending on the strain, from 45 to 90 per cent of the infective centers survive freezing and thawing and that, except in the case of storage of T2 at —20°C., the fraction recovered is independent of the length of time of storage (Sanderson, 1925; Rivers, 1927). It was found that 284 446 INACTIVATION OF BACTERIOPHAGES at these lower temperatures the rate of inactivation by P® decay of all five strains was significantly reduced. Since the rate of radioactive decay is in- dependent of temperature, it follows that a reduction in @ by the altered environmental conditions must be responsible for the reduced rate of bacteri- ophage inactivation. Table II lists the observed values of the slope of the inactivation curves at +4, —20, and —196°C. and the fractional reduction of a compared to its magnitude at +4°C. It is seen that radioactive decay proceeding at — 20°C. inactivates the phages with an efficiency of only 70 per cent of decay proceeding at +4°C. Lowering the temperature to —196°C. TABLE II The Relative Efficiency of P® Inactivation at Low Temperatures | ee Storage at —20° Storage at —196° Phage strain Ao | Slope* Slope* — Slope* aa ) mce./mg. T2 160 —10.5 —6.8 0.65 130 —8.6 —5.6 0.65 125 —8.3 —5.7 0.69 a5 130 —8.5 —5.8 0.68 —4.5 0.53 125 —8.1 —5.6 0.69 —4.6 0.57 AGil 270 | —7.0 —4.8 0.69 —3.9 0.56 ARS} 160 —3.1 —1.6 0.52 Abe 270 —6.4 —4.6 0.72 —3.6 0.56 ON 220 | —4.8 —3.4 0.71 —3.3 0.54 | \ * Refers to the value of —1.48 & 10-®A gal. reduces the fraction of lethal disintegrations even further. At this temperature the efficiency of killing in T1, T3, T5, T7, and 2 is only 55 per cent and in T2 only 65 per cent of its value at +4°C. Since low temperatures appear to reduce the efficiency a, it seemed possible that radioactive decay occurring at temperatures higher than +4°C. might inactivate bacteriophages with greater efficiency. At elevated temperatures, however, bacteriophages are subject to thermal inactivation, and it is only possible to study the combined effects of heat inactivation and radioactive decay. To examine, therefore, the efficiency a at reasonably high temperatures, a heat-stable mutant, T5,¢, was first selected from our strain of TS by the pro- cedure of Adams (1953). When stored in glycerol—casamino acid medium at 285 G. S. STENT AND C. R. FUERST 447 65°C. a stock of T5,, loses 90 per cent of its titer in 5 hours. T5;, is inacti- vated by P® decay at 4°C. with the same specific death rate as the wild type TS. One stock of T5,, was grown in H medium containing radioactive phos- O 12 24 36 O 12 24 36 HOURS SURVIVING FRACTION ie} 002 004 006 0.08 0 0.02 004 006 0.08 l-e-At (9) 12 24 0 12 24 0 12 24 HOURS 10°§ $6 f (0) ro. = OSs Oo ° ° oxo) Rn =) O° ~h e ° af 10 z aXe : (oe) od q 2 SG e ac !0 Ak Le e u g NV > -3 ° = 10 Ge = Tp) Vo eto in Oe 60°C. 65°C. e fr) 002 004 0060 002 004 0 002 004 I-e-M Fic. 2. Inactivation of T5,¢ at different temperatures. Filled circles, radioactive lysate, 4g = 300 mc./mg. Open circles, non-radioactive control lysate. phorus at specific activity of 300 mc./mg. (at which level 0.1 per cent of all phosphorus is P*) and one in non-radioactive H medium. After dilution into glycerol-casamino acid medium, aliquots of both lysates were stored at 4, 50, 55, 60, and 65°C. and assays of the number of infective centers made from time to time. The result of this experiment is presented in Fig. 2. It is seen that the rate of inactivation of the radioactive lysate is almost the same at 4, 286 448 INACTIVATION OF BACTERIOPHAGES 50, and 55°C., at which temperatures the non-radioactive control lysates exhibited little or no heat inactivation. At 60 and 65°C., however, considerable increases in the rate of inactivation of the radioactive T5,¢ lysate are observed, at which temperatures the non-radioactive control lysate now also exhibits an increasing instability. Since the rate of loss of titer of the radioactive lysate may be presumed to be the sum of the rate of death due to heat and to radio- active decay, the rate of P* inactivation can be estimated at any temperature by subtraction of the slope of the survival curve of the non-radioactive con- of 0.4 OS 0.2 0.1 — 0 “196° -20° 0 +20° +40° +60" TEMPERATURE DURING DECAY Fic. 3. The efficiency of killing, a, in T5 at different temperatures. trol from that of the radioactive lysate. (This subtraction of slopes is justified only in experiments of short duration, while (1 — e’) is still approximated by Mt.) The efficiency of killing a at that temperature can then be computed from this difference of rates by means of equation (2). The result of such calculations based on the slopes of Fig. 2 is presented graphically in Fig. 3, in which a has been plotted against the temperature of decay. It is evident that a increases slowly between 4 and 55°C. and begins to rise sharply after that point. At 65°C., @ has reached the value 0.31, which means that now almost one in every three P® disintegrations is lethal to T5,:. Also included in Fig. 3 are the results of the estimations of a in T5 at low temperatures. 287 G. S. STENT AND C. R. FUERST 449 Evidently, it is possible to effect at least a fourfold variation.in a by varying the temperature of storage from the lowest to the highest practicable range. It is to be noted that the increase in a per degree is greater between — 20 and +4°C. than between +4 and +50°C. This, no doubt, implies that a is affected not only by the ambient thermal energy, but also by the change of phase from liquid to solid state. P® Decay after Infection.— Hershey and Chase (1952) have shown that when T2 infects a sensitive bacterium, the phosphorus, and hence the DNA, of the bacteriophage particle enters the host cell, whereas the bulk of the phage protein remains outside. It may then be asked whether P*® decay can still prevent the reproduction of the parental phage and the ultimate emergence of infective progeny if such decay occurs only after the introduction of the DNA of a radioactive T2 particle into the interior of the bacterial cell. In order to study the effect of P® decay after infection, it is necessary to arrest intracellular phage development reversibly for days or weeks so that the slow radioactive decay may proceed at an early stage of the brief 20 minute latent period. This can be achieved by quick-freezing the bacterial cells shortly after infection and storing them at — 196°C. in liquid nitrogen. As in the case of free phages, non-radioactive controls show that more than half of the infected centers survive freezing and thawing, and that the fraction recovered is independent of the length of storage at — 196°C. In those infected bacteria which survive, phage development resumes upon thawing where it had left off at the moment of freezing. A culture of strain B/r was grown in nutrient broth to a density of 108/ml., centri- fuged, and resuspended in fresh broth at one-fourth of its original volume. The sus- pension was then infected with 3 X 10’/ml. radioactive T2 particles, containing P*® at a specific activity of 88 mc./mg. Phage development was again arrested 2.5 minutes after infection by chilling the culture in ice. The infected bacteria were separated from the small fraction of unadsorbed free phage by centrifugation and resuspended in cold glycerol-casamino acid medium. Aliquots of 0.1 ml. of this final suspension were frozen and stored in liquid nitrogen. From day to day, one of the aliquots was thawed by addition of 1.9 ml. of warm medium and plated at once for the number of surviv- ing infective centers. A control culture infected with non-radioactive T2 under other- wise identical conditions was similarly frozen, stored, and assayed. Aliquots of the initial radioactive stock of free T2 and a non-radioactive control stock were also stored in liquid nitrogen and assayed for their survival from day to day. The results of this experiment are presented in Fig. 4. It is seen that in the population of bacteria infected for 2.5 minutes with a multiplicity of 0.075 radioactive T2, per cell, the logarithm of the fraction of individuals capable of giving rise to a plaque when plated after thawing decreases linearly with (1 — e*). The slope of the survival curve is about three-fourth that of the 288 450 INACTIVATION OF BACTERIOPHAGES rate of inactivation of the free radioactive T2 stored at the same tempera- ture. (Neither the control culture infected with non-radioactive T2 nor the DAYS 0) 2 at 64 iS 12 16, 420) 25 10"! SURVIVING FRACTION 6) 0.2 0.4 0.6 er At Fic. 4. P®? inactivation of T2 (Ao = 88 mc./mg.) inside infected bacteria at —196°C. Filled triangles, multiplicity of infection: 0.075 (monocomplexes). Open triangles, multiplicity of infection: 2.2 (multicomplexes). Filled circles, free T2. The dashed curve indicates the expected survival of infective centers at a multiplicity of 2.2 in the absence of multiplicity reactivation. corresponding free phage showed any significant loss of titer.) Hence P® decay occurring in the DNA after it has been separated from the protein “coat”? and exchanged its place in the phage head for the protoplasm of the host cell is still capable of destroying the reproductive capacity of the parent phage, although this inactivation now proceeds with a slightly reduced effi- 289 G. S. STENT AND C. R. FUERST 451 ciency. Results similar to those presented in Fig. 4 have also been obtained after infection of bacteria with radioactive T3 and \ phages. State of the Phage after Decay.— Cross-Reactivation.—The lethal damage sustained by the phage upon decay of one of its phosphorus atoms thus appears to prevent a step of the reproduc- tive cycle which occurs after the invasion of the host. In accordance with this view, we observed that T2 particles inactivated by P® decay are still adsorbed to bacterial cells. In fact, such phages are still able to participate in the re- productive processes occurring inside bacteria infected with a normal, non- inactivated related phage. In experiments already presented elsewhere (Stent, 1953 6) it was found that a radioactive stock of the double mutant strain T2hr, could still contribute its genetic markers to the progeny of a cross with non-radioactive wild type T2+-+ after P*® decay had destroyed the ability of the T2hr, particles to reproduce themselves in solo (cross-reactivation). It appeared, furthermore, that the ability of a radioactive T2 particle to donate either one of these two unlinked loci # and 7 is destroyed separately by P® decay, each locus disappearing at about one-third the rate of the plaque- forming ability of the whole particle. In those infected bacteria in which only one of the two radioactive loci has been inactivated, the surviving locus appears among the progeny in nearly normal yield. Stahl (1954) also discovered the existence of cross-reactivation of genetic markers after inactivation of T4 phage by P® decay. Stahl observed, furthermore, that the likelihood that a P® disintegration prevents both of two markers from appearing among the progeny of a cross with an active phage is inversely related to the genetic linkage distance of their loci. Hence it may be inferred that the lethal damage of P® decay affects the reproduction of only part of the hereditary substance of the bacteriophage particle, leaving the rest intact to reproduce itself in mixed infection with an active phage. Multiplicity Reactivation—The presence of an active phage particle in the same bacterial cell, however, appears to be necessary for the survival of the undamaged parts of a P*-inactivated T2 phage. Contrary to ultraviolet-in- activated T2 (Luria, 1947), infection of one bacterium by several P*-in- activated particles does not lead to the production of active phage (multi- plicity reactivation). In order to test for multiplicity reactivation following P® decay, the stock of radio- active T2 employed in the experiment presented in Fig. 4 was used to infect B/r bacteria at a multiplicity of 2.2 phage particles per cell. As in the low multiplicity experiment of Fig. 4, the mixture of bacteria and radioactive phage was incubated at 37°C. for 2.5 minutes before being frozen, stored at —196°C., and assayed for surviv- ing infective centers from day to day. At a multiplicity of infection of 2.2, the frac- tion of all infected bacteria to which two or more phages are adsorbed (multicom- plexes) is 0.73. Hence if two or more T2 particles were able to cooperate in the 290 452 INACTIVATION OF BACTERIOPHAGES production of active progeny after each individual had already sustained a “lethal” P®? disintegration, then the rate of inactivation of 0.73 of the plaque formers in this ex- periment should have been significantly reduced over the rate of inactivation of singly infected cells. If, on the other hand, the plaque-forming ability of a multiply infected cell is destroyed as soon as each of the infecting particles has been inactivated by P® decay, then the infective centers in this experiment should have disappeared with the “multiple hit”’ kinetics indicated in Fig. 4 by a dashed curve. The result of this ex- periment is also shown in Fig. 4. It is seen that inactivation of multicomplexes pro- ceeds at roughly the same rate as inactivation of singly infected bacteria, indicating the absence of any appreciable multiplicity reactivation. Experiments in which P® decay was first allowed to take place in free T2 and in which bacteria were then mul- tiply infected with the inactivated phages likewise failed to reveal any multiplicity reactivation. Latent Period of Survivors.—Since the efficiency of killing, a, is less than 0.1 at low temperatures, it is apparent that after an amount of decay which TABLE III Photoreactivation of T2 Assayed in dark Assayed in light Treatment of T2 Titer Titer Before P® decay 17 <5108 1.7 XX 108 After P® decay LX, 10% 1.3 XX 10% Before ultraviolet irradiation 229 10° Phas) >< MOP After ultraviolet irradiation | 1.4 & 105 1.3 X 107 leaves only a small fraction of the initial phage population still active has taken place under these conditions there have occurred many non-lethal pP® disintegrations in the survivors. In the case of T2, these survivors, how- ever, exhibit no evident effects of this non-lethal decay and reproduce with normal latent period and burst size. This is in contrast to the survivors of ultraviolet light irradiation whose multiplication is significantly retarded (Luria, 1944). Photoreactivation.—T2 bacteriophages inactivated by ultraviolet light can be “‘photoreactivated” by exposure of bacteria infected with such phages to visible light (Dulbecco, 1949). To examine whether phage inactivated by decay of incorporated P* could be similarly reactivated by light, assays were made of a radioactive T2 stock before and after decay to 0.0001 of the initial titer, incubating the assay plates either in the dark or under a strong fluo- rescent light. A non-radioactive control stock of T2 was inactivated with ultra- violet light to a survival of 0.000056 and similarly assayed in dark and light. The result of this experiment is presented in Table III, in which it may be seen 291 G. S. STENT AND C. R. FUERST 453 that no photoreactivation of the P%-inactivated T2 took place, although the titer of the ultraviolet-inactivated control was raised by nearly a factor of 100 by exposure to visible light. DISCUSSION Cause of Death.— An atom of P® decays into the stable isotope of sulfur, S*, upon ejection of a beta electron-neutrino pair of total kinetic energy 1.7 mev. The beta particle produces ionizations along its path, which are capable of damaging biologica] materials in a way similar to x-rays. Hershey, Kamen, Kennedy, and Gest, however, showed by means of calculations based on the volume of the T2 particle, the density of ionizations along the beta track and the known efficiency of killing per x-ray ionization, or by reconstruction experiments in which non-radioactive phage particles were irradiated with beta particles emitted by external, non-incorporated P® atoms, that beta particle ioniza- tions could not be the principal cause of the inactivation of radioactive bacteri- ophage particles. Hershey ef al. concluded, rather, that a short range conse- quence of the nuclear reaction, e.g. the recoil sustained by the disintegrating nucleus upon ejection of beta electron and neutrino, or the transmutation of phosphorus into sulfur, was responsible for death. The present finding that the sensitivity of radioactive phages to P® decay is reduced only slightly after infection supports this view. For, it appears likely that the state of ag- gregation of the phage DNA is more compact in the phage head than in the protoplasm of the host cell (Watanabe, Stent, and Schachman, 1954). Hence the chance of irradiation of one part of the phage DNA by distant P® atoms of another would have been seriously reduced once infection was under way. Efficiency of Killing — Hershey ef al. suggested that the fact that only one P*® disintegration in about ten was lethal to T2 or T4 might reflect a division of the phage DNA into 10 per cent “‘essential’’ and 90 per cent ‘‘non-essential’’ structures. Under this view, any P*® disintegration in the former would be surely lethal and any in the latter generally harmless. The present finding that a is nearly the same in various phage strains of greatly different size, morphology, and biological properties makes this hypothesis less likely. The dependence of a on tem- perature, furthermore, excludes the possibility that the anatomy of the phage is the sole factor responsible for the efficiency of killing. It seems, rather, that @ must at least in part reflect some structural aspect of the DNA mole- cule, the substance whose function is presumably destroyed by the decay of its radioactive P® atoms. The lethal effects of P*? decay can perhaps be best understood in terms of the macromolecular structure of DNA, recently uncovered by Watson and 292 454 INACTIVATION OF BACTERIOPHAGES Crick (1953), of which a schematic diagram is presented in Fig. 5. This struc- ture reveals DNA as a double helix composed of two intertwined polynucleotide chains of opposite polarity held together laterally by specific hydrogen bonds between purine and pyrimidine bases of opposite strands. The radioactive P® atoms are located in the diester bonds responsible for the continuity of interrupted chain interrupted chain Fic. 5. Schema of the Watson-Crick structure of DNA. The two ribbons sym- bolize the two phosphate-sugar chains, and the horizontal rods represent the pairs of bases holding the chains together through a pair of hydrogen bonds. The breaks in the ribbons indicate the spontaneous interruptions of the polynucleotide chains proposed by Dekker and Schachman. the polynucleotide chains. It appears almost inevitable that every ester linkage is destroyed upon decay of its radioactive phosphorus atom. First of all, the maximum recoil sustained by the phosphorus nucleus is of the order of 80 ev. (the average value being somewhat lower owing to the random orientation of neutrino and beta electron), whereas the energy of the P-O bond holding the atom in place is less than 5 ev. The ester bond is, therefore, probably broken by the Szilard-Chalmers reaction (cf. Libby, 1947). Secondly, even if the 293 G. S. STENT AND C. R. FUERST 455 recoil does not rupture the phosphate ester linkage, 7.e. if the phosphorus nucleus remained in place after all, then the two deoxyribose residues are forth- with linked by a sulfate diester, which should undergo spontaneous hydrolysis in aqueous medium (Kremann, 1907). Inspection of the structure shown in Fig. 5 indicates, however, that breakage of one ester link would not neces- sarily lead to the disruption of the DNA molecule, since the multitude of hydrogen bonds still hold the two sister strands together. This has recently been pointed out by Dekker and Schachman (1954), who propose on the basis of physicochemical evidence that the polynucleotide strands of “‘native”’ DNA are not actually continuous throughout the length of the macromolecule but are already interrupted in such a fashion that on the average one out of twenty to fifty phosphate links is singly instead of doubly esterified, as indicated in Fig. 5. Thus, if there already exist spontaneous breaks within intact DNA, it is not unreasonable to suppose that the low efficiency of killing per P® disintegration means that the DNA molecule can continue to function even after a few additional interruptions of the polynucleotide chains have been generated by radioactive decay. An event secondary to the disruption of the phosphate diester must then attend the lethal fraction a of P* disintegrations. The most reasonable hy- pothesis would appear to be that inactivation is caused by a complete cut of the DNA double helix. One way in which this could occur is that enough energy liberated by the decaying P® atom has been transmitted by a sequence of elastic collisions to the other strand to also cause a break there. Another possibility, in view of the proposal by Dekker and Schachman, would be that the lethal de- cay takes place in an atom situated nearly in apposition to one of the few in- complete ester links on the other strand. In either case, a complete cut results because few or no hydrogen bonds remain between the spots where both sister strands are broken to oppose the dissociation of the macromolecule into two smaller pieces. The effect of heat on the efficiency a is readily explained in terms of this hypothesis. The rapid rise of a above 55°C. must be due to the dissociation of the hydrogen bonds at these temperatures (cf. Dekker and Schachman), thus causing less and less resistance to separation of the two strands by the energy of the radioactive disintegration. A greater and greater fraction of the P® decays can, therefore, result in a complete cut of the double helix. The effect of freezing and of low temperatures on reducing a might be explained by the increase of viscosity of the medium in which the two pieces involved in the break have to move; 7.e., that when the DNA is embedded in ice there exists a greater chance that the energy of the P® transmutation has already been dissipated before the cut has actually taken place. Action of Ionizing Radiations.— It would be possible, though technically rather difficult, to ascertain whether, in agreement with the hypothesis just proposed, decay of incorporated P® 294 456 INACTIVATION OF BACTERIOPHAGES actually depolymerizes highly radioactive DNA molecules with an efficiency similar to a. It is known, however, that x-rays and other ionizing radiations do break down DNA to random fragments of progressively smaller molecular weight at doses comparable to those necessary for the ‘“‘direct’’ inactivation of bacteriophages (Taylor, Greenstein, and Hollaender, 1948; Conway, Gilbert, and Butler, 1950). Hence it is not unlikely that the lethal effect of x-ray ioniza- tions inside the phage particle is also one of cutting DNA molecules, similar to that postulated above for P® decay. Two sets of facts would appear to make this comparison useful: (a) The efficiency of killing per x-ray ionization inside the volume of the phage particle is only of the order of 0.05 in the bacteriophage strains studied here (Watson, 1950); 7.e., similar in magnitude to a. The energy released by each x-ray ionization is thought to be 32 ev., 7.e. similar in magnitude to that of the P® recoil, and to be confined to a radius of a few Angstrom units (Lea, 1947). (The average energy available locally may actually be either more or less than 32 ev. because, on one hand, the ionizations tend to occur in clusters but, on the other hand, their energy has been determined only in air and not in a condensed phase.) Since the two polynucleotide chains of the DNA macro- molecules are separated by at least 10 A (Watson and Crick, 1953), it would appear possible that many of the ionizations, like many of the P® disintegra- tions, damage only one of the strands without causing a complete rupture of the double helix. (b) The x-ray sensitivity of T1 depends on temperature very much like a. At temperatures below freezing, the rate of inactivation by x-rays is only 65 per cent of that just above freezing (Bachofer et al., 1953). At higher tem- peratures, the sensitivity first remains relatively constant and then increases sharply above 50°C., reaching a sixfold greater value at 60°C. (Adams and Pollard, 1952). These observations had already suggested to Adams and Pollard that the weakening of secondary, interchain bonds by heat at the moment of the x-ray ionization might be responsible for increasing the chance of causing lethal damage at higher temperatures. As in the case of P® decay, it is apparent that the greater the extent to which the hydrogen bonds of the DNA macromolecule are dissociated, the more likely will a cut of the double helix result from an energetic rupture of a single polynucleotide strand. SUMMARY The inactivation of the phages T1, T2, T3, T5, T7, and A by decay of in- corporated P* has been studied. It was found that these phages fall into two classes of sensitivity to P*® decay: at the same specific activity of P® in their deoxyribonucleic acid (DNA), T2 and TS are inactivated three times as rapidly as T1, T3, T7, and X. Since the strains of the first class were found to contain 295 G. S. STENT AND C. R. FUERST 457 about three times as much total phosphorus per phage particle as those of the second, it appears that the fraction of all P*® disintegrations which are lethal is very nearly the same in all the strains. This fraction a depends on the tem- perature at which decay is allowed to proceed, being 0.05 at —196°C., 0.1 at +4°C., and 0.3 at 65°C. Decay of P® taking place only after the penetration of the DNA of a radio- active phage particle into the interior of the bacterial cell can still prevent the reproduction of the parental phage, albeit inactivation now proceeds at a slightly reduced rate. T2 phages inactivated by decay of P® can be cross- reactivated; z.e., donate some of their genetic characters to the progeny of a mixed infection with a non-radioactive phage. They do not, however, exhibit any multiplicity reactivation or photoreactivation. The fact that at low temperatures less than one-tenth of the P® disintegra- tions are lethal to the phage particle and the dependence of the fraction of lethal disintegrations on temperature can be accounted for by the double stranded structure of the DNA macromolecule. REFERENCES Adams, M. H., Methods of Medical Research, (I.H. Comroe, editor), Chicago, The Year Book Publishers, 1950, 2, 1. Adams, M. H., Ann. Inst. Pasteur, 1953, 84, 1. Adams, W. R., and Pollard, E. C., Arch. Biochem. and Biophysic., 1952, 36, 311. Bachofer, C. S., Ehret, C. F., Mayer, S., and Powers, E. L., Proc. Nat. Acad. Sc., 1953, 39, 744. Conway, B. E., Gilbert, L., and Butler, J. A. V., J. Chem. Soc., 1950, 3421. Dekker, C. A., and Schachman, H. K., Proc. Nat. Acad. Sc., 1954, 40, 894. Dulbecco, R., J. Bact., 1949, 59, 329. Fraser, D., and Jerrel, E. A., J. Biol. Chem., 1953, 205, 291. Hershey, A. D., and Chase, M., J. Gen. Physiol., 1952, 36, 39. Hershey, A. D., Kamen, M. D., Kennedy, J. W., and Gest, H., J. Gen. Physiol., 1951, 34, 305. Kremann, R., Monatsh. Chem., 1907, 28, 13. Labaw, L. W., J. Bact., 1951, 62, 169. Lea, D. E., Action of Radiations on Living Cells, Cambridge, University Press, 1947, Libby, W. F., J. Am. Chem. Soc., 1947, 69, 2523. Luria, S. E., Proc. Nat. Acad. Sc., 1944, 30, 393. Luria, S. E., Proc. Nat. Acad. Sc., 1947, 33, 253. Rivers, T. M., J. Exp. Med., 1927, 45, 11. Sanderson, E. S., Science, 1925, 62, 377. Stahl, F. W., in a discussion of a paper by Doermann, Chase, and Stahl, J. Cell. and Comp. Physiol., 1954, in press. Stent, G. S., Cold Spring Harbor Symp. Quant. Biol., 1953 a, 18, 255. 296 458 INACTIVATION OF BACTERIOPHAGES Stent, G. S., Proc. Nat. Acad. Sc., 1953 6, 39, 1234. Taylor, B., Greenstein, J. P., and Hollaender, A., Arch. Biochem., 1948, 16, 19. Watanabe, I., Stent, G. S., and Schachman, H. K., Biochim. et Biophysic. Acta, 1954, 15, 38. Watson, J. D., J. Bact., 1950, 60, 697. Watson, J. D., and Crick, F. H. C., Nature, 1953, 171, 737; Cold Spring Harbor Symp. Quant. Biol., 1953, 18, 123. 297 RESISTANCE TO ULTRAVIOLET LIGHT AS AN INDEX TO THE REPRODUCTION OF BACTERIOPHAGE S. BENZER! California Institute of Technology, Pasadena, California Received for publication July 18, 1951 Infection of a susceptible bacterium by a single phage particle initiates a series of events climaxed, after a time called the latent period, by bursting of the cell and the release of a number (burst size) of replicas of the initial phage. We are here concerned primarily with the intervening process of phage replica- tion which takes place behind the cloak of the cell wall. By prematurely dis- rupting infected cells, Doermann (1948) found that infective phage replicas are already present well before the time at which the bacterium bursts, ie., about two-thirds of the way through the latent period. At earlier times, however, no plaque-forming particles are recovered, not even the initial phage. Our atten- tion is, therefore, focused upon this ‘‘dark”’ period, during which the infecting phage must undergo some modification, and the key processes of phage repro- duction come to pass. Luria and Latarjet (1947) conceived the following experiment in an attempt to use target theory for an analysis of the intracellular developments. It had been shown by Anderson (1948) that a bacterium (Escherichia coli, strain B) could be subjected to rather heavy doses of ultraviolet light and still survive in its ability to support the growth of phage T2. Thus, if one were to infect cells of strain B with single particles of T2 and irradiate the phage-bacterium com- plexes, the survival of infectivity (the ability to release at least one phage particle, thereby forming a plaque) of the complex should be determined by the survival of the phage part. If the irradiation is done immediately after infection, one should obtain the same survival curve for complexes as for the free phage ir- radiated before addition to bacteria, i.e., an approximately exponential or ‘‘one- hit” curve. If complexes are allowed to develop to the point where several intra- cellular phage particles are present, the inactivation of the complex requires at least one “hit” in each phage, and a multiple-target survival curve should be obtained. The set of curves for samples irradiated at different stages in the latent period would be expected to resemble the theoretical curves of figure 1. These multiple-target curves, plotted on a semilogarithmic graph, are characterized by asymptotes of constant slope equal to that for the single phage particle. The intercept of the asymptote, extrapolated to zero dose, corresponds to the loga- rithm of the number of targets. 1 On leave of absence from the Department of Physics, Purdue University. This investi- gation was conducted in part at Oak Ridge National Laboratory and continued while the author was at the California Institute of Technology as a Postdoctoral Fellow of the Atomic Energy Commission, and a Fellow in Cancer Research of the American Cancer Society, recommended by the Committee on Growth. Present Address: Institut Pasteur, Paris 15, France. Reprinted by permission of the author and the Williams and Wilkins Co., from the JouRNAL OF BACTERIOLOGY, 63 (1), 59-72, January, 1952. 298 60 S. BENZER [voL. 63 In experiments with #. colz, strain B, and phage T2, Luria and Latarjet found that immediately after infection the survival curve agreed with that of free phage. However, at later times, instead of showing a progressive increase in multiplicity with constant slope, the curves during the first half of the latent period showed a progressive decrease in slope (1.e., a decrease in sensitivity to ultraviolet) while remaining essentially exponential in character. At mid-latent period the curves became multiple-target in character, and thereafter the sensitivity increased again. Since the results did not resemble the family of curves ro} SURVIVAL We I 2) 5) BlOVZOR5OR100 OF ees!) 4S BO ila Seno! O DOSE Figure 1. Theoretical survival curves for complexes (after Luria and Latarjet, 1947). For an individual target the survival y is given by e-?, where D is the dose in arbitrary units. For a complex containing n identical, independent targets, the survival of any one of which is sufficient for survival of the complex, y = 1 — (1 — e-?)r. in figure 1, it was not possible to perform a target-theory determination of the number of intracellular phage particles. Latarjet (1948) repeated these studies, using X-rays, and observed changes which were similar in character although differing in degree. Further investigation of this problem was suggested by the discovery of “multiplicity reactivation” (Luria, 1947; Luria and Dulbecco, 1949). In this phenomenon, two ultraviolet inactivated phage particles, when infecting the same cell, can combine their resources, leading to the production of active phage. 299 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 61 This result may also be stated in the following way: complexes formed by infec- tion of a cell with two phage particles do not have a survival curve corresponding to the two-target curve of figure 1; at any given dose of ultraviolet the probability of the complex being infective is greater than given by that curve. This effect occurs to a marked degree with phage T2 and would be expected to cause anom- alous results in an experiment of the Luria-Latarjet type, where intracellular multiplication is going on. However, there are other phages, T7 for instance, with which multiplicity reactivation does not occur, and it seemed of interest to extend the experiments to such a phage. Another possible cause of anomalous results is ‘“photoreactivation” of phage (Dulbecco, 1950). Ultraviolet inactivated phage particles may be reactivated, after adsorption to a sensitive bacterium, by exposure to white light. Thus, we may expect the infectivity of a phage-bacterium complex to have a higher resistance to ultraviolet if exposed to light (after ultraviolet irradiation) than if kept in the dark. Precautions are therefore necessary in order to avoid this effect. MATERIALS Phages: T7, T2, and T2r, prepared from lysates in broth, purified by centrif- ugation, and resuspended in buffer. Bacterium: Escherichia coli, strain B, grown in broth. Growth medium (broth): bacto-tryptone, 1 per cent plus 0.1 m NaCl. Buffer: 1/15 m phosphate buffer, pH 7, plus 0.1 m NaCl, plus 10-3 m MgSO,. METHODS A Luria-Latarjet experiment involves the following steps: 1. Phage particles are added to a suspension of bacteria and time is allowed for infection of the cells to occur, then unadsorbed phage is eliminated. 2. The complexes are allowed to develop and samples are removed at various stages of the latent period. 3. Each sample is exposed to several doses of ultraviolet. 4. Aliquots are plated to determine the fraction of infective centers surviving each dose. These operations must be completed before the end of the latent period. Furthermore, since the radiation resistance of the infective centers changes rapidly with time, it is essential for accurate results that growth start almost simultaneously in all cells and that it be halted during irradiation. In an attempt to best satisfy these requirements, the following procedure is used: 1. Adsorption of phage without growth. The bacteria are prepared from an aerated broth culture in the logarithmic phase (1 X 10° cells per ml). The cells are centrifuged, resuspended in buffer, centrifuged again, resuspended in buffer at a concentration of 1 X 10° per ml, and aerated by bubbling at 37 C for one hour in order to exhaust intracellular nutrients and bring the bacteria to a starved condition. A purified suspension of phage particles in buffer is then added. Under these conditions adsorption takes place, but no lysis or phage liberation 300 62 S. BENZER [voL. 63 (Dulbecco, 1950). However, the absence of phage liberation is not sufficient to exclude the possibility that intracellular growth progresses to a fairly advanced intermediate stage without reaching completion. We can test for this by making use of the large change in resistance to ultra- violet of T2r complexes. If intracellular development proceeds, the resistance should increase with time. In figure 2, it can be seen that so long as the infected cells are kept in buffer and no nutrient is added, the resistance remains constant. The value of the resistance is only slightly higher than that of the free phage \va— in broth at 37° c in buffer at 37° SURVIVAL AFTER UV DOSE OF 55 SECONDS 0 10 20 30 40 50 60 TIME IN MINUTES Figure 2. Effect of addition of nutrient upon the radiation resistance of T2r complexes formed in buffer. The ordinate is obtained by exposing a sample of complexes to ultraviolet for 55 seconds and determining the fraction which survives. (A) No nutrient added; (B) broth added at time zero. (see later). Thus, the intracellular development does not progress beyond a very early stage. Even if the adsorption period extends over many minutes, each complex is arrested in its development, and adsorption is effectively simul- taneous. In experiments with bacteria grown in synthetic medium (instead of broth) there was a slow increase of resistance, even in buffer, as if considerable intracellular nutrient reserves remained even after starving for an hour. This resistance reached a maximum value corre- sponding to that attained after several minutes of growth in the presence of ample extra- cellular nutrient. 301 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 63 T2r is adsorbed to the extent of over 90 per cent in 10 minutes in buffer. At the same buffer concentration, T7 adsorption is extremely slow, but the rate is greatly increased by using buffer diluted 10 times with distilled water (Watson, personal communication). Therefore, throughout the experiments with T7 diluted buffer was used. The number of infective centers obtained (with either phage) was usually found to be less, by one-third to one-half, than the number of phages adsorbed. The cause of this ‘abortive adsorption” is not understood. To elimi- nate unadsorbed phage, the cells are washed by centrifugation and resuspended in fresh buffer. 100 CENTERS 10 burst size260 INFECTIVE RELATIVE NUMBER OF 0 10 20 30 40 TIME IN MINUTES AFTER ADDING BROTH Figure 3. One-step growth curve at 37 C for T2r complexes formed in buffer. Broth added at time zero. ‘‘Infective center’’ signifies a plaque-forming unit, either an infected cell or a free phage particle. 2. Growth. To the suspension of infected cells in buffer at 37 C, an equal volume of broth at the same temperature is added, and time is reckoned from this moment. In the case of phage T2r the radiation resistance promptly begins to rise (figure 2). From the one step growth curve in figure 3, it may be seen that the (minimum) latent period for T2r is 19 minutes, and the average burst size is 60. For T7, the latent period is 12 minutes, and the burst size is 150. To stop development at any chosen time during the latent period, a sample is removed and diluted rapidly (by blowing out of a pipette) into buffer chilled in an ice bath. The chilling brings the change in radiation resistance to an immediate 302 64 S. BENZER [voL. 63 halt; the resistance remains constant for hours provided the sample is kept chilled. The pipettes used for addition of broth and removal of samples are previously equilibrated at 37 C in an incubator. In this manner, growth may be started and stopped in all cells simultaneously, and the timing controlled to within a few seconds. Furthermore, many samples may be taken at close intervals within the same growth experiment and the irradiation conducted afterwards at leisure. For samples chilled very close to the end of the latent period (after 16 minutes for T2r or 9 minutes for T7) lysis is not prevented by chilling. After a delay, there appears a gradual increase in plaque count, presumably due to slow lysis of cells containing completed phage particles. 3. Irradiation. One or two ml of the suspension to be irradiated are placed in a shallow watch glass. The suspension is transparent to ultraviolet by virtue of the large dilution (100) of the broth. Ice in a small dish is used to chill the suspension from below (figure 4). This prevents growth during the irradiation. Chilling also serves to reduce greatly the rate of photoreactivation (Dulbecco, 1950) which might otherwise be caused by the visible light emitted by the ultraviolet lamp. Figure 4. Arrangement for chilling sample during irradiation. Ultraviolet is supplied by a 15 watt “germicidal lamp” (General Electric Company). The energy emitted in the 2537 A line accounts for almost all the antiphage activity. The intensity of ultraviolet is such that T2r survives to the extent of 10-2 after an exposure of 40 seconds. This same intensity is used throughout the experiments, and the doses are, therefore, given in units of sec- onds. All manipulations after irradiation are conducted in dim yellow light to minimize the possibility of photoreactivation. 4. Plating. Aliquots of samples subjected to various doses of ultraviolet are plated in a top layer of soft agar, seeded with unirradiated B, on broth agar plates. The resultant plaques are counted to determine the fraction of complexes whose infectivity has survived the irradiation. Note on multiplicity of infection. It is essential that the proportion of phage particles to bacteria be kept quite small in order that very few multicomplexes (i.e., bacteria infected with more than one phage particle) be formed. This is particularly important in the case of T2, where the phage exhibits multiplicity reactivation and the multicomplexes are much more resistant to ultraviolet than monocomplexes. Thus, if one assumes a Poisson distribution of phages per bacterium, and a survival value of 10~° is to be accurate within 10 per cent, the average multiplicity of infection must be 2 X 10~ or less. In order to have 303 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 65 such a low multiplicity of infection and still have a measurable concentration of survivors after growth, dilution, and irradiation, it is necessary to start with a high concentration of bacteria (1 X 10° per ml). Concentrations either half or twice this value were found to give the same results. PRELIMINARY CONSIDERATIONS 1. Ability of irradiated bacteria to support phage growth. It is necessary to know the validity, under the conditions used, of the basic assumption that the irradia- tion of the bacteria does not affect their ability to support phage growth. For this reason Anderson’s experiments were repeated, using phage T2r. Starved bacteria, prepared as previously described, were irradiated in buffer before addi- tion of phage. The number of infective centers produced per adsorbed phage was determined by plating and compared to the result with unirradiated bacteria. At the intensity of ultraviolet here used, 4,000 seconds of irradiation are required to reduce to one-half the number of bacteria capable of liberating phage after infection with T2r. Since the largest doses used in irradiating infected cells were 1,000 seconds, inactivation of the bacterial part of the complex should have had a negligible effect upon the survival curve. It is conceivable, however, that the sensitivity of the bacterial part of the complex does not remain unchanged throughout the latent period. Indeed, it becomes difficult to separate the complex into phage and bacterial components once growth has started. For T7, it is likewise found that the highest dose used (850 seconds) has negligible effect upon the ability of the bacteria to yield phage after infection. 2. Effect of irradiation of complexes upon the latent period and burst size of the survivors. It is found that a progressive lengthening of the latent period and decrease in burst size are produced by increasing doses of ultraviolet. For T2 monocomplexes irradiated at mid-latent period with a dose such that 5 per cent survive to form visible plaques, the latent period of the survivors is doubled and the burst size is reduced to 10 per cent of normal. This effect results in a smaller and more variable size of the plaques formed by surviving monocom- plexes. The degree of the effect appears to be determined primarily by the dose of ultraviolet rather than the percentage inactivation of the complexes. There- fore, it is most apparent at times during the latent period when the largest doses are required for obtaining survival curves. In plotting survival curves and at- tempting to analyze them by target theory, we are assuming that inactivation of a complex is an all-or-none phenomenon. It must be realized that the delay in lysis and reduction in burst size of the surviving infective centers could also lead to failure of some of them to produce visible plaques. However, for the doses used in this paper, the plaques observed with T2r and T7 do not taper gradually down to zero in diameter; probably few infective centers fail to be counted for this reason. RESULTS Experiments with T2r.'T2 (used by Luria and Latarjet) and its mutant T2r, which has the same sensitivity to ultraviolet as T2, gave similar results. It is 304 66 . S. BENZER [voL. 63 preferable to work with T2r because it produces larger plaques, thereby obviating in some measure the difficulty created by the decrease in plaque size at large doses. In figure 5 a complete set of curves for T2r (latent period 19 minutes) is given. These qualitatively confirm the observations of Luria and Latarjet with T2. The free phage does not have a strictly exponential survival curve, but the changes in resistance during the latent period are so large that this may be ignored for our purposes. At f = 0 (1.e., for a sample irradiated before the addi- tion of nutrient) the resistance of monocomplexes is slightly higher (by 20 per cent) than that of free phage. This may be partially due to a small amount of development of the phage which can take place in buffer. 5, SURVIVAL OF INFECTIVE CENTERS 3 SURVIVAL OF INFECTIVE CENTERS te) 100 200 300 Oo 100 200 300 400 500 600 uv DOSE IN SECONDS UV DOSE IN SECONDS Figure 6. Survival curves for T2r complexes irradiated at different times during the latent period. Each curve is marked with the time in minutes after adding broth to the complexes formed in buffer. All data are from the same growth experiment at 37 C (latent period = 19 minutes). Average multiplicity of infection = 2 107%. As development proceeds, the resistance of all complexes increases. An es- pecially rapid rise occurs between 6 and 8 minutes, the curves still remaining essentially exponential. The smallness of the bend (at large doses) in the survival curve taken at 7 minutes is very significant, since it implies that, at least up to this point (one-third of the way into the latent period), development has pro- gressed rather uniformly in all infected cells. If, at 7 minutes, 10 per cent of the infective centers had progressed to a stage corresponding to 8 minutes (by virtue of 15 per cent more rapid development), the bend in the 7 minute curve would have been greater than observed. At 9 or 10 minutes the resistance reaches a maximum phase, and the curve has now become “‘multiple-target”’ in character. In figure 6 data are given which were obtained at 10 minutes. The points fit very well a theoretical curve for a 305 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 67 double target. However, the fit may be fortuitous, since slight inhomogeneities in the stage of development are to be expected. At the time of maximum resis- tance, those complexes which are either slightly ahead or behind in stage of de- velopment have lower resistances, causing a distortion of the true curve at the moment of maximum resistance. Thereafter, the curve retains a multiple-target shape, but the resistance of the individual targets, as judged by the final slope, decreases progressively as the end of the latent period 1s approached. CENTERS INFECTIVE theoretical curve SURVIVAL OF 0 200 400 ‘600 800 1000 UV DOSE IN SECONDS Figure 6. Circles: Survival data obtained for T2r complexes irradiated at the phase of highest resistance to ultraviolet (¢ = 10 minutes). Dashed curve: Theoretical survival curve for a double-target complex. The late curves have slight ‘‘tails’”’ which may be due to a small fraction of cells in which the development is retarded. In some experiments, peculiar composite survival curves were obtained as though the development in about half the cells was arrested at a stage corresponding to around 8 minutes, while the remaining cells continued normally. It has not been found possible to clarify the conditions leading to this result. According to Doermann’s findings, there may be a few infective phage particles per cell at the latest time here studied (15 minutes). These would presumably have the resistance of free phages. If survival as an infective center depends upon survival of either the complex or at least one completed phage, the effect 306 68 S. BENZER [voL. 63 of the phages upon the observed survival curve will be negligible, since at 15 minutes the complex has a much higher survival than have several free phages. By reference to figure 1 it can be seen that even a cluster of 100 phages would be inactivated more rapidly than the 15 minute complexes. Experiments with T7. A complete set of survival curves for monocomplexes of T7 and strain B irradiated at intervals during the latent period (12 minutes) is given in figure 7. It will be observed that the resistance remains unchanged (at the same value as for free phage) for 3 minutes in the presence of nutrient. INFECTIVE CENTERS SURVIVAL OF (0) 100 200 300 UV OOSE IN SECONDS Figure 7. Survival curves for T7 complexes irradiated at different times during the latent period. Each curve is marked with the time in minutes after adding broth to the complexes formed in buffer. All data are from the same growth experiment at 37 C (latent period = 12 minutes). Average multiplicity of infection = 2 X 107°. As time goes on, the curves become multiple-target in character, the average multiplicity continually increasing, while the final slope changes only slightly. This result stands in marked contrast to T2r and resembles that predicted by target theory. The exact shapes of these curves are not consistent with an as- sumption of equal multiplicities for all complexes at a particular time. However, the shape may be explained by a distribution of the number of targets, which may be expected as a consequence of different rates of phage multiplication in different cells. It is well known (Delbriick, 1945) that the number of phage particles released by lysis of individual cells has an extremely wide distribution. 307 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE 69 To illustrate the effect of such a wide distribution upon our survival curves, a composite curve is plotted in figure 8, assuming a population consisting of equal numbers of cells having the various multiplicities in figure 1. By extrapolation to zero dose of the asymptotic slope of such a curve, it is possible, in principle, to determine the average multiplicity. However, this requires precise data at low survival values and cannot be done accurately with the points in figure 7. SS < = > « > 7) 10) (162) oeoeeO eNO o> 29) 0 DOSE Figure 8. - --- Theoretical survival curves for complexes containing various numbers of targets (as in figure 1). Theoretical survival curve for a mixed population of complexes. The mixture is assumed to contain equal proportions of complexes having multiplicities 1, 2, 5, 10, 20, 50, and 100. Note: It is possible, by assumption of a suitable population containing a very wide dis- tribution of multiplicities, to obtain a composite theoretical survival curve which is expo- nential, simulating a ‘‘one-hit’’ curve (Dulbecco, personal communication). DISCUSSION The original intent of Luria and Latarjet’s experiment, namely to observe the increase in the number of intracellular phage particles during the latent period, has been achieved with T7. The pattern of growth, so far as radiation resistance is concerned, does not exhibit the anomalies of (T2 and) T2r. There appears to be simply an increase with time of the average number of targets per cell, each target being similar radiologically to a T7 particle. This result shows that irradiation of intracellular phage, in the case of T7, 308 70 S. BENZER [voL. 63 enables us to discern the existence of multiple intracellular entities at a time when fully infective phages are not yet detectable with the Doermann technique. The anomalous results with T2r may be of far greater interest, however. During the critical phase between the entrance of one infecting particle and the appearance of intracellular progeny, enormous changes are reflected by the survival curves. It seems likely that these resistance changes may bear a close relation to the phenomenon of multiplicity reactivation. While the resistance changes do not tell us what is actually happening, they at least give us something easy to measure which serves as an empirical index to development. The value of this index has already been demonstrated previously in the justification of the techniques used in these experiments (adsorption in buffer, chilling to stop growth). In the case of T2r, the maximum change in slope is by a factor of over 20 (free phage compared with t = 10 minutes). As pointed out by Luria and Latarjet, intracellular accumu- lation of ultraviolet-absorbing materials (nucleic acid components) must be considered as a possible contributing factor. By microspectrography of uninfected cells (at 2570 A) Hedén (1951) finds an average extinction through the thickness of a cell of around 0.1, corresponding to 80 per cent trans- mission. A phage particle which is adsorbed on the surface of a bacterium is therefore shielded from ultraviolet to the extent of 20 per cent in one direction. This should lead to an average increase by only 10 per cent in the resistance (at ¢ = 0) compared with free phage (since the cell rotates in all directions during exposure). Penetration of the phage into the cell should have little effect on the magnitude of this average shielding. In order to account for a subsequent change by a factor of 20 in the intensity of ultraviolet reaching the phage, a coating of nucleic acid, 1 » in diameter, would have to be produced. The meas- urements of Luria and Human (1950) and Cohen and Arbogast (1950) on optical density and nucleic acid content in suspensions of cells multiple infected with T2 reveal increases during growth, but these are far too small to account for the observed resistance changes during the first 10 minutes. In the case of T7, however, the small change in slope may well be due to a screening effect. A plausible interpretation of the increase in resistance of T2r complexes during the first half of the latent period may be the following: A T2r particle, after adsorption to a sensitive bacterium, must undergo a series of successive steps A—B—-C-— D -—, ete. in the course of reproduction. Each of these steps has a certain cross section for being blocked by ultraviolet (e.g., by inactivation of an enzyme which is concerned with the step). Blockade of any one of these steps prevents normal development and causes inactivation of the phage. At time zero, the total cross section of the phage is therefore the sum of these individual cross sections, and the survival curve is exponential. As development proceeds, the steps which have already been passed are no longer needed, and the effective cross section decreases progressively while inactivation of the remaining steps retains the characteristics of a one-hit phenomenon. The absence of this behavior in the case of T7 suggests that there may be great differences in the mode of reproduction of T7 and T2r. These two phages are also dissimilar in other respects. T2r is a relatively large particle with a head and a tail and appears to have a kind of membrane (Anderson, 1949); it 309 1952] ULTRAVIOLET LIGHT INDEX IN BACTERIOPHAGE Vil shows multiplicity reactivation with other particles of T2r, and also undergoes genetic recombination with mutants of T2 and other closely related phages. T7, on the other hand, is much smaller in size, spherical in shape, does not appear to have a membrane, and does not show multiplicity reactivation. Whether it undergoes genetic recombination is not known. Therefore, the sequence of events in the intracellular development of T2r, which is reflected in the resistance changes, may be characteristic for the mul- tiplication of only certain types of phage. We stand to learn a great deal about the growth of T2r by making use of the radiation resistance index. If conditions are suitable for phage development, the index (i.e., the survival of infective centers after a given standard dose of ultra- violet) goes up (or down, in the latter half of the latent period). If, for any reason, phage development is blocked, the index remains constant; its value marks the stage at which the block occurs. This makes it possible to study the effect, at any chosen part of the latent period (except near the end), of many factors, for example temperature, growth requirements, and specific chemical inhibitors. The results of these investigations will be reported separately. This tool offers promise not only for studying phage growth but for certain problems in bacterial physiology as well. The growth of phage in a bacterium is dependent upon the metabolic well-being of the cell and on its ability to make use of the substrates supplied to it. By infecting the bacterium with a T2r particle and using the radiation resistance index, the metabolic capabilities of the bacterium under particular conditions can be measured. A unique feature of this technique is that one can determine not only the average rate of metab- olism but the distribution of rates (from analysis of the survival curves) among the individual cells in the population. This idea is currently being applied to the problem of the kinetics of enzymatic adaptation in bacteria. SUMMARY Techniques are described for simultaneous starting and stopping of the growth of bacteriophage in all the host cells of a culture. Using these techniques, a comparison is made of the changes in resistance to ultraviolet of phages T2r and T7 during intracellular growth, following the method of Luria and Latarjet. Phage T7 gives results similar to expectations from target theory, while the results with T2r confirm the (anomalous) behavior observed by Luria and Latarjet, indicating that there may be large differences in the modes of repro- duction of different phages. The utility of the change in resistance as a tool in studying bacteriophage reproduction and certain problems in bacterial physiology is pointed out. REFERENCES ANDERSON, T. F. 1948 The growth of T2 virus on UV killed host cells. J. Bact., 56, 403-410. ANDERSON, T. F. 1949 The reactions of bacterial viruses with their host cells. Botan. Revs., 15, 464-505. 310 Bs S. BENZER [voL. 63 ConHEN, S.S., AND Arpocast, R. 1950 The mutual reactivation of T2r* virus inactivated by ultraviolet light and the synthesis of desoxyribose nucleic acid. J. Exptl. Med., 91, 637-650. Devprick, M. 1945 The burst size distribution in the growth of bacterial viruses. J. Bact., 50, 131-135. DoreRMANN, A. H. 1948 Intracellular growth of bacteriophage. Carnegie Inst. of Wash. Year Book, 47, 176-182. Duusecco, R. 1950 Experiments on photoreactivation of UV-inactivated bacteriophage. J. Bact., 59, 329-347. Hepén, C. 1951 Studies of the infection of EF. Coli B with the bacteriophage T2. Acta Path. Microbiol. Scand., Supplementum LX XXVIII. LaTaRJET, R. 1948 Intracellular growth of bacteriophage studied by Roentgen ir- radiation. J. Gen. Physiol., 31, 529-546. Luria, 8S. E. 1947 Reactivation of irradiated bacteriophage by transfer of self-repro- ducing units. Proc. Natl. Acad. Sci., 33, 253-264. Luria, 8S. E., anp Dux.secco, R. 1949 Genetic recombinations leading to production of active bacteriophage from ultraviolet inactivated bacteriophage particles. Genetics, 34, 93-125. Luria, 8. E., anp Human, M. L. 1950 Chromatin staining of bacteria during bacterio- phage infection. J. Bact., 59, 551-560. Luria, S. E., anp LararsetT, R. 1947 Ultraviolet irradiation of bacteriophage during intracellular growth. J. Bact., 58, 149-1638. 311 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM by André Lwoff and Antoinette Gutmann Department of Microbial Physiology of the Institut Pasteur I. INTRODUCTION We call lysogenic bacteria those bacteria which perpetuate the capacity to form bacteriophages without intervention of exogenous bacteriophages. This definition will be discussed and justified in the course of this work. Thanks to the investigations of J. Bordet, of F. Burnet and M. McKie, of Den Dooren de Jong, of E. and E. Wollman, and of J. Gratia, it is known: 1. that in a lysogenic strain lysogeny is an attribute of every bacterium and, in the case of sporogenic species, of every spore; 2. that lysogeny persists after repeated passages in the presence of a specific anti-bacteriophage serum; 3. that lysogenic bacteria adsorb the bacteriophages which they produce; 4. that lysozyme lysis does not liberate bacteriophages from potentially lysogenic bacteria; 5. that, starting from certain sensitive strains infected by a given bacteriophage, one can isolate lysogenic bacteria which produce a bacteriophage identical to the original bacteriophage. It thus seems strange that the very existence of lysogenic bacteria could have been put in doubt by A. D. Hershey and J. Bronfenbrenner (1948), who wrote: “How virus is transmitted from cell to cell in lysogenic cultures seemingly refractive to lysis remains to be clarified. It must be concluded, however, that the phenomenon of lysogenesis, frequently cited as evidence for the spontaneous intracellular origin of virus, can equally well be explained as some type of association between exogenous virus and incompletely susceptible bacterium.” It seems that one must look for the origin of the skepticism in a possibly too narrow definition of ‘true’ lysogeny due to M. Delbriick (1946), which restricts lysogeny to the case where the bacteriophage is liberated by bacteria in the absence of lysis. Since no example of this phenomenon is known, lysogeny vanishes. This “true” lysogeny was contrasted by M. Delbriick with “pseudo- lysogeny,” in which bacteriophages are liberated following the lysis of sensitive bacteria, the latter being produced by mutation in the course of the growth of a Translated from the French and reprinted by permission of the authors and the Institut Pasteur from the ANNALEs DE L’ INSTITUT Pasteur, 78, 711-739 (1950). (N.B. A part of the section “Materials and Techniques” has been omitted from the translation of this paper.) 312 ANDRE LWOFF AND ANTOINETTE GUTMANN resistant culture contaminated by bacteriophages. But these arbitrary defini- tions do not exclude lysogeny in the sense in which we have defined it; lysogeny in which the property to produce bacteriophage is hereditary. In 1925 J. Bordet wrote: “The faculty to reproduce bacteriophages is inscribed into the very hereditary weft of the microbe,” and Burnet and McKie (1929b) expressed exactly the same idea: ‘‘The permanence of the lysogenic character makes it necessary to assume the presence of bacteriophage, or its anlage, in every cell of the culture, i.e., it is a part of the hereditary constitution of the strain.” No doubt the experiments on which this conclusion was based — which shall be discussed in the course of this work — however suggestive they were, did not bring the element of certainty. It is true that as late as 1949, in spite of numerous works published on lysogenic bacteria, one still did not know the manner in which lysogenic bacteria liberate the bacteriophage that they produce. Is the bacterio- phage secreted by the bacteria in a continuous manner? Is it secreted at the moment of division? Is it liberated by the lysis of certain bacteria? In the absence of any facts, the majority of microbiologists envisaged, along with Bordet and Renaux (1928), and with Burnet and McKie (1929b), that the bacteriophage is ‘‘secreted”’ by the bacterium. But it is really of little importance for the definition of lysogeny whether the bacteriophage is liberated in this or any other manner. The important fact is the reproduction of a bacterium potentially capable of producing a bacteriophage, that is to say the hereditary capacity to produce a bacteriophage. Naturally this does not exclude the mode of liberation of the bacteriophage from being worthy of our attention. J. Northrop (1939) established a parallelism between enzymes and bacterio- phages, based on the observation that the concentration of bacteriophages in a culture of lysogenic bacteria increases, like that of the enzyme gelatinase, in an essentially parallel manner to the multiplication of the bacteria. But it is quite evident that the interpretation which one can draw from such facts will be very different according to whether, for example, in the interval of two divisions every’ one of 100 individuals of a bacterial population liberates a bacteriophage, or whether a single bacterium liberates 100 bacteriophages. The principal problems posed by the existence of lysogenic strains are the following: 1. Can the faculty of producing bacteriophages really be perpetuated without intervention of exogenous bacteriophages? 2. How do lysogenic bacteria liberate the bacteriophages which they produce? 3. If the production of bacterio- phages appertains to only a certain proportion of the bacteria, then what factors induce the production of bacteriophages in a population of potentially lysogenic bacteria? The experiments presented here in detail were summarized in four preliminary notes (Lwoff and Gutmann, 1949-1950); they furnish a reply to the first two questions. The third question was studied in collaboration with L. Siminovitch and N. Kjeldgaard, and the relevant results shall be the subject of a separate paper. II. MATERIALS AND TECHNIQUES 5) It was evident a priori that the study of ‘‘mass cultures,” as it is usually 313 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM practiced, can only furnish partial solutions to the problems of lysogenic bacteria. Only observations carried out on individual bacteria, or on microcultures containing a small number of individuals, are capable of leading to definite conclusions. Strain. Our experiments were carried out on a lysogenic strain of Bacillus megaterium. We have used the classic strain ‘‘899” of den Dooren and de Jong and an asporogenic sensitive mutilat strain. Lysogenic and mutilat strains were subcultured by passages on peptone broth agar. Technique of Micromanipulation. One utilizes an incubator box made from transparent plastic material. An electric heater is placed under the bottom of this box, into which 20 or so holes have been drilled. The temperature is thus the same in all parts of the incubator box and a thermoregulator in one corner insures a constant temperature of 37°. The microscope and the receptor for the de Fonbrune micromanipulator are fixed on the base of the Zeiss-Peterfi micro- manipulator. We have utilized the oil chamber of de Fonbrune. It is unnecessary to go into details concerning this device, of which one can find a description in the monograph of de Fonbrune (1949). A knowledge of these technical details, however, can prevent considerable loss of time. Two pieces of glass of 1.5 X 12 X 7mm. are glued to either side of the depres- sion slide. Their separation is so calculated that it exceeds the length of the coverslips by about .8 mm. The latter, made from neutral glass, are washed in nitric acid, rinsed in double distilled water, wiped with fine cloth and sterilized (as well as the slide) at 150°. For the preparation of the experiment: 1. one sets a cover slip on the slide and places several drops of paraffin oil on the cover slip, so that it is covered by a layer of oil. 2. One draws out the fine end of a Pyrex glass Pasteur pipette so that the diameter of the terminal capillary allows passage of culture medium and that when the tip of the pipette filled with liquid comes in contact with the cover slip under the oil, one drop of liquid comes out and spreads out on the glass. This drop rust have a diameter of about 500 to 800 u. Nine rows of 9 drops can thus be regularly placed. The second and third rows are interrupted at the point of the fourth, fifth, and sixth drop. It is of course possibie to place several different media on the cover slip. The cover slip is then turned upside down and oil is poured between it and the slide. On the bottom of the oil chamber one places drops of distilled water in a manner so as not to disturb the observations; this will prevent any significant evaporation of drops of less than 100 u diameter. The oil chamber is then placed on the stage of the microscope. Technique of Sampling. It is necessary that one can transfer one bacterium, or a minimum quantity of liquid of the order of 10~° mm.*, into a tube or onto a petri dish. The dimensions of the liquid column sampled can be measured with the micrometer objective. In order to transfer the content of a pipette into the tube, one can proceed in the following manner: 1. The field of the microscope is disengaged by placing the oil chamber towards the rear. 2. The needle and the needle carrier are turned by 90°, in such a manner that the point of the needle points towards the right. The angle formed by the micropipette is then in a plane 314 ANDRE LWOFF AND ANTOINETTE GUTMANN parallel to that of the stage of the microscope. The pipette is then displaced towards the left, out of the microscope field. 3. A Pasteur pipette whose tip has been bent at a right angle about two to three mm. from the end is introduced into the right needle carrier of the Peterfi micromanipulator. The pipette contains dilution medium for a length of about 2 cm. It is so oriented that the angle formed by the terminal bend is in a plane parallel to the plane of the stage. 4. The tip of this pipette is brought into the center of the microscope field. The micropipette is then moved towards the right and its level regulated in such a manner that its point is opposite the liquid meniscus of the Pasteur pipette. One introduces the micropipette into the liquid. One inoculates the content of the micropipette into the liquid column of the Pasteur pipette. If one manipulates the bacterium, it is possible to see that bacterium pass from micropipette to Pasteur pipette. In any case, one knows that the operation is finished when a drop of oil appears at the point of the micropipette. All these manipulations are carried out in the incubator box. 5. The Pasteur pipette is then detached from its support, removed from the box and its contents transferred either into a tube or onto a petri plate. After a little practice, these operations can be performed very quickly and with great assurance. All of these manipulations are naturally carried out aseptically. According to our experience, which includes almost 1000 samplings, the chances of contamination are practically nil, since one manipulates liquid quantities of the order of 107° to 107-* mm*. III. BacrertaAL MULTIPLICATION WITHOUT LIBERATION OF BACTERIOPHAGE a. A diplo-bacillus, after five washings, is inoculated into a drop of peptone water. After each division, one of the daughter cells is removed with a minimum of the liquid. Twenty-two daughter cells are thus successively removed. The entire drop is then removed: no bacteriophages. b. In another experiment of the same type, one removes successively 42 daughter cells: the result is identical. Furthermore, assays of four samplings in the course of the experiment and of the total sample at the end show no bacterio- phages. c. The washed filament of three bacteria is observed. One removes successively 14 daughter cells and makes 5 samplings. After the last division, the entire fluid is assayed: no bacteriophages. d. Three diplo-bacilli are washed and inoculated separately into drops of peptone water. When the number of bacteria has attained respectively 24, 47, and 54, the entire fluid is sampled. There are no bacteriophages in the fluid nor in any of the samples previously taken. e. Three diplo-bacilli are washed and transferred into drops of peptone water of 72 to 100 mu diameter. Multiplication stops spontaneously when the number of bacteria reaches respectively 8, 12 and 16. All of the fluid is removed and sampled: no bacteriophages. f. A filament of 7 bacteria is washed and inoculated into a synthetic medium. 315 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM After two hours, there are 82 bacteria: all of the liquid is sampled, no bacterio- phages. Discussion. These experiments, as well as those which shall be described in the two following paragraphs, lead to the thought that a lysogenic bacterium multiplies without producing bacteriophages. They demonstrate that B. megatertum 899 can grow and multiply without liberating bacteriophages. Thus, liberation of bacteriophages is not a phenomenon which necessarily accompanies growth of a population of a lysogenic bacteria; it appears, rather, to be a contin- gent phenomenon. IV. MAINTENANCE OF LyYSOGENIC POWER IN THE ABSENCE OF FREE BACTERIOPHAGE A motile diplo-bacillus is sampled from a culture in peptone water (proteose peptone at 1%). This bacillus is successively transferred in six drops of the same medium. It is left in the sixth. During the entire length of this experiment the diplo-bacillus has remained motile. With B. megaterium the separation of the daughter members generally takes place when the daughter bacteria are already partitioned off. Once this separation releases two daughter diplo-bacilli, one injects into the drop approximately +4 of its volume of new medium. One assures a mixing of the fluids by repeated aspiration back and forth of approximately half of the fluid and then removes one of the bacteria in a volume essentially equal to the volume of fluid which has been added. This sample is transferred into a suspension of sensitive mutilat in soft agar and this suspension is then spread on an agar surface. This operation was repeated until the 10th division. At this point, the two daughter bacteria were sampled separately, the latter with the total of the residual fluid. The 11 plates that had been inoculated all showed a single plaque centered by a bacterial colony, with the exception of the first and the eighth, which showed no plaque whatsoever. The absence of the lysogenic colony on the two negative ‘‘plates” in this experiment, as in the following, probably results from an accidental loss of the bacterium. This experiment was repeated in peptone water to which glucose and a liver extract had been added. The original diplo-bacillus was followed until the 19th division, after which the two final bacteria were plated. The 20 plates, with the exception of the 4th and the 16th which showed no plaque whatsoever, each had, as in the previous series, one plaque centered by a lysogenic colony. One of these centered colonies, after being picked and isolated, proved itself to be lysogenic: the filtrate of its culture contained bacteriophages. The absence of any plaque, except for the single plaque centered by a bacterial colony, shows that no free bacteriophages ever appeared in these microcultures. During the experiments, there was never any noticeable decrease in the size of the diplo-bacillus, as would have occurred if one of its members had lysed, nor was lysis ever observed. Incidentally any lysis would have brought some disturbance in the rhythm of the divisions; but the 19 divisions took place in 486 minutes at regular intervals of 25 + 3 minutes. Discussion. It was established by Bordet and Renaux (1928) that lysogenic 316 ANDRE LWOFF AND ANTOINETTE GUTMANN strains of H. coli conserve their lysogenic nature after numerous passages in the presence of an antibacteriophage serum. Analogous results had been obtained by F. Burnet and M. McKie for various bacteria (1929a,b) and by E. and E. Wollman (1936) for B. megatertum. Since the antibacteriophage serum neutral- izes free bacteriophages, these experiments demonstrate that lysogeny can maintain itself in the absence of free bacteriophages, provided that the concen- tration of antibacteriophage serum during the entire length of the experiment is sufficient to immediately inactivate all bacteriophages. In other words, one must be sure that, under the conditions of these experiments, there is no possibility of a free bacteriophage infecting a bacterium before being neutralized by the serum. This condition was probably met in some of these experiments, but this is not a priori certain. And nothing enables us to state categorically that bacterio- phages which adsorb to bacteria are actually inactivated by serum. It is also known that lysogeny maintains itself equally well in cultures growing in media supposedly deprived of calcium; in fact, in media containing oxylate or citrate (KE. and E. Wollman, A. Gratia). But numerous organic substances possess the power to form complexes with calcium; these complexes could then dissociate, and, in organic media, it is difficult to ascertain the absence of free Ca** ions. The fact that lysogeny of a culture maintains itself in a citrate medium, whereas the adsorption of bacteriophage does not take place except in the presence of calcium, is but an argument in favor of its endomicrobial perpet- uation. Finally, it was shown by Den Dooren de Jong that the lysogenic function of B. megaterium is transmitted in cultures which have been derived from a spore, and even from a heated spore. This is true for all sporogenic lysogenic strains. This is, thus, an additional argument in favor of the endomicrobial transmission of the bacteriophage. But, unfortunately, the form of the bacteriophage within the interior of the spore is not known. If one adds to all of these considerations the fact that in a lysogenic culture every colony — supposedly obtained from a single cell —is lysogenic, one sees that a considerable accumulation of facts favors the conception that the faculty to form bacteriophages is hereditary, that is to say, independent of any exogenous reinfection. In our experiments, lysogeny is maintained in the course of 19 divisions in the absence of free bacteriophage. If the bacteriophage does not multiply within the bacterium and if the mainte- nance of lysogeny proceeds from particles adsorbed on the original diplo-bacillus and distributed equally over the daughter bacteria, then that diplo-bacillus must have carried a minimum of 2!° or 524,288 bacteriophages at the onset. The chance of an unequal repartition over the daughter cells in combination with our selection of that bacterium which happened to have inherited the smallest number of bacteriophages after each such division is so small that it need not be taken into consideration. S. Bayne-Jones and L. A. Sandholzer (1933) estimated the average volume of a single B. megaterium as 2.5 u*. Let us assume the value of 6 y* for the average diplo-bacillus. According to McLauchlan, E. M. Clark, and F. W. Boswell (1947), the dimensions of the head of the bacteriophage of Bacillus megatertum 317 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM are 40 by 100 muy, that is to say, a volume of approximately .9 X 1074 u*. Let us suppose that the polyhedral bacteriophages completely fill the entire diplo- bacillus, which could then contain no more than 66,000 bacteriophages. But, in any case, the maximum number of bacteriophages produced by any single bacterium never seems to surpass 1000. One can consider, without much chance of error, that in a viable lysogenic bacterium the space occupied by the bacteriophages must not surpass 50% of the total bacterial volume. An average viable lysogenic B. megateriuwm diplo- bacillus could thus contain maximally 33,000 bacteriophages. This figure is significantly lower than the value of 524,288 which represents the minimum number of particles which must have been present in the original diplo-bacillus in order to sustain the hypothesis of the perpetuation of the lysogenic function by a simple distribution of pre-existing exogenous particles. This hypothesis thus can be rejected. The hypothesis, according to which lysogeny perpetuates itself by an endo- microbial route without the necessary intervention of exogenous bacteriophages, seems to us, for the moment, the only one capable of accounting satisfactorily for the experimental facts. The problem of the form in which the lysogeny perpet- uates itself shall be discussed in the course of this work. Another conclusion can be drawn from our experiments. One knows (J. Bordet and J. Renaux, 1928; I’. Burnet and M. McKie, 1929a,b) that all colonies issuing from a lysogenic culture are lysogenic. Our experiments confirm and validate these data: every diplo-bacillus from a population of B. megatertum 899 isolated by means of the macromanipulator gives rise to a lysogenic colony. V. Lysozyme Lysis Dors Not LIBERATE BACTERIOPHAGE F. Burnet and M. McKie (1929a), F. Burnet and D. Lush (1936) observed that lysis by a bacteriophage X of a lysogenic bacterium sensitive to this bacterio- phage X and normally producing a bacteriophage Y does not liberate the bacteriophage Y. I’. Burnet and M. McKie understood the whole significance of this phenomenon as early as 1929 and concluded that all bacteria of a perma- nently lysogenic strain possess in their hereditary constitution some unit which is potentially capable of liberating phage. Burnet and McKie found, nevertheless, that a small proportion (about 1%) of the bacteria, of a 24-hour-old agar culture harbors bacteriophages which can be liberated by treatment with distilled water. E. and E. Wollman and A. Gratia (1936), independently and within a few weeks of each other, made a similar observation: lysozyme lysis of lysogenic B. megatervum 899 does not liberate bacteriophages. E. and E. Wollman found one bacterio- phage for several thousand bacterial colonies and concluded that the bacterio- phage exists in two different forms, phases, or states: the “mature’’ bacterio- phage which one could call corpuscular, or, more simply, bacteriophage-virus as it appears in the free state in the culture medium, and the non-virulent, latent ‘Gntracellular bacteriophage” as it exists inside lysogenic bacteria and which we call probacteriophage. Furthermore, it is known that it is impossible to liberate by lysozyme lysis bacteriophages adsorbed to living B. megaterium cells. 318 ANDRE LWOFF AND ANTOINETTE GUTMANN In contrast, lysozyme lysis does liberate bacteriophages adsorbed to killed bacteria (A. Pirie, 1940). The bacteriophage virus does not transform itself into probacteriophage except in living bacteria. The importance of these facts stimulated us to verify the action of lysozyme lysis. Washed bacteria are placed into microdrops. In the course of exponential multiplication, one injects into the microcultures a solution of lysozyme of such concentration that its final dilution is 1 to 50,000. The bacteria are lysed in several minutes. In different experiments 12, 34, 80, and 208 bacteria were thus lysed. All of the liquid was sampled: no bacteriophages. The presence of lysozyme at the concentration of 1 to 50,000 in a microdrop does not interfere with the assay because the medium is considerably diluted. These observations thus extend to exponentially growing bacteria the results of E. and E. Wollman and of A. Gratia on B. megateriwm and they confirm the fundamental observation of F. Burnet and M. McKie (1929b). VI. How Do LysoGeEenic BaAacTeRIA LIBERATE BACTERIOPHAGES? We are thus in possession of the following facts: 1. In the lysogenic strain of B. megaterium 899, every bacterium isolated in the micromanipulator and washed gives rise to a lysogenic colony. 2. The faculty of producing bacterio- phages perpetuates itself in the absence of exogenous bacteriophages, that is to say by an endomicrobial route. 3. Bacteria can multiply without liberating bacteriophages. 4. Lysozyme lysis does not liberate bacteriophages. Nevertheless, a filtrate of a culture of lysogenic B. megateritum, indeed the filtrate of every lysogenic culture, always contains some bacteriophages. We have verified the fact that this is also true for dense cultures in microdrops: one always finds bacteriophages in the liquid of a small drop of peptone water which contains several thousand bacteria. One can thus entertain the hypothesis that only a small percentage of the bacteria produce bacteriophages. We were encouraged to persevere in the investigation of the mode of liberation of bacterio- phages by the observation of bacterial ‘“ghosts.”’ The formation of the ghosts was observed in the microdrops. It is the result of a relatively slow degeneration of the bacterium which can take several minutes or several seconds. The ghosts persist for several hours. We thought that we had observed bacteriophage lysis. But assay of the medium after the slow lysis did not reveal any bacteriophages. Nevertheless, we continued to follow the development of the microclones, assaying the medium from time to time. We give here the results of a first series of experiments published previously in a preliminary note (A. Lwoff and A. Gutmann, 1949b). A. A filament of free bacteria is washed and brought into one drop of medium S. The following table gives the time in minutes (m), and the number of bacteria (b): m b m b 0 3 65 Zs 30 4 105 25 55 6 123 45 319 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM After the 123rd minute one removes 28 X 10° u* of medium, representing approximately 90% of the volume of the drop: 480 bacteriophages. The bacteria remained united in the long filament. No lysis had been observed. No ghost was visible. We thus concluded that it might be possible that the production of bacteriophages proceeds in the absence of bacterial lysis. At this time we knew only one type of lysis: the slow lysis in which a well recognizable ghost persists and we thought that the absence of such a ghost implies the absence of lysis. Since then, we have recognized another rapid type of lysis, in the course of which the bacteria disappear in less than one second without leaving a recog- nizable residue. This is the type of lysis which liberates bacteriophages. Several terminal bacteria could well have lysed in the course of this experiment, and this lysis could have gone unnoticed. This experiment can thus not be retained to support the thesis of a liberation of bacteriophage without bacterial lysis. B. A diplo-bacillus is washed and placed in peptone water. One notices, according to the table below, that one bacteria disappeared between the 50th and the 55th minute, two disappeared between the 61st and the 62nd minute: m b 2 4 4 14 4 31 6 35 vi 50 11 55 10 61 13 62 11 83 a, 109 43 Samples were taken from the microdrops after 14, 48, and 55 minutes respec- tively 2.5 X 108, 4 X 10%, and 2.4 X 10%: no bacteriophages. At the 63rd minute, one samples 2.4 X 10%’: 18 bacteriophages. At the 83rd minute, 3.2 X 10% y*: 17 bacteriophages. At the 115th minute 5.15 X 10% y*: 68 bacteriophages. The residual volume was 92 X 10’ yu’. The initial volume was thus 149 & 10% u*. One can thus calculate that the number of bacteriophages at the time of the three samplings was 1130, 725, and 190. It is to be noted that the sampling made at the 55th minute, after the disappearance of one bacterium, did not reveal any bacteriophages. In this experiment the bacteria were motile. It is possible that the first lysis was a slow lysis which did not produce bacteriophages and in which the bacterial ghost remained unnoticed. One also notices that the calculated number of bacteriophages was 1,130, a number which seems to us too 320 ANDRE LWOFF AND ANTOINETTE GUTMANN high to correspond to the lysis of only two bacteria. It is possible that, in this experiment, the mixing before the first sampling was insufficient. C. A filament of three bacteria is washed and transferred into peptone water. The history of these three bacteria, a, 6, and c, which remained in a single filament, is indicated in the table below. One notices that bacterium a divided twice, and that the four bacteria which issued from its division lysed between the 88th and the 114th minute. m a b c 0 1 1 1 25 1 1 2 50 2 1 2 77 2 1 2 82 4 1 3 88 4 1 4 107 2 1 4 114 0 2 4 120 2 6 134 Z 9 142 2 9 155 2 iil A sampling of 1 X 10° u* undertaken at the 114th minute did not reveal bacteriophages. A sampling of 1.4 * 10° at the 142nd minute revealed the presence of 71 bacteriophages. Sampling the total fluid at the 155th minute (116.3 X 10° u*) revealed 400 bacteriophages. If one takes into account the ratio of these volumes, one concludes that the total number of bacteriophages must have been approximately 900 at the instant of the 2nd sampling. It is to be noted that the first sampling at the 114th minute was negative while at the 142nd minute there was the appearance of numerous bacteriophages without there having been any lysis of other bacteria. It is possible that also here, as in the preceding experiment, the mixing of the culture medium was insufficient. It is also possible that after lysis the bacteriophages remained engulfed in the viscous cellular debris for some minutes and thus escaped the action of mixing currents. Another experiment should allow us to recognize the character of the lysis by the bacteriophage. A diplo-bacillus is washed and inoculated in one drop of proteose peptone. At the 26th minute, there are two diplo-bacilli, of which one is removed. At the 72nd minute, two other diplo-bacilli are removed. There a filament containing 4 bacteria remains in the drop. A sampling of 6.5 X 10% py made at this moment is negative; at the 143rd minute there is a filament of 8 and a filament of 10 bacteria. One sees one of the bacteria — perhaps one diplo- bacillus? — disappear in about 1 second without leaving any trace. One samples 321 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM 8.5 X 104 uv’ of fluid, corresponding to about half the volume of the drop: 110 bacteriophages. The total number of bacteriophages produced is thus about 220. It is this observation which convinced us of the existence of a rapid lysis followed by the appearance of bacteriophages. Subsequently, we have often observed lysis followed by the appearance of bacteriophages. We have noticed that this lysis does not leave any visible ghost and that it often occurs in less than one second. It could thus very well pass unobserved in a drop which contained several diplo-bacilli in the process of multiplication. We had thought (1949b) that we observed the liberation of bacteriophages without lysis, but after having recognized the two types of lysis — slow and rapid — and having analyzed the possible causes of error which can arise in the course of experiments in microdrops, we have attempted experiments in which a very small number of bacteria shall be present at the same time in a single microdrop. Each time that this was possible, we have worked in such a manner that only a single diplo-bacillus was present in a drop. It is only under these conditions that one can be assured that bacterial lysis is not overlooked. We have also tried to achieve conditions under which a single diplo-bacillus, or a single bacterium, lyses in one drop. In this way, one can sample the totality of the microdrop, thus avoiding having to measure the dimensions of the micro- pipette and calculating its volume and reducing the causes of error in the quanti- tative determinations to a minimum. We took care to stir the drop repeatedly if only a part of the drop was to be sampled. One will see in the following that no liberation of bacteriophage was observed in the absence of bacterial lysis. Second Series of Experiments. a. A filament of 14 bacteria is washed and transferred into a microdrop of peptone water at time h; at h + 20 minutes, there are 18 bacteria. A diplo-bacillus detaches itself and is transferred into a micro- drop. Its two members lyse at h + 40 minutes, at 40-second intervals. At h + 68 minutes, 8 bacteria of the original drop are transferred into microdrops, where they lyse without leaving a trace in less than 10 minutes. There remained 8 bacteria in the original drop. Seven of them lysed under our eyes between h +67 andh+ 71. The residual bacterium is eliminated. All of the liquid is then sampled. The plaques on the petri dish are almost confluent, impossible to count. It looked as if there were about 2000 plaques on the plate. b. A filament of three bacteria is washed and brought into a drop A. At time h + 45, there are about 4 bacteria. A sampling made then is negative: no bacteriophages. At h + 60 minutes, there are 6 bacteria, only 4 at h + 72. These four residual bacteria are transferred with a minimum of fluid into one drop B. All of drop A is sampled: 138 phages, corresponding to the lysis of two bacteria. In drop B two out of four bacteria lysed between h + 72 and h + 79. The two residual bacteria are transferred with a minimum of fluid into a drop C. All of drop B is sampled, where two bacteria have lysed: 38 bacteriophages. The two bacteria remain for 28 minutes in drop C. They are then transferred into D. The total sampling of C does not reveal any bacteriophages. In drop D the two bacteria disappear 4 minutes after their transfer: total sampling indicates 162 bacteriophages. One will notice that in drop C, where two bacteria remained 322 ANDRE LWOFF AND ANTOINETTE GUTMANN until the fourth minute preceding lysis, there was no liberation of bacteriophages. c. A diplo-bacillus was washed and brought into a drop A. Samplings made at h + 35 and h + 70 minutes are negative. At h + 120 minutes there are four bacteria. At the 140th minute, lysis of two members of a diplo-bacillus is observed within a 10-second interval. The other two bacteria are transferred into drops B and C. Total sampling of A: 27 bacteriophages. The bacterium in drop B disappears after the 12th minute: 9 bacteriophages. After 17 minutes in drop C, the remaining bacterium is transferred to D, from where, after an additional 17 minutes, it is transferred into drop E. All of drops C and D are sampled: no bacteriophages. In drop E, the bacterium has disappeared after the third minute: 15 bacteriophages. During the 34 minutes that had preceded lysis there was thus no liberation of bacteriophages by the bacterium which had liberated bacteriophages in drop E. d. A diplo-bacillus is washed and put under observation in a microdrop. At h + 3 minutes there are three bacteria, at h + 48 minutes: 7 bacteria. At the 65th minute, each of the bacteria has divded in two. There are eight bacteria which have remained together in one filament. At h + 83, one of the bacteria is lysed. Within 6 minutes, three other bacteria have lysed. The four bacteria which have disappeared belong to the same mother bacterium. All of the fluid is sampled: 579 bacteriophages. The four remaining bacteria were transferred into another drop: after 22 minutes there were 10 bacteria whose fate was not followed further. Lyses were observed in numerous other experiments, whose details are unneces- sary to give here. The number of phages liberated by the lysis of a single bac- terium can be inferred from the following data, where the italicized numbers represent the number of bacteria lysed and the romanized numbers the number of bacteriophages liberated: 2: 138, 2: 38, 2: 162, 2: 27, 1:9, 1: 15, 4: 89, 4: 452, DE2a0 £2158, 2: 108, 12178, 4:579, 6: 600, 227106; this corresponds to an average of 72 phages per bacterium lysed. The lowest number was 9, the highest 178. It should be noted that under our observational conditions of utilizing an objective ocular lens of magnification 40 and 12.5 respectively, the septum which separates the two daughter bacteria is not visible at the time when it is already functional. We often happened to observe the lysis of a bacterium of unique appearance. Half of that bacterium, in fact a diplo-bacillus, disappeared under our eyes, leaving for a short time a mutilated bacterium bounded by a circular surface. Bacteriophage lysis thus reveals the existence of the septum although the latter is not yet visible under the conditions of our observations. It is quite exceptional to see single independent bacteria in the case of B. megateriwm. Nevertheless, the separation of the two members of a diplo-bacillus was frequently observed before the lysis by the bacteriophage. We thus believe that certain of the numbers which we have given really correspond to the production of phage by a single bacterium. It remains possible, however, that other values which we have reported for one bacterium really correspond to a diplo-bacillus. An important statement must be made at this time. We have seen under our 323 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM eyes some hundreds of bacteriophage lyses. We have thus observed the lysis of 16, 8, 4, or 2 contiguous bacteria. But we have never observed the lysis of a single member of the diplo-bacillus. This question shall be discussed in paragraph VII. Discussion. J. Bordet and Renaux (1928), F. Burnet and M. McKie (1929b), and E. Wollman (1936) envisaged that the bacteriophage is secreted by lysogenic bacteria without the latter being destroyed. The problem of the mode of liberation of bacteriophage by lysogenic bacteria was also discussed by J. Northrop (1939) who, having noticed the appearance of bacteriophages during the growth of lysogenic cultures, concluded that the bacteriophage ‘is produced during the growth of bacteria and not during the lysis.” M. Delbriick and S. E. Luria (1942), on the basis of the observations of E. Cordts, which, to our knowledge, have never been published, thought that lysogenic bacteria can produce bacteriophages without lysis. It should be noted that in the experiments of J. Northrop the value of the ratio of bacteriophages to bacteria was approximately 2. If, in the interval between two divisions, three bacteria out of every hundred lyse, each liberating 72 bacteriophages, then the value of the ratio of bacteriophages to bacteria will be 216 to 97; that is to say, approximately 2. Lysis of 3% of the bacteria during an interval of two divisions does suffice to account for the relative number of bacteriophages and bacteria observed by Northrop. This lysis would entail only a 3% reduction in the theoretical ‘‘growth rate.” But it is difficult to measure growth rate with greater accuracy than 5%. Unless a very high proportion of the “bacteria lyses, no study of growth curves can thus furnish valid data relevant to the mode of liberation of the bacteriophages. All that can be said is that, in the experiments of Northrop, the bacteriophages are liberated during the growth of a bacterial population. But it is impossible to rule out the lysis of a certain percent- age of the bacteria. Our experiments thus bring proof that the bacteriophage produced by the lysogenic B. megaterium is liberated by bacterial lysis. Until the third minute prior to lysis no bacteriophage is liberated. For a long time bacteriologists have observed the partial lysis of colonies of lysogenic bacteria. The majority of these workers thought that this lysis was due to the bacteriophage. But we know that lysogenic bacteria can lyse without necessarily producing bacteriophage. Cultures of B. megaterium 899 lyse completely in peptone medium if they are deprived of oxygen. This lysis begins as soon as anaerobiosis is established. The lysis does not liberate bacteriophages. The fact that the colonies of a lysogenic bacteria lyse partially or totally does not imply that this lysis corresponds to the production of bacteriophages. A fortiori, it is not possible to conclude that under these condi- tions the bacteriophage is liberated by the lysis of bacteria. Is it possible to exclude the hypothesis of a secretion of bacteriophages by viable bacteria from the fact that in our experiments liberation of bacteriophages took place only by bacterial lysis? In the case of sensitive Escherichia coli infected by bacteriophage T2, the nucleus disappears between the fifth and tenth minute after infection (Luria and Palmer, 1945-1946). If the disintegration of 324 ANDRE LWOFF AND ANTOINETTE GUTMANN the nucleus obligatorily accompanies the production of bacteriophages, then it is evident that no bacterium could survive phage production. But it is possible to conceive of production of bacteriophages without destruction of an essential bacterial organelle. It is necessary, however, to think of a way for the bacterio- phages to come out. One knows that lysogenic bacteria adsorb the bacterio- phages. One is thus obliged to envisage a variation of bacterial properties which allows certain bacteria to adsorb bacteriophages at certain stages and to let them out at other stages. It is also possible to conceive that the bacteriophage is liberated when all the elements capable of fixing the bacteriophage are “satu- rated.” Thus liberation of bacteriophages in the absence of bacterial lysis is theoretically possible, but at the present time we do not know of any such case.! And in the only case in which the mode of liberation of bacteriophages by a lysogenic strain was studied with techniques offering sufficient guarantees, the mode of liberation of bacteriophages was bacterial lysis. VII. Factors oF BACTERIOPHAGE PRODUCTION The reader has no doubt noticed that in certain of our experiments all of the bacteria put into a little drop grew and multiplied, whereas in others all the descendants of one bacteria lysed. We thought at the outset that the sudden change of medium was responsible for the onset of bacteriophage production. In order to explain the fact that only a certain percentage of the bacteria was lysed, the hypothesis was envisaged according to which this change only induced the lysis if it took place at certain stages of the life cycle of the bacterium; for instance, during nuclear division. But, we subsequently observed considerable differences between different series of the experiments. On a certain day, for example, we had put 18 bacteria in three drops; all of them divided. The 153 bacteria from these divisions were lysed by lysozyme: no bacteriophages. There had been no production of bacteriophage. On another day, we started with 38 bacteria divided among 5 drops. The descendants of three groups did not show any lysis. The descendants of two groups did lyse partially, and all of the descendants of two other groups lysed. The following results correspond to the study of 104 groups of bacteria: Nnatial number... ... is. 4306 6 4 8 4 8 4 4 Maximum number............ 17 4] 29 13 34 8 10 Number of lysed bacteria...... 0 0 0 12 14 8 10 Proportion of lysed bacteria.... 0/17 0/41 0/29 12/13 14/34 8/8 10/10 1W. H. Price noticed a massive liberation of bacteriophages by cultures of Staphylococcus muscae of constant optical density. The ratio of bacteriophages to bacteria rises from 0.2 to 26 in about 30 minutes. A similar phenomenon was observed in cultures of Bacillus mega- terium by A. Lwoff, L. Siminovitch, and N. Kjeldgaard (unpublished observations), who interpret the phenomenon in the following manner: The optical density of the culture remains fairly constant, or increases slightly, but the bacteria lyse in the course of the dilutions which precede spreading for assay on the petri dishes. This phenomenon shall be described and discussed in detail in a communication which shall appear soon in this periodical. We thus consider that the experiments of Price do not bring any proof of liberation of bacteriophages in the absence of bacterial lysis. 325 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM The results presented below correspond to a study of 104 groups of bacteria: Number of drapsne:. Meno ee hee eee, oe) are eee 104 Fotial number of bacteria nn cre sees aie eee 295 Binalnumbers: (itt eee eed Cee ee ee 947 Number of drops where lysis was observed....................... 3 Numberoiglysed!bacteria 22 sop a tyra ort Cae 44+2+4+8 Hotaltotbactentaslysed tty Lye: Meee sk es kp ay a 14 The number of bacteria used was relatively restricted, but one can nevertheless infer that, in certain experiments, the proportion of bacteria producing phage was less than 5%, while in others, this proportion represented a quarter, or even a third, of all the bacteria used. Discussion. The Mutation Hypothesis. The fact that only a certain proportion of bacteria produced bacteriophages poses the problem of the nature of the factors which induced a lysogenic bacterium to produce bacteriophages. If a sensitive mutilat is mixed with bacteriophages, one observes the lysis of some of the cells and the growth of resistant lysogenic colonies. The statistical analysis of the phenomenon, such as has been carried out by M. Dulbriick and 8. E. Luria (1942), for H. coli, has not been carried out, and it is impossible to state that the resistant bacteria represent spontaneous mutants. This is possible, or even probable. One could envisage the hypothesis that this mutation from sensitiv- ity to resistance is reversible. In the case of a lysogenic bacterium the mutation from resistance to sensitivity would create conditions which permit “inactivation” of the potential bacteriophage and of the lysis of the bacterium. One, thus, conceives the idea that the mutation could intervene in determining the production of bacteriophages by certain lysogenic bacteria. The mutation rate is generally independent of the conditions of the medium, and we have seen that the percentage of bacteria which lyse varies from .5 to 30%. But this does not necessarily exclude the mutation hypothesis. It is, in fact, conceivable that this variability is the result of the simultaneous coming into play of a high mutation rate and of selection. We would invoke the clonal character of the lysis in support of this hypothesis. Let us recall that we never observed the lysis of a single member of a diplo-bacillus, and that we have sometimes seen the lysis of all of the bacteria in the same filament. This could be interpreted in the following manner: Only the descendants of certain mutants lysed, while there occur from 1 to 4 divisions between mutation and lysis. This hypothesis is not incompatible with the hypothesis of induction. One could see, in effect, that the mutation affects only a genotypic factor, and that the latter does not express itself by the production of bacteriophages except under specific conditions of the medium. It is, however, the study of the kinetics of the production of bacteriophages, which shall now be presented, which allows one to exclude the hypothesis of the intervention of a mutation. The Induction Hypothesis. The behavior the B. megaterium in microdrops has given us the impression that the production of bacteriophages must depend, in part, on the previous history of the bacteria, and the hypothesis was envisaged 326 ANDRE LWOFF AND ANTOINETTE GUTMANN that the inducing factor intervenes in the course of the development of the mother culture. The drop technique, in spite of long and laborious experiments, did not bring the solution to this problem, which was then taken up with ordinary cultures. Since that time, experiments (A. Lwoff, L. Siminovitch, and N. Kjeldgaard) have shown that it is possible to achieve conditions which induce the lysis of 20 to 30% of a bacterial culture and that this lysis is accompanied by the liberation of bacteriophages. These experiments shall be described in a separate communication. We shall content ourselves here with a discussion of the conclusions to which these experiments led, that is to say, that the production of bacteriophages is induced by external factors. The Hypothesis of Burnet and McKie. F. Burnet and M. McKie (1929b) realized that all bacteria of the lysogenic strain contain in their hereditary constitution a unit which is potentially capable of liberating bacteriophages; that these bacteriophages can be liberated during the process of normal growth; that activation of the hereditary structure must take place spontaneously for there to be any liberation of bacteriophages; that the data do not allow one to infer whether or not there is an intracellular activation; that the conditions are probably analogous to those which intervene in monomolecular reactions or in radioactive disintegrations. Undoubtedly, some of these concepts did not stand up to the test of time. But the problem was remarkably well stated. And the hypothesis of “activation” should have merited better than a complete indif- ference. Everything seems to indicate that the bacteriophage possesses a remarkable attractive power. It inhibits, in effect, bacterial growth, detours to its own end the metabolites necessary for enzymatic adaptation (J. Monod and E. Woll- man, 1947) and growth of a different bacteriophage (M. Delbriick and S. E. Luria, 1942; A. D. Hershey and R. Rotman, 1948). H. J. Muller (1947), while discussing the problem of the reproduction of the gene, concluded that only the phenomena of long range forces could account for the duplication. Perhaps the attractive power of bacteriophages is of this same type. Let us suppose that in a lysogenic bacterium the probacteriophage, under whatever form it might exist, is in competition with a certain number of other particles. In potentially lysogenic bacteria that equilibrium is stable. But if, for one reason or another, the equilibrium is perturbed possibly because the attractive power of the bacterial particles is diminished or because the attractive power of the probacteriophage is increased, the latter will develop and give rise to bacterial virus particles. More concretely and simply, one might envisage that the “‘key enzyme’ necessary for bacteriophage synthesis, which M. Delbriick and 8. E. Luria (1942) have postulated in order to explain the phenomenon of interference, becomes available; perhaps, because the normal substrate of this enzyme ceases to be synthesized. Let us note in passing that this defect of sub- strate synthesis could as well be the consequence of a mutation as a change of the conditions of the medium. In any case, the association or combination of this key enzyme with the probacteriophage would then set off the process of mul- tiplication. 327 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM We can now discuss the “activation” theory of Burnet and McKie. The activation could be the consequence of a mutation. But since, as in the case of B. megaterium, this activation is induced by conditions of the medium, it is simplest to envisage, at least in the case of the bacterium which we have studied, that the activation is the result of the suppression of a competition between the pro- bacteriophage and a specific substance or particle. This is obviously but a working hypothesis. According to unpublished observations of A. Lwoff, Louis Siminovitch, and N. Kjeldgaard, the length of the latent period of the bacteriophage of B. meg- atervum in the sensitive strain under the conditions of these experiments is 45 minutes. Thus, in the experiments of these authors, 45 minutes elapsed between the moment of the inflection of the bacterial growth curves and the start of the production of the bacteriophage, which lasted for 20 to 30 minutes. “It seems,”’ they write, “‘as if a factor, or an ensemble of factors, among which we know that the aeration of the medium plays an important role, determines the condition which sets off the production of the bacteriophages. This sudden drop in the rate of growth is evidently related to the development of the bacteriophage, but one does not know whether it is the effect or the cause or both cause and effect.” A. Lwoff, L. Siminovitch, and N. Kjeldgaard envisage that the factors of induction act by modifying the relative speed of multiplication of the bacterium and of the probacteriophage. The equilibrium is modified in an irreversible man- ner once the number of probacteriophages surpasses a critical value. In order to explain its clonal characteristics, it suffices to suppose that lysis does not take place until several divisions have occurred. The problem of the nature of the factors of induction and of the mechanism of their action shall be discussed in a future communication which shall appear in these Annales. VIII. Derrnirion or LysoGenic BACTERIA Since the liberation of bacteriophages by the lysogenic strain 899 of B. meg- atervum occurs by bacterial lysis, there exists an incompatibility between the pro- duction of bacteriophages on the one hand and the survival and, a fortiori, perpetuation of the bacterial individual on the other hand. In a “lysogenic” strain, there are bacteria which multiply and other bacteria which produce and liberate bacteriophages. A lysogenic culture producing bacteriophages can be transferred indefinitely, but only under the condition that it includes bacteria which do not produce bacteriophages. On the contrary, a bacterium producing bacteriophages is a bacterium condemned to death. It is thus appropriate to distinguish between a lysogenic culture and a lysogenic bacterium; in case of the latter the production of bacteriophages is but a potential faculty. For that reason we have proposed the following definition: ‘A lysogenic bacterium is a bacterium which perpetuates the capacity to form bacteriophages without intervention of exogenous bacterio- phages.” In the case of lysogenic bacteria, the bacteriophage perpetuates itself in the form of the probacteriophages and not in the form of bacteriophage virus par- 328 ANDRE LWOFF AND ANTOINETTE GUTMANN ticles. One can thus compare a lysogenic bacterium to a healthy carrier of germs. What constitutes the particularity of lysogenic bacterium is that they per- petuate a potentially lethal character. The expression of this character, which can be induced by exogenous factors, involves the death of the bacterium. e bacteriophage o probacteriophage lysogenic bacteria ; , ] ‘ : sensitive (4 «induction easton infection->{ Rectan) ~ / a soc tee ae BS e) e ee eet Soe co ee | e* @ | *ece ‘ pee Ne Lo PES) i we Diagram showing the evolution of bacteriophage and probacteriophage (prophage) in sensitive and lysogenic megaterium bacilli. Both in the case of the sensitive bacterium, as well as in the case of a resistant bacterium, the penetration of the bacteriophage is followed by a disappearance of the infectious character. In both cases, the infectious corpuscle is not re- generated until a short time prior to the bacterial lysis. In both cases, finally, the multiplication of the bacteriophage involves the lysis of the bacterium. But in the case of a sensitive bacterium the penetration of the bacteriophage and its transformation into a non-infectious form is followed immediately by an 329 INVESTIGATIONS ON A LYSOGENIC BACILLUS MEGATERIUM important process of multiplication involving the death of the bacterium. In contrast, after the penetration of the bacteriophage into a resistant bacterium destined to become lysogenic the probacteriophage behaves as a nonpathogenic, normal cellular unit, which does not assume a pathological development except as a consequence of an “activation.” This development in a bacterium of a particle which ultimately becomes “malignant” recalls in some respect the multiplication of neoplastic cells in a cancerous organism. One could be tempted to write, as perhaps we ourselves have done, that potentially lysogenic bacteria live “in equilibrium”’ with their bacteriophage. But, in fact, they live in equilibrium with a probacteriophage. There exists an incompatibility between the structure “bacteriophage-virus” and the survival of the bacterium. It is true that lysogenic bacteria are resistant to the bacteriophages which their sisters produce. But no more than a sensitive bacterium does a lysogenic bacterium resist the multiplication of bacteriophages. It is thus the multiplica- tion of bacteriophages which constitute the pathologic fact. IX. RESuME AND CONCLUSIONS 1. Bacillus megaterium cells washed and isolated in microdrops were utilized in an attempt to resolve some of the problems posed by lysogenic bacteria. 2. B. megatertum can multiply without liberating bacteriophage: production of bacteriophages thus does not obligatorily accompany the growth and the division of the bacteria. 3. The capacity to produce bacteriophages was maintained in the course of 19 divisions in the absence of all free bacteriophages: the faculty to produce bacteriophages is thus perpetuated intrabacterially. 4. Rapid lysis of lysogenic bacteria was observed under the microscope. This lysis is followed, and never preceded, by liberation of bacteriophages. B. megateritum thus liberates bacteriophages by lysis. 5. The proportion of bacteria which liberate bacteriophages varies within considerable proportions: from less than 5% to 30%. All indications are that the production of bacteriophages is induced by external factors. 6. Lysogenic bacteria are defined as bacteria in which the capacity to produce bacteriophages is perpetuated without intervention of exogenous bacteriophages. 7. The theory of the activation of an intracellular anlage of the bacteriophage (F. Burnet and M. McKie) has been discussed, developed, and amended. BIBLIOGRAPHY Apams (M. H.). J. Immuznol., 1949, 62, 505-516. BayYNE-JONES (S.) et SANDHOLZER (L. A.). J. exp. Med., 1933, 57, 279-304. Borpet (J.). Ces Annales, 1925a, 39, 717; C. R. Soc. Biol. Belge, 1925b, 93, 1054. 330 ANDRE LWOFF AND ANTOINETTE GUTMANN Borpet (J.) et Renaux (E.). Ces Annales, 1928, 42, 1283. Burnet (F. M.) et Lusu (D.). Austr. J. exp. Biol., 1936, 14, 27-38. Burnet (F. M.) et McKrx (M.). Austr. J. exp. Biol., 1929a, 6, 21-31 et 1929b, 6, 277-284. FonBRUNE (P. DE). Technique de micromanipulation. Masson, edit., Paris, 1949. Devsrick (M.). Biol. Rev., 1946, 21, 30-40. Dewprick (M.) et Luria (8. E.). Arch. Biochem., 1942, 1, 111-141. Den Dooren ve Jone (L. E.). Zentralbl. Bakt., 1931a, 120, 1-23; [bid., 1931b, 122, 277-286; [bid., 1934, 131, 401-410. Duaear (B. M.) et Anmsrrone (J..K.). Ann. Missouri Bot. Garden, 1923, 10, 191. Foster (R.). J. Bact., 1948, 56, 795-809. Gorin! (L.) et FromaGenot (C.). C: R. Acad. Sci., 1949, 229, 559-561. GratiA (A.). Ces Annales, 1936a, 56, 307; C. R. Soc. Biol. Belge, 19366, 122, 812; Ibid., 1936c, 123, 506; ces Annales, 1936d, 57, 652. Gratia (A.) et Wetcu (M.) C. R. Soc. Biol. Belge, 1939; 132, 330. Hersuey (A. D.). 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Biol., 1937, 124, 931; Ces Annales, 1938, 60, 13. 331 MICROBIOLOGY INDUCTION OF BACTERIOPHAGE LYSIS OF AN ENTIRE POPULATION OF LYSOGENIC BACTERIA! by André Lwoff, Louis Siminovitch, and Niels Kjeldgaard Presented by Robert Courrier In a culture of lysogenic Bacillus megaterium, some bacteria multiply without liberating bacteriophages and perpetuate the lysogenic strain, while others produce bacteriophages which are liberated by lysis.2- Under normal conditions of exponential growth, only a very small percentage of bacteria produce bacteriophages. *» We have succeeded in inducing bacteriophage lysis of an entire popula- tion of lysogenic bacteria by means of irradiation by ultraviolet light. B. megaterium is grown in a yeast extract medium.? During the expo- nential growth phase, when the number of bacteria has attained 34 x 10° per ml., a 2 mm layer of the culture is irradiated with ultraviolet light from a high pressure mercury vapor lamp, which delivers to the surface of the liquid an energy of 2000 ergs per mm? per minute of radiation of wave length 2537 A. The culture is then shaken at 37° and its optical density (O.D.) increases for about 80 minutes. The normal growth rate, that is to say the number of doublings per hour, is 3. Immediately after irradiation the growth rate is in the neighborhood of 1.5. At about the 80th minute, when the O.D. has increased by a factor of 3 to 4, bacterial lysis takes place: the culture clears in 40 to 80 minutes. The fraction of bacteria surviving is usually less than 10~‘. Lysis is accompanied by the liberation of about 70 to 150 bacteriophages per bacterium. Analogous results have been obtained with irradiation for 20, 30, 90, or 120 seconds. A culture growing in synthetic medium and similarly irradiated from 1 to 60 seconds does not lyse. On the other hand, if a culture growing in yeast medium is centrifuged, resuspended in synthetic medium, and then ir- radiated for 5, 10, 20, or 30 seconds, bacteriophage lysis does take place. Translated from the French and reprinted by permission of the authors and the Imprimérie Gauthier-Villars from the ComprTss RENDUS DES SKANCES DE L’ACADEMIE DES SCIENCES, 231, 190-191 (1950). 332 Microbiology Finally if yeast extract is added to a culture growing in synthetic medium and the culture irradiated immediately for 10, 20, 30, or 60 seconds, no lysis takes place. But irradiation carried out 20 to 40 minutes after addition of the yeast extract (by which time the O.D. has increased by about 50%) induces bacteriophage lysis. UV irradiation thus induces production of bacteriophages only with bacteria which have grown for 20 to 40 minutes in a complex organic medium such as yeast extract. Irradiation for 30 to 60 seconds of a non-lysogenic culture of B. mega- tertum growing in yeast extract does not appear to affect bacterial growth. The lysis of the lysogenic strain is thus probably not a direct effect of the UV irradiation. It has been demonstrated previously that all bacteria of a lysogenic strain are capable of perpetuating the lysogenic character.?. The experi- ments described here, which have been complemented by studies of bacteria irradiated and then isolated in microdrops, now demonstrate that under certain conditions, all the bacteria of a lysogenic population are capable of undergoing lysis with liberation of bacteriophages. ‘Supported by a grant of the National Institutes of Health of the United States of America. 2A. Lwoff and A. Gutmann, Ann. Inst. Pasteur, 78, 711-739, (1950). 3A. Lwoff, A. Siminovitch, and N. Kjeldgaard, Comptes rendus, 230, 1219-1221, (1950); Ann. Inst. Pasteur, 79, 815 (1950). 333 GENETICS LYSOGENY AND GENETIC RECOMBINATION IN ESCHERICHIA COLI K12 by Elie L. Wollman and Francois Jacob Presented by Jacques Tréfouel Crosses between F. coli K12 Hfr and F-, both lysogenic, show that the ) pro- phage and Gal, are situated on the segment TL Lac,. Spontaneous induction of the prophage suppresses those of the zygotes which received a A prophage whenever the Hfr parent is lysogenic and the F~ parent non-lysogenic. This modifies the distribution among the recombinants of characters linked to the d prophage. It had been supposed until now that the presence of the prophage does not alter the results of genetic analysis of EH. cola K12. The phe- nomenon of spontaneous induction observed in the course of crosses between lysogenic Hfr (ly*) bacteria and non-lysogenic EF (ly~) bacteria established the fact that in the course of certain crosses an 1m- portant fraction of the zygotes can be eliminated.! One might ask whether this elimination does not affect preferentially some segments of genetic material, thus exercising a counterselection with respect to certain classes of recombinants. In particular, it might be possible to explain in this way the anomalies found in crosses of the type Ft ly* & F7 ly~.? In crosses of Hfr Tt Lt S* & F- T- L’ S& in which the bacteria are mixed in the proportion of 1 Hfr bacterium for 20 F~ bacteria the fre- quency of recombination (the number of Tt L* S" recombinants divided by the initial number of Hfr) varies considerably according to the actual cross carried cut. This frequency, which is of the order of 5 to 10% for crosses Hfr lyt X F~ ly-, surpasses 50% in crosses of Hfr lyt X F- ly anc of Hirdy=>GR-lys. Translated from the French and reprinted by permission of the authors and the Imprimérie Gauthier-Villars from the ComprTEs RENDUS DES SEANCES DE L’ACADEMIE DES SCIENCES, 239, 455-456 (1954). 334 Genetics The distribution of non-selected markers among the recombinants is also affected by the nature of the crosses. In a cross of Hfr ly~ Lact Galt x F- ly~ Lacy Gal; 55% of the recombinants are Lact, 45% are Gal*. The simultaneous passage of lact and Gal* occurs in 40% of the cases, which indicates linkage between Lac, and Gal, In a cross of Hfr lyt Lact Galt X F- ly~ Lacy Galj, in contrast, 25% of the recombinants are Lac,* but only 3% are Galt, of which about half are also Lact. Among these recombinants, only 0.2% are lysogenic. It is known that there exists a direct linkage between lysogeny and the character Gal,.2? The extreme rarity of lysogenic recombinants, as well as the considerable diminution of the proportion of Galt recombinants, is the consequence of the spontaneous induction of the prophage in a large fraction of the zygotes. The relatively smaller diminution of the proportion of Lact recombinants similarly reflects this phenomenon, but shows at the same time that the character Lac, is much less linked to the characters Gal, and ly than the latter are linked to each other. Lac, is apparently located between TL and Gal,. The localization of the prophage in this region of the bacterial chromosome is demonstrated by crosses of the type Hfr lyt Lact T1* xX F- lyt Lac, TI’, in which the two lysogenic strains perpetuate different \ prophages. The prophage of the Hfr parent is transmitted to 30% of the recombinants while the Lac and the T1* characters are each transmitted in 50 and 75% of the cases. It should be noted that the establishment of the prophage of the Hfr parent in the recombinants is one third as frequent as the development of this prophage in the zygote in the course of crosses Hfr lyt X IF” ly~. It is known that in a cross of Hfr X F~ the high frequency of recombina- tion observed concerns only the segment TL Lac,. We have verified this fact in all of our crosses. The relatively high frequency of recombination of the character \ lysogeny thus localizes the \ prophage in this segment. The elimination of a chromosomal segment by spontaneous induction of the \ prophage could also occur, though to an extent difficult to evaluate, in crosses of the type lyt x ly* or F* ly X F ly™. It offers a model which might conceivably explain the segmental eliminations des- cribed by J. Lederberg in heterozygote diploids’ and by W. Hayes‘ in recombinants of Hfr strains. 1F. Jacob and E. L. Wollman, Comptes rendus, 239, 317, (1954). 2h. L. Wollman, Ann. Inst. Pasteur, 84, 281, (1953); R. K. Appleyard, Cold Spring Harbor Symp., 18, 95, (1953). 3E. M. and J. Lederberg, Genetics, 38, 51, (1953). 4W. Hayes, Cold Spring Harbor Symp., 18, 75, (1953). 5J. Lederberg, Proc. Natl. Acad. Sci., 35, 178, (1949). 335 GENETICS SPONTANEOUS INDUCTION OF THE DEVELOPMENT OF BACTERIOPHAGE }) IN GENETIC RECOMBINATION OF ESCHERICHIA COLI K12 by Francois Jacob and Elie Wollman Transmitted by Jacques Tréfouel In the course of crosses between lysogenic Hfr and non-lysogenic F- bacteria of F. coli K12, the prophage passes from the lysogenic to the non- lysogenic parent becomes induced, and develops in the latter. This process leads to the destruction of the zygote formed. In crosses between lysogenic (lyt) and non-lysogenic (ly~) Escherichia coli K12 we have not been able to demonstrate the transfer of the lysogeny of the Ft parent to a recombinant possessing essentially F~ genome.! Nevertheless, the lysogenic character segregated and appeared to be linked to another genetic factor.*»! Since the \ prophage is inducible, it appeared possible that recombination between F*ly* and F-ly~ involves the development of the phage and the destruction of the zygotes formed’ The discovery of strains giving a high frequency of recombination (Hfr)* makes it possible to verify this hypothesis. Hir lv* S* bacteria (lysogenic and sensitive to streptomycin) and F- lv-/X S™ bacteria (non-lysogenic and resistant to and to streptomycin) are mixed in broth in the proportion of 1 Hfr to 20 F~. This mixture is shaken at 37° and at various times samples are removed and spread on strep- tomycin containing agar seeded with indicator bacteria resistant to strep- tomycin but sensitive to phage \. A control culture of Hfr is treated in the same manner. One observes that the number of infective centers in the mixture in- creases linearly as a function of time until about the 40th minute, when a plateau is reached which corresponds to approximately half of the initial 1K. L. Wollman, Ann. Inst. Pasteur, 84, 281, (1953). 2K. M. and J. Lederberg, Genetics, 38, 51, (1953). 3L. L. Cavalli and H. Heslot, Nature, 164, 1057, (1949); W. Hayes, Cold Spring Harb. Symp. 18, 75, (1953); W. Hayes has sent us his strains of Hfr. Translated from the French and reprinted by permission of the authors and the Imprimérie Gauthier-Villars from the ComprTEs RENDUS DES SHANCES DE L’ACADEMIE DES SCIENCES, 239, 317-319 (1954). 336 Francois Jacob and Elie Wollman number of Hfr. This number is 100 times greater than the number of infective centers found in the control. After the 60th minute, the number of infective centers suddenly increases and at about the 150th minute reaches a value 50 to 100 times greater than that of the first plateau. The difference between the mixture and the control Hfr is further ac- centuated if anti-phage serum and streptomycin are added to the two cul- tures at the beginning of the experiment. For, the number of infective centers in the control falls rapidly to zero while the number of infective centers in the mixture is only reduced by one-half. The development of phage \ has thus been induced by putting Hfr lyt in contact with F- ly~. This development can only take place in F7 bacteria (or in the zygotes), since the Hfr bacteria were sterilized by streptomycin. If the same cross is repeated with an I~ Ss strain, addition of streptomycin during the latent period arrests the development of the phage. An even more direct demonstration of the passage of the prophage from Hfr to F~ bacteria (or to the zygote) and of its development in the latter is furnished by the following experiment. It is known that certain bacterial strains confer a phenotypic modification to the bacteriophages which they produce.‘ Thus bacteriophage »(K12) produced by FE. coli K12()), whose efficiency of plating is identical on FE. coli K12 8 and on FE. coli C, has a greatly reduced efficiency of plating (10? to 107%) on K12 8 after passage on EL. coli C. If development of the prophage actually takes place in the non-lysogenic F~ bacteria, then in a cross of K12 Hfr(A) S* with C/X F- S the bacteriophages produced should be A(C) and not A(K12). Experiments show this to be the case. When F~ bacteria perpetuate a prophage, normal or defective, the immunity which the prophage confers on them® protects them equally against the development of a prophage coming from the Hfr parent. If one crosses Hfr bacteria superinfected with virulent mutants of the type d,,° one observes little or no passage of the superinfecting phages into the F~ bacteria. This result indicates that the transfer of phage material from Hfr to F~ takes place in the prophage state and not in the vegetative state. It also indicates that the induction of phage development in the course of recombination takes place in the F~ bacterium (or in the zygote) and not in the Hfr bacterium. It is evident that the effect described here must introduce important distortions in the genetic analysis of HL. colt K12: in effect, only those zygotes give rise to recombinants in which no multiplication of the phage has taken place. The proportion of these survivors will vary considerably depending on the nature of the cross. Thus in the cross of Hfr ly By 5S* by W678 T- L~ B; ly~/AS* the number of recombinants of the type 4G. Bertani and J. J. Weigle, J. Bact., 65, 113, (1953). 5F. Jacob and E. L. Wollman, Cold Spring Harb. Symp., 18, 101, (1953). 337 Genetics Tt L* S* does not exceed 5% of the initial number of Hfr bacteria, although more than 50% of these bacteria give rise to infective centers. Among 200 recombinants examined not one was lysogenic. In a compar- able cross in which the F~ strain perpetuates a defective prophage, the number of [* L* S" recombinants reaches 50% of the initial number of Hfr, although the number of infective centers does not surpass 5%. Among the recombinants about 30% are normal lysogenics. The phenomenon of spontaneous induction of the prophage is also found in crosses of F*+ ly* by F~ ly~ and thus explains the observed absence of the transmission of lysogeny in these crosses. It could also be the origin of other anomalies found in the study of the genetics of E. coli K12. 338 Transduction of Lysogeny in Escherichia coli FRANGOIS JACOB Service de Physiologie microbienne, Institut Pasteur, Paris Received, March 2, 1955 SUMMARY Transduction provides a new tool for a genetic analysis of lysogeny. Re- cently, transduction has been shown by Lennox to occur, through phage P1, in various strains of Escherichia coli. Another strain of temperate phage has been isolated, which is also able to transfer genetic characters from a donor to an acceptor strain of #. coli K12. Linked characters can be transduced simul- taneously. Lysogeny or nonlysogeny, with respect to each of three different prophages is transduced together with a galactose marker, to which these three prophages are linked as shown by bacterial recombination evidence. When two genetically different and complementary prophages—one in the donor and one in the acceptor cells—are used in transduction experiments, recombination of prophages has been shown to occur. INTRODUCTION A new mechanism allowing the transfer of genetic characters from one bacterial strain to another was found in Salmonella by Zinder and Lederberg (1952). This mechanism, for which the term ‘‘transduction”’ was coined, appears to involve phage particles as vectors of the genetic material of the bacteria (Zinder, 1953; Stocker et al., 1953). Transduction can be observed when phage-sensitive bacteria, acting as acceptor cells, are infected with phage particles grown on donor bacteria which differ from the acceptor by one or several genetic characters. Some of the in- fected bacteria that survive phage infection acquire and transmit to their progeny one or several characters of the donor strain. The trans- ducing ability seems to be restricted to some strains of temperate phages. It has recently been shown by Lennox (1955) that phage P1 is able to transduce various markers from one strain of HE. coli to another and that linked characters can be transduced together. This finding makes it possible to compare linkage data provided by transduction and by bacterial recombination which was demonstrated by Tatum and Leder- berg (1947) to occur in strain K12 of E. colt. Reprinted by permission of the author and Academic Press, Inc. from ViroLoey, 1 (2), 207-220 (1955). 339 208 FRANCOIS JACOB This paper is mainly concerned with preliminary results on trans- duction of lysogeny in EL. cola K12 by the use of a strain of temperate phage isolated in our laboratory. Previously, information on the genetics of X lysogeny was gained through bacterial recombination experiments with EH. coli K12 (Lederberg and Lederberg, 1953; Wollman, 1953; Appleyard, 1954a; Wollman and Jacob, 1954). The results indicated that the d-lysogenic character segregates in crosses between lysogenic and nonlysogenic bacteria and, moreover, that this character is linked to a marker that plays a role in galactose utilization. Nevertheless, whereas the presence of a nuclear unit controlling lysogeny was thus demonstrated, it had not yet been proved fully that this bacterial unit was identical to the prophage, i.e., to the genetic material of the phage in the lysogenic condition (Lwoff, 1953). Transduction of lysogeny offers a new way of analyzing the relations between prophage and bacterium, as well as the size and orientation of the prophage. MATERIAL AND METHODS Transducing phage. Various kinds of temperate phages have been tested for their transducing ability in EL. coli K12. Of twenty-three phages released by different lysogenic strains of FE. coli isolated from patients, only one was active: phage 363. It forms tiny turbid plaques on FE. coli K12 (Fig. 1A). Its latent period in broth is about 50 minutes, and its burst size is 200 to 300. Its frequency of lysogenization is higher at 20° than at 37°. Phage 363 appears to belong to the same group as phage P1, previously described by Bertani and Nice (1954) and used by Lennox (1955) in his transduction experiments. Bacteria lysogenic for 363 are immune against P1, and vice versa. Phage 363 is inactivated, although at a lower rate than P1, by an anti-P1 serum.! Transduced prophage. The properties of phage d and its relationship with the bacterial host have been described (Lederberg and Lederberg, 1953; Weigle and Delbriick, 1951). Two other temperate and ultraviolet- inducible phages, released by lysogenic LH. coli isolated from patients, have also been used in these experiments: phages 82 and 434 (Fig. 1B, D). The properties of these two phages will be described elsewhere. Phages 82, \, and 434 can easily be distinguished by their host range, as shown in Table 1. Bacterial strains. The bacterial strain used as a donor was a proto- troph, K12, sensitive to 363, 82, 2, and 434. 1A sample of anti Pl serum (anti H~) was kindly supplied by Dr. J. Beumer. 340 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 209 a : f BO a Ci © % 92 oe" °e Oo e ° oe e <8 Fra. 1. Plaques formed on £. coli K12 by (A) phage 363, (B) phage 82, (C) phage Ams, and (D) phage 434. The acceptor strain was a derivative of W677 (Lederberg, 1947) pre- pared by Dr. E. L. Wollman. This strain, called P678, is unable to syn- thesize threonine (7'-), leucine (L~), or vitamin B, (B,) or to ferment lactose (Lac; ), xylose (Xyl-), mannitol (Man-), maltose (Mal-), or galactose (Gali, Gal; ). It is not lysogenic for \ (A)~ and is resistant to streptomycin (St). The Gal, marker of this strain is closely linked to the locus. Its relationship to other galactose loci in the same region is unknown. Substrains of K12 and P678, lysogenic for one or more of the four prophages (363, 82, 4, and 434), have been isolated. In the description of the following experiments, P678(A)* will refer to a substrain of P678 lysogenic for \. Variants resistant to one or more of the three phages have also been used: P678/) will refer to a variant of P678 resistant to X. Preliminary crosses between Gal; SsHfr, nonlysogenic, and Gal, S'F-, lysogenic for one of the prophages, indicate that both prophages 82 and 434, as well as X, are located in the Gal, region. When S™ and Gal} are used as selective markers, the proportion of nonlysogenic recombinants is about 90% with phage 82, 80% with phage \, and 70% with phage 434. Prophage 363 is located in a different region. 341 210 FRANCOIS JACOB TABLE 1 Host RANGE OF PHAGES 82, \, AND 434 Phages Bacterial strain 82 N 434 K12(82)* 0 ai Bre K12(A)t ar 0 a5 K12(434)+ =F ae 0 K12/r =F 0 ae K12/82, 434 0 air 0 Media. For infection experiments, broth supplemented with 107? M CaCl, was used. The selective medium was the following: KH:PO.—13.6 g; (NH4)2SO, —2 g; Ca (NO;)2—0.001 g; MgSO,-:7 H,O—0.02 g; Difco agar—20 g; H,O—1000 ml; KOH added to pH 7.0. Sugars were added separately at a concentration of 1%. Amino acids were added at a concentration of 100 ug/ml; vitamin B, at 5 ug/ml. Transduction experiments. Phage 363 was grown on the prototroph strain K12, either lysogenic or not. The preparations were sterilized with chloroform. Their titer usually reached between 2 and 8 X 10? particles per milliliter. For transduction experiments, about 5 X 10° particles of phage 363 were added to 4 ml of a broth culture containing about 2 * 10° bacteria per milliliter of the acceptor strain, P678 Sr". This mixture was shaken for 2 hours at room temperature and then centrifuged and washed. The bacterial pellet was resuspended in buffer, and aliquots were plated on selective minimal medium supplemented with streptomycin to avoid contamination. In each experiment, the phage preparation was tested for sterility as well as for plaque count, and a noninfected culture was assayed as a control for spontaneous mutants. EXPERIMENTAL RESULTS Evidence for transfer of bacterial genetic material through phage 363. Phage 363 can transfer various kinds of characters from donor to ac- ceptor cells. As previously found by Lennox (1955), unlinked characters are transduced independently, whereas characters which are known to be linked can be transduced simultaneously. In Table 2 are reported the results of an experiment in which several characters have been trans- 342 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 211 TABLE 2 TRANSDUCTION OF VaRIOUS MARKERS WITH PHAGE 363 Colonies Colonies per 5.6 108 icine Pepin” Baten pected with Threoninet 4 730 Leucine* 1 1280 Threonine* + leucinet 0 23 Lactoset 0 63 Galactose? 0 129 Acceptor P678 bacteria infected with phage 363 grown on prototrophic K12 (multiplicity of infection, 0.3), and uninfected control bacteria were shaken for 2 hours at room temperature. After centrifugation, the bacteria were resus- pended in buffer and plated on various selective media. duced. Aliquots of noninfected P678 bacteria and of bacteria infected with phage 363 grown on a prototrophic strain were plated on various selective media. It is seen in Table 2 that the nutritional characters threonine* or leucine* are transferred separately with a frequency of about 10~* per adsorbed particle. These two markers, which are known to be linked from bacterial recombination experiments (Lederberg, 1947), can be transferred simultaneously with a frequency of about 1077 per phage particle. The two fermentative markers, Lact and Gal}, are transmitted with a lower efficiency than 7+ or L+. Gal}, with which the experiments re- ported in this paper will be mainly concerned, is transferred with a fre- quency of about 0.7 to 1.5 X 10-® per phage particle. Four hundred colonies selected for transduction of Gal; were assayed for threonine, leucine, lactose, mannitol, and xylose. None of them had acquired any of these characters other than galactose utilization. The efficiency of transduction varied widely from one experiment to another and from one preparation of phage 363 to another. In various experiments, the frequency of lysogenization with phage 363 in the transduced clones was found to vary from 30 to 70%. Nevertheless, in a given experiment, this proportion was about the same for each class of transduced cells, independently of the selective marker. Transfer of nonlysogeny. In order to demonstrate transfer of non- lysogeny, bacteria lysogenic for one of the three prophages—82, \, or 434—and resistant (nonabsorber) to the homologous phage were infected 343 212 FRANCOIS JACOB TABLE 3 TRANSFER OF NONLYSOGENY Selection for Experi- Threonine* Lactose* Galactose ment Acceptor strain + No. Number | Number | Number | Number | Number | Number of of of of of of colonies non- colonies non- colonies |nonlyso- % tested | lysogenic} tested | lysogenic] tested genic 1 P678(82)+/82 200 0 200 0 200 19 9.5 P678(82)+/82 200 0 400 53 13 1 P678(A)t/r 200 0 200 0 200 12 6 P678(A)*/r 200 ) 200 0 400 32 8 1 P678 (484) */434 160 0) 160 0 160 3 1.8 2 P678 (434) */434 400 2 0.5 Acceptor bacteria P678 lysogenic for one of the three prophages—82, \, or 434— and resistant to the homologous phage were infected with a preparation of phage 363 grown on nonlysogenic K12. After plating on various selective media, col- onies were tested for their ability to release the original phage, 82, \, or 434. with a preparation of 363 grown on prototrophic nonlysogenic bacteria. Infected bacteria were plated on various selective media, and colonies were tested for their ability to release the phage of the parent strain (82, \, or 434). The results of such experiments are reported in Table 3. It is seen, on the one hand, that among the 7*, L*, or Lac* colonies none was found to be nonlysogenic. On the other hand, a small fraction of the Galt clones was found to be nonlysogenic, the proportion of non- lysogenic clones depending on the nature of the prophage involved This result is in agreement with bacterial recombination experiments which show that each of these prophages exhibits a different linkage to the Gal, locus. A question raised by the transduction of nonlysogeny is whether only a piece of the prophage can be replaced by an homologous region of nonlysogenic bacteria. Such a partial substitution could eventually result in an immune defective clone, i.e. a clone in which bacteria possess a fragment of the prophage which does not allow the synthesis of infective particles, but prevents phage multiplication after infection with homol- ogous particles. In order to detect such clones, transduction of non- lysogeny was also performed on lysogenic bacteria able to adsorb homolo- 344 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 213 gous phage particles. With prophage \, no defective immune clone was found. With phage 82, it was found that 2 out of 21 Gal+ clones which did not produce infective phage were immune against phage 82 and its virulent mutant, 82c. After ultraviolet induction, these two strains ex- hibited a small degree of lysis, but no infective phage was released. After several generations, both strains had become sensitive to phage 82. These results suggest that recombination might have occurred between the prophage and an homologous genetic fragment issued from nonlysogenic bacteria. Unfortunately, because of the lack of markers on phage 82, no genetic analysis was undertaken. Transfer of lysogeny. In order to demonstrate transfer of lysogeny, nonlysogenic bacteria 678/\/82, 434 were infected with preparations of 363 grown on prototrophic bacteria lysogenic for one of the three phages (82, \, or 484), and the bacteria were plated on various selective media. The results of such experiments are listed in Table 4. As in the case of nonlysogeny, lysogenic colonies were found only among those selected for galactose utilization. For each prophage, the frequency of transfer of lysogeny appears to be of the same order of magnitude as that found for the transfer of non-lysogeny. TABLE 4 TRANSFER OF LYSOGENY Selection for eeperinient Phage 363 Threoninet Lactose* Galactose No. grown on strain Number Namber Number Number Number |Number of | Se of. at of. of % “Festal” | tysogenic] °PecPag® | lysogenic] “piotaa® | geste 1 K12(82)* 200 0) 200 0 200 21 10.5 K12(82)* 400 49 12.25 1 K12(A)t 200 0 200 0 200 11 5.5 K12(A)t 400 24 6 1 K12(434)+ 200 0 200 0 200 2 1 2 K12(434)+ 400 1 0.5 Acceptor nonlysogenic P678/\/82, 434 bacteria were infected with one of three preparations of phage 363 grown on prototrophic lysogenic strain, K12(82)*, K12(a)t, and K12(434)*, respectively. After plating of each mixture on various selective media, colonies were tested for their ability to release the correspond- ing phage, 82, \, or 434. 345 214 FRANGOIS JACOB In another experiment, a preparation of phage 363 was grown on a double lysogenic prototroph, K12(82)+(A)+, and used for infecting non- lysogenic P678/82/\. The colonies having acquired the Galy character were tested for their ability to release phages 82 or \, and some were found to release both. As has already been mentioned, in a given experiment, the proportion of transduced clones which are found to be lysogenic for 363 is inde- pendent of the selective marker. This is also true in transduction experi- ments involving the transfer of lysogeny for one or more of the three prophages—82, \, and 434. The proportion of clones found to be lyso- genic for 363 is the same, whether or not the transduced clones have be- come lysogenic for 82, \, or 434. Since such transduction experiments were performed with a multiplicity of infection lower than one, it is clear that the transfer of lysogeny for 82, \, or 434 is performed through phage 363 and is not the result of some kind of “phenotypic mixing.” Reconstruction experiments show that no appreciable reinfections and lysogenizations with phage 363 occur on the selective plates. It appears therefore that the genetic material of several viruses can be included in a single phage coat. In transduction experiments, the transfer of lysogeny has been found to be about as frequent as that of nonlysogeny. This finding is not in agreement with the results found in bacterial recombination. In crosses between lysogenic Hfr(\)+ and nonlysogenic F~, \ prophage develops in most of the zygotes (Jacob and Wollman, 1954a). This development brings out the destruction of such zygotes which lyse and release phage particles. Lysogenic recombinants are therefore very rare. On the con- trary, little or no development of the prophage is observed in the reverse cross between nonlysogenic Hfr and lysogenic F~(A)*, and the frequency of nonlysogenic recombinants found agrees with expectations. The dis- crepancy between the results obtained by transduction and those ob- tained by bacterial recombination can be due to the fact that trans- duction experiments were performed at 20°, a temperature at which phages 82, \, and 434 do not multiply, whereas bacterial crosses were performed at 37°. It is therefore possible that, at 37°, a fraction of the transduced prophages is able to develop during transduction experi- ments. A temperature effect on the frequency of \-lysogeny transfer was observed by Appleyard (1954a). In order to test this possibility, the following experiment was designed (see Table 5). Phage d (as well as 82 or 434) forms plaques with the same 346 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 215 TABLE 5 DEVELOPMENT OF TRANSDUCED \ PROPHAGE umibertot particlesiner auraber of particles per tee pees conta milliliter forming a 363 plaque pines bs due wea petet on when plated on Ki2/A K12(363)* K12/363 Untreated 1.8 xX 108 Pos yk OP 1b << 51108 10 minutes after addition low XX 108 iy, $< Ale Io 102 of anti-A serum 30 minutes after addition 1.9 X 108 14 <8 a9 S< 10} of anti-A serum Same preparation, previ- aah, Xi 10° 4.1 X 10! 4.9 X 10! ously treated with anti-\ serum, 30 minutes after addition of anti-363 serum To a preparation of phage 363 grown on K12(A)t, anti-A serum was added at time 0 and the mixture was incubated at 37°. After 30 minutes, anti-363 serum was added. Before addition of any serum, and at various times during the ex- periment, samples were removed and diluted. Aliquots were plated as follows (after 30 minutes preadsorption at 37°): (1) on K12/d (for plaques of 363); (2) on K12(363)+, which adsorbs both \ and 363 without multiplication of the latter (for total \ plaques); (3) on K12/363, which adsorbs » but not 363 (for free \ phage). Figures indicate the number of plaques found without treatment, after treatment with anti-A serum alone, or after successive treatments with both sera. efficiency of plating on K12, K12(363)t, or K12/363. In the prepara- tions of 363 grown on lysogenic K12(\)t, there are always some free particles which have been released during the growth of the bacteria. Such preparations give the same number of \ plaques on K12, K12(363)+, and K12/363. When most of the free \ particles are neutralized by anti-A serum, a higher number of \ plaques is found by plating on K12(863)*, which adsorbs 363 without allowing its multiplication, than on K12/363 phage, which does not adsorb phage 363. If, now, the preparation pre- viously treated with anti-A serum is exposed to anti-363 serum, the number of \ plaques on K12(363)* is reduced at a rate similar to the one found for inactivation of total plaques of 363. After about 30 minutes of exposure to anti-363 serum, the same number of \ plaques is found by plating on K12(363)* or on K12/363. The results of this experiment show, therefore, that at 37° the \ ma- terial carried in particles of phage 363 is able to multiply vegetatively 347 216 FRANCOIS JACOB if injected in \ nonlysogenic bacteria. If it is assumed that, as in the case of bacterial recombination, most of the transduced \ prophages develop when transduction is performed at high temperature, such an experi- ment enables one to estimate the fraction of particles of 363 carrying \ material as between 10-6 and 10-°. This proportion is of the same order of magnitude as the fraction of particles of 363 found to be able to transduce threonine or leucine characters. Prophage recombination. Prophage recombination between two geneti- cally different and complementary prophages has been shown to occur in transduction. In such experiments, phage \ was used because its genetic system had been investigated extensively (Jacob and Wollman, 1954b; Kaiser, 1954). All the known markers of \ are located on a single linkage group, which has been represented in Fig. 2. When sensitive bacteria are infected with genetically marked phages, some become lyso- genic. The phage particles released by such bacteria and their progeny remain genetically identical to the original particles used for infection. The markers available for the study of \ genetics during the vegetative phase can therefore be used for studying the prophage. In the experiments described below, three markers have been used: ms (medium-sized plaques), co (cocarde) and m; (minute plaques). These markers cover most of the known length of the \ linkage group; in crosses involving these three markers the two parental and the six recombinant types can be distinguished easily and all are temperate. During the vegetative phase, the recombination frequency is about 7 to 9% between m; and c, and 4 to 5% between c, and mi. In the experiment reported in Table 6, lysogenic bacteria P678 (Amsc.m? )/A were infected with particles of 363 grown on K12(Amscemi). The mixture was plated on galactose medium. After reisolation on galac- tose agar individual colonies were grown in broth and analyzed for the type of phage spontaneously released. It is seen in Table 6 that, of 240 colonies, 213 released only the original type Amsc.mj. The other 27 can be classified in two groups. In the first group, corresponding to 18 colonies (7.5%), one or several of the original prophage markers have (Pq) Me ms 9) Si) CenC Co mi 3 3 15 15 Ol 5 Fria. 2. The linkage group of bacteriophage \. Figures indicate percentage of recombinants found in two factor crosses. 348 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 217 TABLE 6 \-PROPHAGE RECOMBINATION THROUGH TRANSDUCTION Number of clones Types of phage released Phage alleles present |Phage alleles transduced PALS} m;com; MsComi 2 M5CoMj Ms5CoMj mj 6 m;¢csm; mscem; com; 9 mietm; micemi mictm; 1 m;c¢m; MsCaM; ct {m;com* MsComi 1 mi mM5€,9MNj Mj m;com? mM;Comj MsComi ny 3 Taney 4 CoMi m;CoM;j CoM: M5C,Mj m;c,m? MsCoMi ++ +.+. + = 4 m5CoMj m5 CoMji Ms5CoMi ce cd oy. nena Ms Co Mj M5CoMj M5CoMij M5 CoMj mc,m? me,mi 1 {m;com? MsComit mt \mfeomi me ‘ Lysogenic acceptor bacteria P678(Am;c.mj )+/A were infected with phage 363 grown on prototrophic lysogenic K12(Am;csmj;)*. After plating on galactose medium, colonies were reisolated and tested for the type of phage released. been replaced by markers of the transduced prophage. In spite of the small numbers involved, it is clear that the markers of the prophage \ are not independently substituted, the frequency of substitution de- creasing progressively from the m; to the ms end of the ) linkage group. In the second group, corresponding to 9 cases (3.8%), the bacteria were found to release, besides the original m;c.m; type, one or more other types of \ particles, even in single-burst experiments. This finding shows that alleles issued from the transduced prophages have been added to the original prophage. Here, again, the mj region is the most fre- quently added. These clones, in which bacteria carry two phage alleles at one or several loci, appear to be rather stable, since they were tested for phage production after two successive isolations on agar and growth in broth, corresponding to at least 50 generations. Nevertheless, single-burst ex- periments show that phage markers are very heterogeneously distributed 349 218 FRANCOIS JACOB in the population. During the multiplication of such strains, bacterial clones segregate which have lost one or more prophage alleles. Very often, clones are found in which only the original m;c,m; prophage re- mains. Other clones exhibit various types of phage allele combinations. Attempts to demonstrate some kind of diploidy for the Gal, marker in these strains were unsuccessful. After two successive isolations on galactose-selective medium, colonies were grown in broth and streaked on EMB galactose agar. All contained only Gal+ bacteria. These results seem analogous to those found after mixed infection with two geneti- cally labeled \ phages (Appleyard, 1954b; Jacob, unpublished results). The results of this experiment show conclusively that the transduced prophage can either recombine with, or be added to, the prophage of the acceptor bacteria. Since the probability of being incorporated decreases from the m; to the m; end of the \ linkage group and since, in addition, m; has no selective advantage over ms; in establishing lysogeny after in- fection, it seems likely (although the data are statistically not very sig- nificant) that the m; end of the \ prophage is the closest to the Gal, locus. Although no definite conclusion can be drawn from the small numbers of cases, the ratio of the probabilities of bringing either m; alone or m; and c, together (3:9) is not in disagreement with the ratio of the frequencies of recombination mjc,/c,m; found in crosses between vegetative phages (4:9). DISCUSSION The experiments reported in this paper show that the phage material present as a prophage in the donor bacterium can be transferred to an acceptor bacterium by means of a transducing phage. The transduced phage material can develop in the nonlysogenic acceptor bactertum when experiments are performed at 37°. It seems likely, however, that this phage material is transferred as a constituent of the donor bacterial chromosome, 1.€., aS a prophage, since in transduction, as well as in bacterial recombination, the same linkages between lysogeny and nutri- tional markers are found. Moreover, at 20°, the transfer of lysogeny oc- curs with about the same frequency as the transfer of nonlysogeny. In E. coli K12, two relative measures of linkage between two given characters, such as threonine and leucine, can be obtained, one by trans- duction and one by recombination. The relationship between these two scales of measure is about the same when lysogeny is one of the char- acters involved. In transduction experiments, lysogeny behaves there- 350 TRANSDUCTION OF LYSOGENY IN ESCHERICHIA COLI 219 fore exactly like any other genetic character. These results confirm pre- vious findings of Lederberg and Lederberg (1953) and of Wollman (1953), gained by means of bacterial recombination and showing that lysogeny is controlled by a genetic determinant of the bacterium. In transduction experiments the same efficiency is found in the transfer of lysogeny to nonlysogenic bacteria and of prophage markers to already lysogenic bacteria, proof that the genetic determinant of the bacterium which controls lysogeny is the prophage itself. The three prophages which have been used in this study are likely to be located at closely linked loci. Their relationship is now the subject of more detailed investigation. Although we know little about the orientation of the prophage with respect to the bacterial chromosome, data concerning prophage re- combination during transduction seem to indicate that the m; end of the \ prophage is the closest to the Gal, locus. If the size of the transducing piece is not too widely distributed and if recombination inside the pro- phage region behaves in the same way as in other regions, such an ex- periment may allow us to estimate the genetic size of the prophage as compared to that of the adjacent bacterial region. In the case of the ex- periment previously described and in spite of the small numbers in- volved, the mi—m; distance may be estimated at about 4 to 6% of the Gal,—m; distance. Such an estimation is probably premature, because we still lack a definite picture of the relationship between the prophage and the bacterial chromosome. The case of bacteria found after transduction or mixed infection, which carry several pairs of prophage alleles that segregate during bacterial multiplication, is unclear. In such bacteria, the two prophages are likely to be located on the same region of the bacterial chromosome, since both additions and substitutions of prophage alleles are found in a single transduction experiment. One of the most striking facts derived from transduction of lysogeny is that a phage coat can contain, besides homologous genetic material, the genetic material of one or more other viruses. ACKNOWLEDGMENT The author wishes to thank Mlle Y. Nicolas for valuable technical assistance and Dr. C. Levinthal for criticisms and help in the preparation of the manuscript. REFERENCES APPLEYARD, R. K. (1954a). Segregation of lambda lysogenicity during bacterial recombination in Escherichia coli K12. Genetics 39, 429-439. 351 220 FRANGOIS JACOB APPLEYARD, R. K. (1954b). Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39, 440-452. Bertani, G., and Nics, 8. J. (1954). Studies on lysogenesis. II. The effect of tem- perature on the lysogenization of Shigella dysenteriae with phage Pl. J. Bacter- iol. 67, 202-209. Jacos, F., and WoutimaN, E. L. (1954a). Induction spontanée du développement du bactériophage \ au cours de la recombinaison génétique chez Escherichia coli K12. Compt. rend. 239, 317-319. Jacos, F., and Woutman, E. L. (1954b). Etude génétique d’un bactériophage tempéré d’Escherichia coli. I. Le systéme génétique du bactériophage. Ann. inst. Pasteur 87, 653-673. Karser, A. D. (1954). A genetic analysis of bacteriophage lambda. Ph. D. Thesis, California Institute of Technology, Pasadena. LEDERBERG, E. H., and LEDERBERG, J. (1953). Genetic studies of lysogenicity in Escherichia coli. Genetics 38, 51-64. LEDERBERG, J. (1947). Gene recombination and linked segregation in Escherichia coli. Genetics 32, 505-525. Lennox, E. (1955). Transduction of linked characters of the host by bacterio- phage P1. Virology 1, 190-206. Lworr, A. (1953). Lysogeny. Bacteriol. Revs. 17, 269-337. Srocxer, B. A. D., Zinprer, N. D., and LeprerserG, J. (1953). Transduction of flagellar characters in Salmonella. J. Gen. Microbiol. 9, 410-433. Tatum, E. L., and Lepersera, J. (1947). Gene recombination in the bacterium Escherichia coli. J. Bacteriol. 53, 673-684. WeIGLE, J. J., and De.sriicx, M. (1951). Mutual exclusion between an infecting phage and a carried phage. J. Bacteriol. 62, 301-318. Wo.uwman, E. L. (1953). Sur le déterminisme génétique de la lysogénie. Ann. inst. Pasteur 84, 281-293. Wo.imaNn, E. L., and Jacos, F. (1954). Lysogénie et recombinaison génétique chez Escherichia coli K12. Compt. rend. 239, 455-456. ZinvER, N. D. (1953). Infective heredity in bacteria. Cold Spring Harbor Sym- posia Quant. Biol. 18, 261-269. ZinvER, N. D., and LepERBERG, J. (1952). Genetic exchange in Salmonella. J. Bac- tertol. 64, 679-699. 352 Recombination between Related Temperate Bacterio- phages and the Genetic Control of Immunity and Prophage Localization’ A. D. Kaiser AND F. JACOB Department of Microbiology, Washington University School of Medicine, St. Louis, Missourt, and Service de Physiologie Microbienne, Institut Pasteur, Paris, France Accepted August 31, 1957 Lysogenic bacteria are immune to infection with phage homologous to the prophage. By means of crosses between phages showing different immunity specificities it is shown that immunity specificity is determined by the same segment of the phage genetic material that controls the ability of the phage to lysogenize. Furthermore, this same segment, called the ‘‘cr region’’, also determines which locus is occupied by the prophage on the chromosome of Escherichia coli K12. INTRODUCTION Lysogenic bacteria possess and transmit to their offspring the power to produce bacteriophage. The phage that lysogenic bacteria produce is capable of making new lysogenic strains by infection of sensitive bacteria. By morphological, serological, and genetic criteria, the phage produced by such a lysogenized strain is identical with the phage used for the lysogenizing infection. Thus each lysogenic bacterium carries all the genetic information present in an infective phage particle. Nevertheless, disruption of lysogenic bacteria fails to reveal infective particles. The structure bearing the genetic information of the phage in a lysogenic bacterium is called a ‘‘prophage”’ (Lwoff, 1953). There is strong evidence that the prophage is the genetic material of the phage bound to a specific site of the bacterial chromosome (Lederberg and Lederberg, 1953; Wollman, 1953; Appleyard, 1953; Jacob, 1955). The presence of the prophage confers upon the host bacterium a re- sistance to infection with the homologous phage and its mutants. This 1 Part of this work was supported by grant E-1275 from the National Insti- tutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland. Reprinted by permission of the authors and Academic Press, Inc. from VrroLoey, 4 (3), 509-521 (1957). 353 510 KAISER AND JACOB property, called immunity, is phage specific since lysogenic bacteria re- main, with some exceptions, sensitive to all other phages that can infect their nonlysogenic counterparts. Immunity is reciprocal, in the sense that a bacterium lysogenic for any temperate mutant of a phage is immune against the wild type and its mutants. Several experiments (Jacob, 1954; Bertani, 1954; Goodgal, 1956) support the idea that the deoxyribonucleic acid (DNA) of homologous phage can penetrate into a lysogenic bacterium, suggesting that immunity is due to a block, which prevents phage multiplication inside the bacterium. Little is known about the mechanism of immunity or the origin of its high degree of specificity. It has been suggested that there is a relation between immunity and the specific location occupied by a prophage on the chromosome of its host (Lwoff, 1953). A new experimental approach to the problem of immunity was opened when a series of temperate phages, all active on FH. coli K12, was isolated. Each of these phages has a different specificity of immunity: bacteria lysogenic for any one of them are still sensitive to the others. Yet, genetic recombination occurs between pairs of these phages when they multiply together in the same bacterium. Thus, the genetic control of immunity could be studied by phage crosses. Not only do these phages differ in their specificity of immunity, but they differ also in their lo- cation as prophages in the host. Each of them appears to occupy a specific location on the bacterial chromosome (Jacob and Wollman, 1957). Phage crosses offer, therefore, the possibility of determining which part of the phage genetic material determines the specificity of im- munity and which part determines the specificity of the prophage lo- cation on the bacterial chromosome. The results of such crosses are the substance of this report. MATERIALS AND METHODS Only those materials and methods which were not described in a previous publication (Kaiser, 1957) will be given here. Media. EMB galactose agar contains 10 gm of Bacto-tryptone, 5 em of yeast extract, 2 gm of K,HPOs, 0.4 gm of eosin Y, 0.065 gm of methylene blue, 5 gm of p-galactose, and 15 of gm Bacto-agar per liter of distilled water. Bacteria. A galactose negative mutant of H. coli KK12 strain C600 was selected by the penicillin method (Davis, 1948; Lederberg and Zinder, 1948) following an irradiation with ultraviolet light. This mutant is 354 IMMUNITY AND PROPHAGE LOCALIZATION oilall TABLE 1 Immunity AND Host RANGE OF PHAGES X, 434, 82, AND 21 Host bacterium Phage K12 K12(a) K12 (434) K12(82) K12(21) K12/r K12/434 N + - + + + - + 434 + + — + + + ~ 82 + + + ~ + + ~ 21 + + + + - — + Key: + indicates ability of phage to grow on the host; — indicates inability to grow. Note: Loopfuls containing 2 X 108 or more phage particles were inoculated onto the surface of an agar plate seeded with bacteria. Lysis of the bacteria within the inoculated area was taken as the sign of phage multiplication. related to the gali- of Morse ef al. (1956), since it can be transduced to galt by AHFT gal. but not by AHFT gal.-. Phages. Coli phages 434, 82, and 21 (Jacob and Wollman, 1956) are capable of lysogenizing #. coli K12. All three are serologically related to phage \. Phage 2 is the wild type described previously (Kaiser, 1957). The ability of phages \, 434, 82, and 21 to grow on various lysogenic and resistant strains of EH. coli K12 is shown in Table 1. It may be seen that each phage is distinguished from the others by its ability to grow on all of the lysogenic strains except the homologous one: each of the four phages has a different immunity specificity. Crosses. The technique of crossing these phages is the same as the one already described for \ X X crosses (Kaiser, 1957). Because many stocks of } mutants are available, crosses were made between \ and each of the other phages. RESULTS 1. Genetic recombination between \, 434, 82, and 21. The first series of crosses involved phages 434, 82, or 21 as one parent, and \ marked either with ms, C02, C1, CO1, or mi as the other parent. A map giving the position of the markers on the \ linkage group is given in Fig. 1. The purpose of these crosses is to see if 434, 82, and 21 carry and are able to transfer to \ the wild type alleles mst, coot, c1*, cot, and mi+. Thus, for example, Ams, Which forms medium-sized plaques, was crossed to 484 and the offspring of the cross plated on K12(434), to examine only the phages with the immunity specificity of 4. The presence of some large plaques, 355 512 KAISER AND JACOB - ome Ms a coo C, CO, mi 1% pa St RECOMBINANTS al S 7 ‘ oe ‘ 7 x peas x 7 XN co. 244 47 30 =| 5042C9, punt I mn Fig. 1. A map representing recombination frequencies observed between pairs of \ mutants (Kaiser, 1957). The c segment is shown on an expanded scale. ms+, indicated that ms;*+ was present in 434 and that recombination can occur between the m; locus and the locus controlling immunity specificity. Similar experiments were carried out with cos, c1, co1, and mz. The markers C0s, C1, and co, cause the production of clear plaques, their corresponding wild alleles controlling the production of turbid plaques. The marker mi causes minute plaques with a halo. The results of these crosses, sum- marized in Table 2, are that in the cross \ X 434 recombination can occur between ms, C02, co, and mi on the one hand, and the loci controlling the specificity of immunity on the other hand. In crosses \ X 21, recombina- tion of ms, co2, and mi with immunity specificity can occur and in crosses X 82 the loci ms, co, and mi can recombine with the loci controlling the specificity of immunity. The pattern of recombination is similar in the three types of crosses of \ with 434, 82, and 21 in that recombination can occur at the ends, m; and mi, but not in the middle, ci, of the » linkage map. The three types of crosses differ, however, in the length of the middle region which fails to recombine with the loci controlling immunity specificity. In the cross \ X 21 this region includes both ¢ and co;, in the cross \ X 82 both co. and ¢:, while in the cross \ X 434 only ¢ fails to recombine. Because the region within which recombina- tion fails to occur is the shortest for the cross \ X 4384, a more detailed study of recombination between \ and 434 was undertaken. Crosses were made between 434 and a selected series of clear mutants of 2. The clear mutants were selected to mark different regions of the c segment. The c segment of d consists of at least three regions. Each region is probably responsible for the performance of a specific function in lyso- genization (Kaiser, 1957). One mutant, number cs, was selected from region III; 4 mutants, c47, c30, C1, and C50, from region I; and two mutants, 356 IMMUNITY AND PROPHAGE LOCALIZATION 5S TABLE 2 OccURRENCE OF WILD TypPpE RECOMBINANTS WITH THE IMMUNE SPECIFICITY OF \ IN CROSSES BETWEEN A SERIES OF A MuTANTS AND THREE RELATED PHAGES » Mutant employed Cross m5 C02 C1 co. mi 434 Xd + + = + + eS Gan a. - ~ - a G2 a + = = +a + * Recombinants arose which were more turbid than Aco, but less turbid than Aco,. Key: + signifies the occurrence of wild type recombinants with the immune specificity of \; — indicates failure to detect wild type recombinants among 1000 offspring with the immune specificity of X. Norte: Several mutants of A (A m5, A C02, \ ci, .cO1, and m2) were crossed to 434 and the phage progeny was plated on K12(434), where plaques with the wild type morphology and the immunity specificity of \ could be detected. Analogous crosses were carried out with phages 21 and 82. Cz, and co, from region II. A map showing the positions of the clear mutants employed is given in Fig. 1. The crosses made were 484 X Acz, where c, represents individual members of the selected series of clear mutants. The progeny of each cross were plated on K12(4384) and on K12(\). The presence of turbid plaques on K12(434) demonstrates the presence in 434 of a c,* allele, which can recombine with the locus controlling immunity specificity of dA. Clear plaques on K12(\) reveal phages with the immunity of 434 which have picked up a c; allele from \}. A comparison of the proportion of turbid plaques on K12(434) with the proportion of clear plaques on K12(A) serves as an internal control, because they are reciprocal re- combinants and should therefore have the same frequency. When 434 alone is plated on K12(A), there are 0.07 % clear plaques due to mutants in the 434 stock. This proportion is, naturally, present as a background in the yield of all of the crosses. The proportion of turbid mutants in a stock of clear is less than 1 in 104 and is therefore too low to be de- tected in the crosses. The results of this series of crosses, given in Table 3, show that recombinants arise from the crosses with mutants C44, C4, and co, but for none of the others. There is, therefore, recombination between 434 and \ in regions II and III of the c segment but not in region I. The 357 514 KAISER AND JACOB TABLE 3 Crosses \ CLEAR X 434 TuRBID Plated on C600(434) Plated on C600(A) Plated on C600 Cross Num- Nuh ; Num- | Num- of Nun- | Num- b umber |% Turbid| b b 2 b b Al firhid clear venue turbid clear clear turbid ales ae 8 ACs XK 434 81 2232 Bot) 29 WULON RES: Cg XK 434 0 12528 | <0.01 | 8166 4 | 0.05 hex X 484 0 3080 | <0.03 | 3632 2 | 0.06 he. & 434 0 9132 | <0.01 | 5518 4 | 0.07 C50 & 434 0 S22 ORO2N i S65: LP OROS ACan K 434 41 7612 0.54 | 6542 32 | 0.49 ACO, X 434 65 5182 SZ 4249 | 170 | 3.8 dct X 434 1550 0 <0.1 434 alone 3000 1 | 0.03 | 2500 3 eal Aces, alone 2, 3744 0.05 Ac4o alone 0 4296 | <0.03 dco, alone 0 2502 | <0.04 Note: Progeny of crosses of the type \ clear by 434 turbid, plated on C600, C600(A), or C600(434) were scored for the number of clear and turbid plaques. The clear mutants of \ employed in these crosses were selected to mark different regions of the c segment: C47, C30, C1, and C59 to mark region I, ca, and co; to mark region II, and ce, to mark region III. limits of recombination between » and 434 lie, on one side, between Cas and cy; and, on the other side, between cso and cy. The precise location of the limits between these pairs of markers is suggested by the fre- quencies of recombination with c44 and cy, 1.e., 3.5% recombination to the right of 44 and 0.54% recombination to the left of ca. The recombination pattern in \ X 434 crosses can be understood if it is postulated that the c; region controls immunity specificity, that there is a c; region specific for the immunity of \ and a c; region specific for the immunity of 434. 2. 434-\ Hybrid. Hybrid phages which have only the immunity de- termining region of 434 and the rest of their chromosome from )\ were isolated from 5 successive backcrosses of 434 to \. The first step was to cross 434, which forms tiny turbid plaques, with \c:, which forms large clear plaques. A few large turbid plaques were found among the progeny. They were tested and all found to have the immunity of 484. The second step was to cross one of the large turbid 434 to Aca and to isolate a 484 C44 358 IMMUNITY AND PROPHAGE LOCALIZATION 515 by plating on K12(A). Step three consisted in replacing ca by ca+ by crossing to Ac; and picking the turbid recombinants on K12. In step four, 484 cast was crossed to Aca. A 434 Castes. was selected by picking a clear plaque on K12(A). The final step consisted in replacing cy. by cat by crossing 434 castc42 to Ac; and plating on K12. At each stage in the backerosses the immunity pattern was checked. This sequence of 5 successive backcrosses would be expected to replace all of the parts of the 434 chromosome capable of recombination with the homologous parts from \. We have, therefore, a phage which should possess the cy region of 434 embedded in an otherwise \ genome. This phage will now be designated 434hy. The hybrid character of 434hy is evidenced by the following properties. Apart from its immunity pattern, which is that of 434, phage 434hy behaves like A. On K12 it forms large turbid plaques identical to those of \ and it has the host range of \. Whereas phage 434 is inactivated by anti-\ serum at a slower rate than is \, the hybrid is inactivated at the rate characteristic of \, as is shown in Fig. 2. Further evidence for the homology of 434hy with \ is shown by a comparison of \ X 434hy with X d crosses. In a cross \msc, X 434hy mi, 12 % recombination was ob- served between m; and c, and 4% between c; and mz. A corresponding cross between Amsc and Ami gave 8% and 5% recombination, respec- SURVIVING FRACTION Minutes — 5 10 Fic. 2. Neutralization was measured by incubating a mixture of phage and a 1/100 dilution of rabbit anti-A serum at 37°. At the times indicated aliquots were withdrawn, diluted at least 1/104 and plated on C600. The surviving fraction is the ratio of the number of plaques at time ¢ to the number at time zero. 359 516 KAISER AND JACOB tively, in the two regions. The absence of a significant difference in recombination frequencies suggests that 434hy is homologous to X. Since the c; region is only about 1% long, absence of homology there would not be expected to depress recombination frequencies significantly in this cross. Bacteria lysogenic for 434hy behave exactly like K12(434) in response to infection by A, 434, Ac, 484 c, and434hy. Thus the 434hy prophage determines the immunity pattern characteristic of the wild type 434. The existence of hybrids with a c; region from 434 and the rest of their chromosome from \, which have the specific immunity pattern of 434 but not that of A, demonstrates that the c; region (or a portion of it) controls the specificity of immunity. Wild type T4 is capable of multiplication on K12(A), but the ry, mutants of T4 are not (Benzer, 1955). T4r;; was tested on K12(434) and on K12(434hy); it was found to multiply on both strains. T4r+ was able to multiply on both also. This suggests that the block in T4ry, multiplication is caused by the c; region of since replacement of the c; region of \ by the c; region of 434 eliminates the block. 3. Chromosomal localization of 434hy prophage in E. coli K12. Prophage \ and prophage 434 occupy distinct but closely linked loci on the linkage map of F. coli K12 near certain galactose loci (Jacob and Wollman, 1957). It must, therefore, be determined whether prophage 434hy oc- cupies the \ locus, the 434 locus, or neither. In K12(\) prophage \ occupies the \ locus and in K12(484) prophage 434 occupies the 434 locus, by definition. Allelism tests between K12(434hy) and K12(\), and between K12(434hy) and K12(434), should localize the 434hy prophage. Allelism tests were performed by means of transduction with phage P1 (Lennox, 1955). The occurrence of transductants nonlysogenic for the prophage of the donor and the prophage of the recipient would indicate that the two prophages are nonallelic. The absence of non- lysogenic transductants indicates allelism, subject of course to the limita- tion in the number of transductants examined. In the experiments to be reported, a stock of Plke (Lennox, 1955) was prepared on a gal*+ donor strain. The gal~ recipient was infected with this Plke stock and galt transductants isolated, purified, and tested for prophage by replica plating on C600, C600(A), and C600(434). The recipient strains had all been rendered /\/434 to prevent infection by free phage d or 434. 360 IMMUNITY AND PROPHAGE LOCALIZATION ol7 TABLE 4 PROPHAGE SEGREGATION IN TRANSDUCTION Transductants gal* Non-_ Lysogenic Total Donor gal* Recipient gal Seuss Produce phage with the immunity specificity of x 434 \ and 434 (434hy) A (A) 75 267 39 16 397 (434hy) B (A) 103 122 114 59 398 (434hy) (434) 0 0 789 0 789 (434) (434hy) 0 0 785 0 785 (A) (434hy) 36 38 320 21 415 (434) A (A) 39 228 25 6 298 (434) B (A) 23 370 7 0 400 (A) (434) 18 29 71 2 120 (A) (A) 0 943 0 0) 943 (434) (434) 0 0 200 0 200 (434hy) (434hy) 0 0 400 0 400 Norte: The transductions were performed by infecting the recipient with phage Pl that had been grown on the donor. The infected mixture was incubated 1 hour at 25°, then spread on EMB galactose agar. Galactose positive transductants were isolated, purified by two serial single-colony isolations and then scored for the type of phage production by replica plating onto K12, K12(\), and K12(434). A and B indicate strains isolated independently using the same stock of phage and sensitive bacterial strain. The donor strains were all prepared from C600 and the recipient strains from C600 gal-. The results of these tests, presented in Table 4, are that nonlysogenic transductants did arise when the pair K12(A), K12(434hy) was tested, independently of which was the donor and which the recipient. How- ever, no nonlysogenic transductants arose when the pair K12(434), K12(434hy) was tested. Therefore, prophage 434 hybrid occupies the 434 locus. Incidentally, the absence of nonlysogenic transductants when the pairs K12(A), K12(A) or K12(434), K12(434) were tested is a strong argument in favor of the uniqueness of the \ and the 434 loci. If prophage 434hy occupied the same locus as prophage 434 in K12, then the same frequencies of recombination would be expected between the locus of prophage 434hy and the locus of prophage \ as between the locus of prophage 434 and the locus of prophage \. Referring to Table 4, it may be seen that when the donor was lysogenic for 434hy and the 361 518 KAISER AND JACOB recipient lysogenic for \ an average of 22 % (75 + 103/397 + 398) of the galt transductants were nonlysogenic. But when the donor was lysogenic for 434 and the recipient lysogenic for A, 9% (39 + 23/298 + 400) of the galt were nonlysogenic. In the reciprocal transductions where the donor was lysogenic for \ and the recipient lysogenic for 434hy or for 434, the frequencies of nonlysogenic strains among the gal+ were 9% (36/415) and 15% (18/120), respectively. The significance of the differ- ences in each case is questionable for two reasons. First, differences of the same order were observed when the same transduction was repeated with independent isolates of the same donor strain, as may be seen by comparing examples A and B in Table 4. Second, the hypothesis that prophage 434hy and prophage 434 occupy different loci would require that a particular order be assignable to the loci of the three prophages involved. Neither the order \ — 434 — 434hy nor the order \ — 434hy — 434 is consistent with the frequencies of nonlysogenic transductants ob- served in reciprocal transductions. For, whereas the order ) — 434 — 434hy might have been indicated when the donor was lysogenic for 434 or for 434hy and the recipient lysogenic for \ due to a greater frequency of nonlysogenic strains in the latter transduction than in the former, the reverse order \ — 434hy — 434 would have been indicated by the reciprocal transductions when the donor was lysogenic for 4, due to a greater frequency of nonlysogenic transductants when the recipient was lysogenic for 434 than when the recipient was lysogenic for 434hy. These data, therefore, are not incompatible with the idea that prophage 434hy occupies the same locus as prophage 434. Allelism of prophage 434hy to prophage 434 was independently con- firmed by the bacterial cross Hfr T+L+Ss gal+(434) & F*T-L-S'(434hy) gal-//434. Among 400 gal+St prototrophs no nonlysogenic strains were found. Bacteria lysogenic for both \ and 434 or for \ and 434hy were found in some of the transduction experiments reported in Table 4. Ten strains of each type were examined in detail; all of them had the following proper- ties. They produced two types of phage, one type which plated on K12(\) and another type which plated on K12(434). When induced with ultra- violet light and plated, before lysis, on C600, they produced mottled plaques like those of C600 mixedly infected with \ and 434. These strains were immune both to \ and to 434 and did not support the growth of T4ryy. 362 IMMUNITY AND PROPHAGE LOCALIZATION 519 DISCUSSION In crosses between \ and any one of the other phages studied, 434, 82, and 21, genetic recombination observed in the ms; and mi regions indi- cates clearly that genetic homology exists between the linkage maps of these phages, at least in their outer parts. That the degree of homology may vary according to the pair of phages is shown by the fact that in crosses with A, the regions of the map within which recombination can occur is different for 434, 82, and 21. The most interesting of these three phages is 434, which appears to exhibit complete homology with \ except for a small region of its linkage map, the c; region. The c; region of the genetic material of phage \ has been defined as that segment of the linkage map within which are lo- cated all mutations (22 out of 22 studied) that suppress the ability of the phage to lysogenize or, at least, reduce the lysogenization frequency to less than 10~° (Kaiser, 1957). Mixed infection with different pairs of c; mutants have indicated that the c; region is probably a single cistron or functional unit as defined by Benzer (1957). The failure to find intra-c; recombinants in a \ X 434 cross may be explained by two kinds of hypotheses. Either the c; regions of \ and 434 fail to pair, or recombination occurs but the recombinants are inviable because of the hybrid character of their c; region. In both models, struc- tural dissimilarity of the c; regions of \ and 434 would seem to be implied by the absence of recombination there. The main result obtained by comparing the properties of \ and 434hy is that these two phages, which have in common the whole linkage map except the c; region, differ both by their immunity pattern and by their specific location as prophages on the bacterial chromosome. The conclu- sion, therefore, seems inescapable that the c; region controls not only a reaction involved in the lysogenization process, but also the immunity pattern and the specificity of prophage location. The question arises, therefore, whether these properties are but different expressions of the same function. To understand what this function might be it is necessary to consider the nature of prophage. Prophage is defined as that element in a lysogenic bacterium which is responsible for the production of a particular bac- teriophage (Lwoff, 1953). Bacterial crosses between K12 strains lysogenic for different mutants of \ (Appleyard, 1953) as well as transduction of prophage (Lennox, 1955; Jacob, 1955) demonstrate that prophage is the 363 520 KAISER AND JACOB genetic material of a phage associated with a particular locus of the bacterial chromosome. If then the c; region of the phage were responsible for the association, the three functions—lysogenization, localization, and immunity—would all depend on the c; region. In the following discussion this idea will be explored in more detail. The simplest way to explain how the c; controls the specificity of prophage localization would seem to be that the prophage is attached to the bacterial chromosome by its c; region. The c; region of \ would attach to the locus in K12, the c; region of 434 to the 434 locus. A slight structural change in a c; region might prevent attachment. Thus it would be easy to see how a mutation in the c; region could prevent lysogenization. The role of the cy region in immunity may be summarized in the fol- lowing way. The fact that \ but not 434hy can reproduce in K12(434) shows that immunity is directed against the c; region of a superinfecting phage. The fact that \ but not 434 can reproduce in K12(434hy) shows that immunity is governed by the c; region of the prophage. Thus the prophage controls the inhibition of vegetative multiplication of superin- fecting particles possessing an homologous c; region. This may be ex- plained by two types of hypothesis. Either immunity operates in the bacterium at the chromosomal level and is due to some steric hindrance, or immunity is a cytoplasmic expression of a genetic function of the pro- phage. This could be either the production of an “immunity substance” (Bertani, 1955) by the prophage-bacterial chromosome system or the inhibition by the prophage of the production by the bacterium of a substance necessary for the multiplication of homologous particles. No decision between these hypotheses can be reached until further attempts have been made to dissociate the genetic control of lysogenization, specificity of prophage location, and specificity of immunity. The c; region of the prophage also controls the ability of T4ry, to multiply on lysogenic K12. The mechanism of this effect remains un- known but the same hypothesis as those proposed for immunity may be considered (Lederberg, 1957). ACKNOWLEDGMENTS We want to thank Dr. André Lwoff in whose laboratory at the Institut Pasteur much of this work was carried out. During this time the senior author was a fellow in cancer research of the American Cancer Society. 364 IMMUNITY AND PROPHAGE LOCALIZATION 521 REFERENCES ApPpLEYARD, R. K. (1953). Segregation of lambda lysogenicity during bacterial recombination in E. coli K12. Cold Spring Harbor Symposia Quant. Biol. 18, 95-97. Benzer, S. (1955). Fine structure of a genetic region in bacteriophage. Proc. Natl. Acad. Sci. U.S. 41, 344-354. 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