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THE ACTINOMYCETES Vol. I NATURE, OCCURRENCE, AND ACTIVITIES by Selman A. Waksman BALTIMORE TILE, WILLIAMS: & WILKINS COMPANY THE ACTINOMYCETES Vou. I: NaTuRE, OCCURRENCE AND ACTIVITIES Copyright ©, 1959 The Williams & Wilkins Company Made in the United States of America Library of Congress Catalog Card Number 59-9962 Composed and printed at the WAVERLY PRESS, INC. Baltimore 2, Md., U.S. A. PREFACE In any attempt to classify and divide liv- ing systems, nay, even living versus non- living systems, certain borderline bodies are encountered which may be considered as transition forms from one group to another. This was recognized by the early students of the microscopic forms of life, who con- sidered the bacteria and similar organisms as “‘protista”’ or primitive bodies, related, on the one hand, to the plants and, on the other hand, to animals. Recently accumu- lated information points also to viruses as transitory between nonliving and _ living bodies. The actinomycetes form such a borderline system, but on a much more specialized scale. Considered by some as_ bacteria (“higher bacteria’), or Eubacteriales, and by others as fungi (‘lower fungi’’), or Hyphomycetes, actinomycetes are often placed in a group by themselves, with some of the properties of both. There are found, among the actinomycetes, certain forms that are more closely related to the bacteria and others that are nearer to the fungi. My personal attention was first directed to the actinomycetes about 45 years ago. In 1914, as a senior in college, specializing in soil microbiology, or, as it was designated at that time, “‘soil bacteriology,” I was as- signed by my professor, Jacob G. Lipman, the task of making a comparative monthly study of the bacterial population of certain soil types located on the experimental grounds of the college. The results obtained in this study were used for a thesis which I presented the following June for my B.Sc. degree.* Throughout the year 1914-1915, [sampled, * “Bacteria, Actinomyces and Fungiin the Soil.” Selman A. Waksman, Thesis, Rutgers College, New Brunswick, N.J., 1915 (Abstract published in J. Bacteriol. 1: 101, 1916). at monthly intervals, several different soil types. Samples taken under sterile condi- tions were obtained from various depths (from the surface to 30 inches). These I brought to the laboratory and plated out, using suitable dilutions and proper culture media. After varying periods of incubation, I counted the colonies of bacteria developing on the plates. I was soon struck by the fact that a fairly large number of the colonies that I could observe did not look exactly like the majority of the others, more typical of bacteria. These particular colonies were compact and leathery in nature, pyramidal in structure, penetrating deep into the agar medium, frequently covered with a surface fuzz that was distinct from the substrate growth. On examination of such colonies even with a low-power microscope, the fuzzy growth proved to be made up of an aerial, branching mycelium that resembled that of fungus colonies. When I brought the plates to my professor, he shook his head, smiled, and said, ‘Yes, I have been aware of the occurrence of these types of bacteria. Frequently they are desig- nated as a special group, under the name actinomyces. You had better go and see our botanist, Professor M. T. Cook. He may be able to tell you more about them.” Professor Cook was indeed familiar with the group, but merely as causative agents of potato scab. He considered them, not as bacteria but as fungi. He referred me to various papers in which further information could be obtained on this group of organisms. I decided in my very early studies, that the organisms could be differentiated from both bacteria and fungi. To my great satisfaction, I learned later that similar suggestions had already been made previously by others. Thus, at the very threshold of my scien- vl THE ACTINOMYCETES, VOL. I tific career, I came in touch with a group of microorganisms that were to occupy a major part of my future scientific life. The final year of my undergraduate studies of these organisms was followed by three years of graduate work,t and by many more years as scientific assistant and finally as micro- biologist at the New Jersey Agricultural Experiment Station. The following treatise is, in part, a sum- mary of these investigations carried out for nearly half a century, mostly in the labora- tories of Rutgers University, first at the College of Agriculture and Experiment Sta- tion, and more recently at the Institute of Microbiology. In a larger sense, however, I + “Proteolytic Activities of the Soil Fungi and Actinomycetes,” Selman A. Waksman, Ph.D. Thesis, University of California, December 1917 (J. Bacteriol. 3: 475-492, 509-530, 1918). wish to give credit to the many other investi- gators who, by their careful and exhaustive studies, have so far advanced our knowledge of the actinomycetes during this first half of the Twentieth Century. In the preparation of this volume, I have drawn freely from the various theses sub- mitted by candidates for their Ph.D. degrees, working under my direct or indirect super- vision. I wish to acknowledge the assistance of my colleagues and collaborators, notably Dr. Ruth E. Gordon, Dr. Hubert A. Le- chevalier, Mr. Robert A. Day, and Mrs. Herminie B. Kitchen. I also wish to thank Dr. C. W. Emmons, of the National Insti- tutes of Health, for reading Chapter 17, and Dr. L. A. Schaal, of the U. S. Department of Agriculture, for reading Chapter 18. Selman A. Waksman INTRODUCTORY No other group of microbes, and for that matter no other group of living systems, whether of plant, animal, or microbial origin, has been in recent years the focus of so much attention by the investigator, especially the microbiologist, the chemist, and the medical scientist, and by the pharmaceutical manu- facturer, as the actinomycetes. Only 20 years ago scarcely a dozen laboratories in the whole world were devoting much atten- tion to this group of organisms, and they were concerned largely with either disease- producing or soil-inhabiting forms. Today, literally thousands of investigators in numer- ous laboratories throughout the world are isolating cultures of actinomycetes from soils and other substrates and = studying their physiological and biochemical activi- ties. This increased attention is due primarily to the discovery that the actinomycetes comprise many forms that have the capacity to produce a large number of chemical sub- stances capable of inhibiting the growth of microorganisms, especially disease-producing forms. These substances have come to be known as antibiotics. The discovery that certain actinomycetes can produce growth- promoting substances or vitamins and cer- tain potent enzyme systems has added greatly to this interest. Many of the anti- biotics produced by the actinomycetes have found extensive practical application in the control of infectious diseases of man, ani- mals, and plants; also in animal nutrition; and in the preservation of biological prod- ucts, including virus preparations, and of human foodstuffs. Our first knowledge of the actinomycetes dates back to 1875, when Ferdinand Cohn named an organism he found in the tear duct of the human eye Streptothrix Foersteri. This was soon followed (1877 to 1878) by a de- vil scription by Harz, of another organism, Actinomyces bovis, found in “lumpy jaw’’ of cattle. Since then, many actinomycetes have been isolated, and a number of genera and hundreds of species have been described. These include organisms causing animal and plant diseases and numerous saprophytes occurring in soils, in dust, in water basins, and in other natural substrates. Because of the above two generic names and for other reasons, the systematic posi- tion of actinomycetes became highly con- fused. Animal and plant pathologists, bota- nists, zoologists, mycologists, bacteriologists, and biochemists were eager to introduce new names in describing as new species freshly isolated cultures of actinomycetes. New genera and new species were thus created, without due regard to previously established names or even previous descriptions. This tended to complicate greatly our knowledge of the taxonomy and classification of the actinomycetes. A number of subsequent milestones in the history of actinomycetes should be noted. Among them were the isolation by Israel of a pure culture of an anaerobic organism, for which the generic name Actinomyces was re- served; the introduction of synthetic media by Krainsky and by Waksman and Curtis; the recognition of the sporulating mecha- nisms of actinomycetes by Orskov; the clas- sification systems of Waksman and Henrici and of Krassilnikov; the isolation of anti- bioties from cultures of actinomycetes; and finally the study of the cell walls of actino- mycetes. These and numerous other mile- stones have marked the development of our knowledge of the actinomycetes from the original concept that they were a small group of negligible organisms causing certain ob- scure diseases to the comprehensive recog- Vill nition that they represent a large and highly important microbial group of universal dis- tribution, possessing numerous biochemical activities, and of great practical potenti- alities. From an ecological point of view, the interest in the actinomycetes has centered largely upon the study of their occurrence in soils, in composts, in water basins, in the atmosphere, and in the infected tissues of living systems. Their role as causative agents of human, animal, and plant diseases at first attracted wide attention, but more recently this interest became of limited significance. Under some conditions, however, the actino- mycetes may play a highly important role in the causation of certain plant diseases, such as potato scab. From a biochemical point of view, interest in the actinomycetes has centered largely upon their role in the transformation of or- ganic matter in the soil and their ability to form antibiotics, vitamins, and enzymes. The interest in the antibiotics produced by actinomycetes has been phenomenal. It all began with the isolation of actinomycin in 1940. This was followed by the isolation of streptothricin in 1942 and of streptomycin in 1948, and later of chloramphenicol, the tetracyclines, the erythromycins, the neo- mycins, novobiocin, oleandomycin, nystatin, and numerous others. To date, more than 500 different antibiotics have been isolated from cultures of actinomycetes. Many of them have been obtained in the form of pure com- pounds, the chemical nature of which has been determined. Others are still of unknown composition. Nearly 25 of these antibiotics have already found extensive practical appli- ‘ation as chemotherapeutic agents. Of the total 2,400,000 pounds of antibiotics pro- duced in the United States in 1955, valued at more than a half a billion dollars, at least two-thirds have been obtained from cultures of actinomycetes. The interest in evoked the antibiotics THE ACTINOMYCETES, VOL. I tremendous interest in these organisms, their distribution in nature, their growth and nu- trition under controlled conditions, and finally their biochemical activities. Among the earlier treatises devoted to the subject of actinomycetes, note should be taken of the work of Lieske (1919), Duché (1935), Kriss (1937), Krassilnikov (1938), and Cope (1938). I have personally contributed to many phases of the study of actinomycetes. Following my work on ‘‘The cultural prop- erties of actinomycetes,”’ published in 1919, I edited the section on actinomycetes in the various editions of Bergey’s Manual, begin- ning with the first in 1923 and including the seventh in 1958. My more recent books in- clude a book on The Actinomycetes published in 1950 and various volumes and papers on the antibiotics of actinomycetes. The rapid accumulation of basic knowl- edge concerning the actinomycetes justifies a comprehensive treatise at this time. In this work, I have made no attempt to review or even to list the extensive literature on this subject. Only certain pertinent references have been selected. In view of the fact that more than 6000 references on the subject of a single antibiotic, streptomycin, had been collected (as of 1952!) one can readily imagine the extensive literature covering the other antibiotics that have found practical application in the treatment of numerous human and animal diseases, in animal feed- ing, and in the preservation of various bio- logical preparations and food materials. And all of these antibiotic references, of course, would be in addition to the thousands of papers that have been published relating to the organisms themselves. This treatise is limited to a review of our knowledge of the true actinomycetes. It does not concern itself with the various bacterial forms frequently included among the Actino- mycetales, namely, the mycobacteria, coryne- bacteria, and mycococci. In view of the frequent references to mem- INTRODUCTORY ix bers of the various genera of the actino- mycetes by vernacular designations, the following comments may be made here: The terms ‘‘actinomycete”’ and ‘‘actino- mycetes”’ will be used in this treatise as inclu- sive terms for any or all of the organisms now included in the Actinomycetales, exclu- sive of the mycobacteria and corynebacteria. The term ‘Actinomyces’ will be used only when referring to the single genus of that name; ‘‘actinomyces”’ will be used as the vernacular expression only for members of this genus, both in singular and in plural senses. The term “‘streptomyces” will be used as a vernacular expression of the genus Streptomyces, both in singular and_ plural The will be used in the vernacular for ae senses. terms ‘‘nocardia’”? and ‘‘no- cardias” members of the genus Nocardia, and ‘‘micro- and monospora”’ “micromonosporas” for * Micromonospora.”’ TABLE OF CONTENTS The Actinomycetes VOLUME I OCCURRENCE, NATURE, AND ACTIVITIES LLCO SR ee en eC EL lg, IE rd es «meg Set Vv LLU LCS) 9 i i bee i Ce ee edad Ry ty a pew A Oe vil Te PISEOR CH AC KOTOUNG » 6 sc ses: ton wo Le ea oe ee. 1 2. Isolation, Identification, Cultivation, and Preservation... .. ant! Net 17 See Ser DUerOmam- NG GUE S = > Oe os acphaoscle oa Se ee Loe 29 4. Nomenclature and General Systems of Classification... ............ 47 5. Morpholory; Cytology and life Cycles:....2:....... 00:2. .02.0 2. 71 6: Variations, Mutations and Adaptations............2.....:..2.. 95 weonbypioloxy Growin and Nutrition. <2 46 esha eee pee eee: 113 See Minerals VECtADOMSEHN 2, 305,saeon mete ek ae ee ee eee 138 SEE NOGHEMNCAM NCHIVIGIES:. As 4 22 Amat vn sictrceees cee ays ee eel ee: 148 EAN NICGHANISINS 2c 2: ays 5 e- ang «4,5 cent 2S el cae] OO tee 168 MPPMELOUUICLON: Ol, EIA VIMNESS 3. icy cles Sts ele ae Eee a eee 183 12. Production of Vitamins and Other Growth-Promoting Substances... 193 a i ee reduction of Pigments: «3.2.5.2 sth Bees ee ee A ee 198 Pim Onigiie PTOPeriles 2 £20 scs5 feces lee eee 207 PSE TOC Mon OF ANTIDIOLICS...4 . A804 08 coe eee ee ks ee ene ae ee: 225 16. Decomposition of Plant and Animal Residues................... 248 hae ancationsol Animal Diseases: .. 0:2 0s.csee cee eee eee een ee ee 251 Pee usin on lant (DISGASGS ! AO. ko). s.c-cc3c Sine ee ee a a 265 OMCs occ Ge a, 4. eb te we opayee PARI eRe Oar ae en ee 277 EVCICTOUMCES Bary nk dss oc Scale re ee ee ee 278 ideson@rannisnis.””) 5 cies tei acte.2.4 eile ampere ee yd ee ee 296 index 16 opeciesiat Actinomycetes:.. 4... --aa. 7. a st - — te ’ sy : : wi ni fh uel Aes ae t) Drert irik - ie a) = : p i ' > “p> 7 _ : i ii ty 2 7 _ 7 ; i : ; J ni i a ¢ Ni 4 aw aftr aba ae La of hit pac 7 4 : ; ; f , . : a Oey oe lae ; j : iM , i of ¢ ' ae ae es a gd 7 5 i i ay sie ay 8 Lj i iy ae ey) eee = Re — “i) a4)" D F iG ") i ~ mee ee ste aa ; if ’ _ An eT Ce 7 Pipi mAs | fate ’ yi j ( 5 sf r = : ( Aft ise Piel 7 i a ~ eh ia - , if =~ { r if ' t pat iy , ; , j wed Lelie Pa Bi ane! a, it pi aw gap ii Py) ier Be ® aay ah 7 ‘ shy ® : To . F ’ 3 we yet j : ] Z ry Me J if * ‘ ; at i" Th war + , init Lawroale [ = ‘ , - = ae - — he C. BA) PP Bo J Historical Background What Are Actinomycetes? Actinomycetes are a group of branching unicellular organisms, which reproduce either by fission or by means of special spores or conidia. They are closely related to the true bacteria; frequently, they are considered as higher, filamentous bacteria. They usually form a mycelium which may be of a single kind, designated as substrate (vegetative), or of two kinds, substrate (vegetative) and aerial (in part sporogenous). In the early descriptions, actinomycetes were often defined as ‘“‘unicellular microor- ganisms, 1 yw in diameter, filamentous; branching monopodial, seldom dichotomous, producing colonies of radiating structure.” Two forms of reproduction have commonly been recognized: (a) fragmentation, or oidia- formation, and (b) segmentation. Both kinds of spores grow in ordinary media to form a filamentous mycelium. Frequently the actinomycetes have been looked upon as a separate group of organisms occupying a position between the filamen- tous fungi and the true bacteria. It has even been said that actinomycetes are the original prototypes from which both fungi and bac- teria have been derived. Some forms of actinomycetes, such as members of the genus Nocardia, are known to have their counterparts among the bacteria; other forms, like some species of Streptomyces, Mi- cromonospora, and some of the other genera, have their counterparts among the fungi. The similarity in diameter between bacteria and the mycelium and spores of actinomy- cetes and certain common chemical and bio- chemical properties, recently discovered, sug- gest that the actinomycetes should be classified with the bacteria. They are usually placed in a separate order, the Actinomyce- tales, which is said to be distinct from the Eubacteriales, or the true bacteria, although this relationship has recently been ques- tioned. The actinomycetes are generally recog- nized to represent a large and heterogeneous group of microorganisms, comprising several genera and numerous species. They vary greatly in their morphology, physiology, biochemical activities, and role in natural processes. They play an important part in the cycle of life in nature by bringing about the decomposition of complex plant and ani- mal residues and the liberation of a continu- ous stream of available elements, notably carbon and nitrogen, essential for fresh plant growth. Some of the biochemical activities of the actinomycetes are now being utilized for the large-scale production of chemical substances essential for public health and human economy. Early Concepts The early history of the actinomycetes revolves around their role as causative agents of disease, especially a disease in cattle known as “‘actinomycosis” or “lumpy jaw.” Ferdinand Cohn’s first description of an actinomycete was based upon his study of an ? 2 THE ACTINOMYCETES, Vol. I Ficure 1. The first illustration of an actinomycete ever published, Streptothrix Foersteri (Reproduced from: Cohn, F. Untersuchungen iiber Bacterien II. Beitr. Biol. Pflanzen 1: 141-207, 1875). organism found in concretions of the lachry- nation. Cohn says: “On April 15, 1874, he mal ducts and which he named Streptothrix transmitted to me a mass which was whitish, Foerstert. The concretions were transmitted like tallow, easily broken down and still to Cohn by Foerster for microscopic exami- consisting of fine, very thin colorless branch- HISTORICAL BACKGROUND 3 ing threads running parallel to one another or in various directions, curving and in places also wavy.” This type of growth re- minded Cohn of the curvatures of spirilla and spirochaetes, although it was more irreg- ular. The threads were found to break up into fragments, some of which reached a length of 50 uw. The branching filaments were surrounded with masses of micrococci, filling the spaces between the threads. These fila- ments were distinctly different from the straight, thick, and unbranched (false- branching) Leptothrix buccalis commonly found in the mouth. The photographs of the organism published by Cohn leave no doubt that this was a true actinomycete. Cohn con- sidered this organism to be a bacterial form with branching mycelium, though all at- tempts to cultivate the organism failed. Two years later, Harz examined a patho- logic specimen, obtained from ‘“‘lumpy jaw” of cattle and submitted to him by Bollinger. He gave to the organism observed in this specimen the generic name Actinomyces and the specific name bovis. No pure culture was obtained. The masses of filaments were found to be arranged radially, which suggested the name “actino-myces” or “ray-fungus.”’ Neither of these two generic descriptions was universally accepted, largely because the first (Streptothrix) had been preempted in 1839 by Corda for a true fungus and the second (Actinomyces) had been meeting with much criticism, because the descrip- tion of the organism was based on its etiol- ogy rather than on its morphology and cul- tural characteristics. The first isolations of pure cultures of ac- tinomycetes from human and animal infec- tions involved some difficult problems in ecology and taxonomy. They were the pri- mary causes of much confusion in the history of actinomycetes. O. Israel claims to have isolated in 1884, from a human infection, an aerobic filamentous organism, the hyphae undergoing ready fragmentation. Bostroem claims to have isolated in 1885, also from human cases, an aerobic, filamentous, spore- forming culture. Nocard isolated an aerobic culture in I888 from an animal infection. This was followed (1889) by the isolation from a human infection of an aerobie culture by Afanassiev. In 1890, Eppinger isolated a nonsporulating aerobic organism, and Wolff and J. Israel isolated, the same year, a non- sporulating microaerophilic form. These cultures came from different sources and, because of their filamentous nature, were considered to represent the isolates of Cohn and Harz. None of the above isola- tions were, however, the cause of as much confusion as the report made by Bostroem of his isolation in 1890 of a pure aerobic cul- ture of an actinomycete from a case of ac- tinomycosis. This culture, now known to be a Streptomyces, rapidly found a place among the various collections and was believed at first to be the true cause of actinomycosis. The general consensus now is that this cul- ture did not represent the causative agent of the disease but was merely an air con- taminant. Unfortunately, this error remained to plague the subsequent literature of the actinomycetes and became a cause of much confusion. First, the claim that Actinomyces bovis was an aerobe rather than an anaerobe was wrong; second, the wide distribution of the contaminant led many to assume that actinomycosis was caused by an aerobic or- ganism similar to the group now designated as Streptomyces. For many years, investigators continued to believe either that the causative agent of actinomycosis Was an aerobe or that there were two forms, one an aerobe and the other an anaerobe. There is no doubt now that Bos- troem never succeeded in growing the true etiologic agent of actinomycosis but that some of his attempted isolates became con- taminated with saprophytic actinomycetes from the dust in the air, and thus resulted in the mistaken isolation. Topley and Wilson { THE ACTINOMYCETES, Vol. I (1929) proposed that this isolate be named Actinomyces graminis. Vuillemin (1931) con- sidered it to be identical with Actinomyces sulphureus Gasperini (1894). In the absence of pure cultures of the causative agent of the disease for compara- tive studies, some of the early workers on actinomycetes had only a limited concept of the growth and life cycle of these organisms. This is illustrated, for example, in the de- scription by MacFayden (1889) of the his- tory of an actinomycete colony: “Tt has its starting point in one or more cocci transported by the plasma currents or by the agency of a carrier cell (leucocyte). The cocci multiply by elongation and subse- quent fission. By elongation some of the cocci give rise directly to short bacillary forms, and through these to long filaments. The further extension of the colony is effected by the growth and multiplication of both threads and cocci. The majority of the threads tend to develop clubs at their outer ends (involution forms).’? For more phan- tasy and inaccuracy, one would have to search widely in microbiological literature. Not much progress in the general under- standing of these organisms seems to have been made during the next 20 years, as illus- trated by reference to them in the Second Edition of H. W. Conn’s (1909) Agricultural Bacteriology. In speaking of the actinomy- cetes, he says: “Under this head are included a few forms of fungi which resemble other bacteria in some respects, but differ in others. They are composed of threads which are commonly larger than the threads of bacteria, and which may show frequent branching, a char- acteristic not usual in bacteria. They also have a peculiar method of forming reproduc- ing bodies. The group is not one of very great importance. One type of Streptothrix is ex- tremely abundant in soil and appears as round, white opaque colonies with an exten- sive brown halo upon the plates.” An important cause of confusion was the fact that the actinomycetes were grown on nitrogen rich organic media, now known to be totally unsuitable for them to form a char- acteristic growth, essential for comparative studies and for proper identification. As a result, a highly complex terminology was developed for the designation of actinomy- cetes; numerous descriptions of ‘‘new’’ spe- cies soon began to appear. This is illustrated by the summary made, as early as 1892 to 1894, by Gasperini (Table 1). There is no wonder, therefore, that the nature and classi- fication of the actinomycetes soon appeared hopeless. The adoption of the name ‘actinomy- cetes” was suggested by Gasperini and Lach- ner-Sandoval. Sanfelice, impressed by the analogy of the biological properties of the actinomycetes and those of the tuberculosis organism, suggested that the relationship of the actinomycetes to the bacteria was closer than to the fungi. Gasperini emphasized that the species or varieties belonging to the ac- tinomycetes, included under one genus Ac- tinomyces, show great variations in form and in behavior, especially in their ability to produce aerial spores and soluble pigments. Some of these properties were recognized to be inconstant and were found to depend on the conditions of culture and the composition of the medium; minor variations of the latter could bring about marked changes in growth and pigmentation. Historical Periods Before we consider in detail the historical background of our knowledge of the actino- mycetes, we must recognize certain distinct periods in which the various concepts con- cerning the nature of these organisms and their importance in the cycle of life became crystallized. There is, of course, considerable overlapping of the various periods, since no one period came to an end before another HISTORICAL BACKGROUND 5 TABLE 1 Observer Rivolta Cohn t. bovis sulphureus t. Foerstert Species of actinomycetes recognized in 1892 to 1894 by Gasperini Name Observer Act. bovis (?) = Streplothrix Foersteri - Act. canis | Vachetta Act. pleuriticus canis famil- | Rivolta | daris Act. canis Rabe Act. bovis farcinicus | Nocard Bacillus farcinicus — Act. catt Rivolta = — Act. bovis albus Gasperini | Streptothrix 1, 2,3 Almquist Streptothrix Albus Rossi-Doria Act. asteroides Hppinger Cladothrix asteroides — Strept. asteroides Gasperini Strept. Eppingerit Rossi-Doria Act. chromogenus Gasperini Strept. chromogenus = Act. bovis luteo-roseus Act cuniculi Act. Hoffmanni Act. albido-flavus Act. violaceus Act. carneus Gasperini Schmorl Gruber Rossi-Doria Rossi-Doria Rossi-Doria Act. citreus Gasperini Act. pluricolor (2) Terni Act. arborescens Edington Act. ferrugineus Naunyn Strept. niger Oospora Metschnikow? (2?) Oospora Guignardi (?) Rossi-Doria Sauvageau & Radais Sauvageau & Radais Streptothrix cunicult - Micromyces Hoffmanni — Streptothrix albido-flava — Streptothrix violacea — Streptothrix carnea -- one began. These periods can be briefly out- lined as follows: 1. Causation of disease. This period began in 1875 and continued to the end of the 19th century. The predominant interest in the ac- tinomycetes during these years was in their role as pathogens, first in human and animal diseases, especially actinomycosis in cattle, and later in plant diseases, particularly po- tato scab (R. Thaxter). 2. Occurrence and importance in soil. Dur- ing the next two decades, beginning about 1900, with the work of Beijerinck, and ending about 1919, with the work of Krainsky, Conn, and Waksman and Curtis, the interest in the actinomycetes was predominantly concerned with their occurrence in soils and in other natural environments. The intro- duction of synthetic media served to broaden greatly our knowledge of the nature and oc- currence of the actinomycetes. 3. Biological period. Between 1919 and 1940, intensive knowledge accumulated con- cerning the cultural properties of the actino- mycetes, their physiology, and their biochem- ical activities, notably their antagonistic effects upon bacteria and fungi. This period may be said to have begun with the work of Waksman in 1919 and Lieske in 1921. It continued with the studies of Gratia and his group on the bacteriolytic effects of certain actinomycetes and of Krassilnikov and his associates on the antibacterial properties of actinomycetes. Problems of variability (Schaal, Tempel, Kriss), decomposition of plant and animal residues (Conn, Waksman 6 THE ACTINOMYCETES, Vol. I ~ 02 a. ete — ~ N. polychromogenes..... + a + N. brasiliensis. ......... + = — N. brasiliensis. ......-.«.. 4. a ao + INS CO RTINCO 5 Ge epee abe = _ ~ — NE eryihiopolis: 96.22. — — _ NEN GUCDONULG:. taaeee eee - = ~ _— NERODOCOm ira nee - _ -- _ Nielevshmaniw. 2 tes nk - fe _ — N. madurae......... oo + _ = NE INGCUT OE 2.8.2 snes ot + + Ne pelletienrtves. c+. at ce - - = _ Wie TONGCONeNSUS..255 8 os > + =| N. intracellularis....... = _ — = VEER OAL GAT UC TUT yg aan ee - — _ - MECN Gs och a caiveaeg te = _ — 68 THE ACTINOMYCETES, Vol. I i B- :3 0) Of O2 Cultural characteristics of strains of Nocardia, Streptomyces, and Mycobacterium (Schneidau and Shaffer) Strain Gross culture Slide aalbaxe Physiologic and biochemical properties Color of substrate growth Starch agar Starch agar Surface growth red White, cream, tan, Yellow, orange, buff, or brown Aerial mycelium Branched mycelium Fragmentation Acid-fastness Growth at 46° C Paraffin utilization Diastasis Gelatin liquefaction Casein hydrolysis Urease Zeya SSS See SSS SS 2 2a Se ee . asteroides Henrici A10A.......... . asterordes ATCC 9504............ 5 CSUGHOUTIES IMMUOO, COO. seh owes oe WASTCROLOES ALL © CO ao0Snee ke we . asterovdes ATCC 9969............ . asteroides NRRL B970........... blackwellii ATCC 6846........... CU DY ACL LOVE Et seas ot ene Ncuniculiy ACC G8645..0-5- 5-00 - . sylvodorifera ATCC 7372......... _ minima ATCC 8674.............. TUOURON OTE BN). 5, coeos cacao sea. polychromogenes ATCC 3409...... brasiliensis Ochoa 409........... ROS HRADSUS ZUNE oo Gao 562500 eae ee IC OMUOLULO MAW © CrAZ omen ns ae NconallinaAdkC C999. 3... ee ae veryihropolissA TCE 4277... .....-: LOVE ULAAG © Cx 985Ommen ae eras PODACOMAWA© CAD Gee nels eat: LOR MN IEE OSDn eran rae (OIG? INIA CONSIGN, Sos bee eoeee oa gardnernt ADCC 96045.........44- . leishmani ATCC 6855.....2..-2:- wmadunae Ochoa Alp .ee sss) eee MINGOUTCewAGa © © 624 555ee se nee pepelleteny Nbacazpe20 ome are on). me nCletier AZ omen ys oe ae . rangoonensis ATCC 6860......... 5 UPUREA TCH US BBW y.6% 686450000606 5 HHO INIUCKO, BBS ao nok amo on OMUSCLLS AVL © © 13 B2 GAN ee ae cere ~ ON PRAM CC) BB sage dees oduss . venezuelae ATCC 10595........... PIO UEUTUCUNUPZOLA 2 Oe caer reese ae 5 Gpontiye WYN NO sean sgeo ucts: DU CUNO) Meera in iho cota. oe MER SINeqmatuseZiO) Levent tees cee. MM. stercorts’262........ 5. : MMR ee ee ptt t+t+tett+t+tet +t | | b++++4+1 | b++4++14+4+1 ++ II" a | as | + + | Diffusible pigment Pttee i per tsi }+++14++ }+++4+4+1 b+ i +++ b++++1 4 }++++++++4++4+ | | | [se Stesrete A =lstrish State staat chest ie taste steel PLL LEHRER LEE LE EEHE HFEF EHE IHEP EL LE EI 1 + | Hemolysis b+ i t+t++tt+i¢+)++4+4+4+14+4+4+ b+t+t+++4++4+4+4+¢4+4++4+4+4+4+4+4 l++4+ > | West sste Stee dle ste SteSste ie Ss Sis sheet steht ote ale eset feria cteeete o | Wteestaen| b+) +++) | b+t+tit++t++t4¢t+ i ats dee! | }+++H+1 leet eagle b+t++ i ++++++++H HHH ++tet+t+et+4etq¢t+ b+t+tit+++4+4+4+4 1 It | +++4+4+4 1 | | fee pete pti ++tsst +++4+4+1 +++++ | | | +++4+++1 NOMENCLATURE AND GENERAL SYSTEMS OF CLASSIFICATION The strains of Nocardia studied were sep- rated, on the basis of the cultural data pre- sented, into “true”? Nocardia and “strepto- myces-like’? Nocardia. The former have been further tentatively divided into the follow- ing groups: (a) V. asteroides group (all strains of N. asteroides plus N. blackwellii, N. cunie- uli, N. caprae, N. sylvodorifera, N. poly- chromogenes, N. minima, and N. paraffinae) ; (b) N. corallina (N. globerula, N. erythrop- olis, and N. convoluta); and (ce) N. opaca group (NV. opaca and N. rubra) (Table 12). Various other criteria have been suggested as aids in the classification of the actino- mycetes, notably members of the genus Streptomyces. It sufficient to mention phage susceptibility (Stocker) and forma- tion of antibiotics (Kurosawa, Kuroya, et al., 1950). According to Krassilnikov (1957), antibiotics may be considered as necessary in the struggle for life between rival organ- isms. They manifest their activity toward competing organisms only, but never against cultures of the same species. He believed that strains of one species produce antibiotics inhibiting the growth of all the strains of rival species. This antagonism may be uni- lateral or bilaterial. He suggested that the specificity of antibiotics produced by various species be used in taxonomy for the differ- entiation of these species. He reported suc- cess in demonstrating, by means of cross is 69 antagonism experiments, the heterogeneity of many species which heretofore were con- sidered as homogeneous. Waksman and Lechevalier found that, on the basis of sensitivity to isoniazide, the phylogenic position of the various Actino- mycetales was indicated as follows: The pathogenic forms of the genus JJ ycobac- tertum are susceptible to less than 1 mg of isoniazide per milliliter, the avian form being least susceptible; the saprophytic forms were less susceptible, some being resistant to 100 and even 1000 mg per milliliter, most of them being susceptible to less than 5 and 10 mg per milliliter. The micromonosporas were more resistant to isoniazid than the myco- bacteria (10 to 1000 mg per milliliter). The nocardias were also moderately more resist- ant, most of them requiring 80 to 300 mg of isoniazide per milliliter for growth inhibi- tion; some were more susceptible (10 mg per milliliter), and others were resistant (more than 1000 mg per milliliter). The strepto- mycetes were most resistant, requiring as a rule more than 1000 mg of isoniazide per milliliter for inhibition. The various gener: of the Actinomycetales could thus be listed in the following order of increasing resistance to isoniazide: pathogenic mycobacteria —> saprophytic mycobacteria — MJicromono- spora — Nocardia — Streptomyces. Among the new approaches to the tax- TABLE 13 Classification of the actinomycetes based on cell wall composition (Commins and Harris) Cell wall components Family Genus Sugars Amino acids Mycobacteriaceae | Nocardia | Arabinose, galac- Alanine, glutamic acid, DL- | Mycobacterium tose | diaminopimelic acid | Corynebacterium Actinomycetaceae Actinomyces Galactose | Alanine, glutamic acid, lysine Streptomyces | Micromonospora | (Propionibacterium) * Streptomycetaceae No characteristic | . . . | Alanine, glutamic acid, gly- cine, LL-diaminopimelie acidt sugar * Position rather doubtful. + In Micromonospora pu-diaminopimelic acid is present in addition, 70 THE ACTINOMYCETES, Vol. I onomy of actinomycetes, that based upon the chemical composition of the cell wall deserves particular consideration (see Chap- ter 9). The results tended to emphasize the close relationship of the actinomycetes to the bacteria and not to the Eumycetes or true fungi. Cummins and Harris (1958) found that the cell walls of the actinomycetes were made up of sugars, amino sugars, and a few amino acids (usually three or four), the general pattern of these components being identical with that of the gram-positive bacteria. The mycelial walls of the fungi are composed entirely of carbohydrate. They proposed a system of classification of the actinomycetes based upon the chemical composition of their cell walls (Table 13). They even went as far as to suggest that the order Actinomycetales be abolished alto- gether and the families of actinomycetes be included with the Eubacteriales. Recognition of Certain Groups Among the Actinomycetes In order to complete the historical back- ground of classification of actinomycetes, one further aspect must be mentioned, and that is the recognition by many of the earlier investigators that certain individual species of actinomycetes may just as well be given a group characteristic. Sanfelice suggested in 1904 that the actinomycetes be divided into three groups as follows: 1. Streptothrix alba. Colonies opaque to white, covered with calcareous powder, and adhering fast to the medium. On potatoes, growth is rapid, white, with a lime-like surface; pigment remains unchanged, or may become gray; occasionally the color may change to black or straw-yellow. San- felice emphasized that ‘‘the superficial observer could create out of a dark culture a new species, without recognizing the original nature of S. alba.” 2. Streptothrix flava, frequently obtained from the dust, shows much variation in pigmentation. Growth lichenoid, intensely yellow. On potatoes, growth is also lichenoid, but color less intense. Color may gradually change on continued transfer, becoming either lighter or deeper orange-red. Ap- parently, this group comprised forms that are now largely considered as Nocardia species. 3. Streptothrix violacea. Opaque, lechenoid growth, brownish in color, occasionally turning black. On potato, growth is of a bluish amethyst color. Foulerton, Chalmers and Christopherson, Langeron, and Brumpt also suggested divi- sion of the pathogenic species into several sections or groups. These were designated as Breviores (A. bovis, A. israeli), Minores (A. asteroides), and Majores (A. albus, A. chro- mogenes). These sections correspond to the first three genera in the Waksman and Hen- ric classification, namely, Actinomyces, No- cardia, and Streptomyces. Further systems of classification of the various genera, notably Streptomyces, into groups, sections, and series, each of which comprises a number of species, will be dis- cussed in detail in Vol. II, Chapters 20 and 21. Cc Le Mer. bik . KR 4 5 Morphology, Cytology, and Life Cycles General Morphological Properties of Ac- tinomycetes As has been pointed out, all the evidence recently submitted concerning the structure and functions of actinomycetes definitely establishes the fact that these organisms are to be classified with the bacteria and not with the fungi. Lehmann and Neumann (1896) were among the first to draw attention to the close morphological relationship between the diphtheria and the tubercle bacillus, on the one hand, and the actinomycetes on the other. Later (1920), they emphasized that the distinction between mycobacteria and actinomycetes is not very sharp, inasmuch as some mycobacteria show only slight re- sistance to decolorization by mineral acids, and some of the actinomycetes possess relatively well-developed acid-fastness. As pointed out previously, Cummins and Har- ris suggested, on the basis of recent chemi- val evidence, that the order Actinomycetales be abolished altogether and that the fami- lies of the actinomycetes be included in the Eubacteriales. Actinomycetes, like the true bacteria, are procaryotes. Their growth (protothallus) is made up of branching filaments, producing a mycelium. This may be of two types, one prostrate, forming a vegetative growth, sometimes referred to as substrate myce- lium; the other, erect or aerial mycelium. The spore-bearing hyphae of the aerial my- celium usually have a somewhat greater diameter than the hyphae of the substrate 71 mycelium. These two types of mycelium, or mycelial stages, are not only structurally different but possess different growth re- quirements. The secondary or aerial myce- lium is considered by most investigators to originate asexually from the primary or substrate mycelium; some consider it as a sexual stage. Actinomycetes produce two types of spores: (a) true conidia, and (b) arthrospores or chlamydospores. The earlier investigators, notably Lachner-Sandoval, recognized “‘frag- mentation”’ spores appearing as spherical to cylindrical segments of old hyphae, pro- duced by the contraction of the protoplasm; and ‘‘segmentation” spores produced by the septation of the tips of the aerial filaments, usually formed in lateral branches of the aerial hyphae but also extending to the main filaments in substrate growths. According to Neukirch, the ‘‘segmentation’? spores are produced not by a process of septation of the aerial mycelium, but by the successive contractions of the protoplasm, until ap- proximately isodiametric portions are sepa- rated by regularly alternating empty spaces (see also Domec). On the basis of a study of a number of saprophytic actinomycetes, belonging to the genus now recognized as Streptomyces, Drechsler summarized their morphological characteristics as follows: 1. The vegetative growth consists of a myce- lium composed of profusely branching hyphae, the terminal growing portions of which are densely 72 THE ACTINOMYCETES, Vol. I filled with protoplasm; toward the center of the thallus, the vacuoles increase in size, and may be associated with the presence of metachromatic granules. The vegetative hyphae of the mycelium are far larger than those of ordinary bacteria of the acid-fast group; the hyphae also lack the uni- formity in diameter generally characteristic of the true bacteria. 2. The aerial mycelium produced on suitable media usually occurs as a mat of discrete fructi- fications. Each of these represents a well-char- acterized sporogenous apparatus, consisting of a sterile axial filament bearing branches in an open racemose, or dense capitate, arrangement. The primary branches may function directly as sporo- genous hyphae, or they may proliferate branches of the second and of higher order; sporogenesis, in the latter case, is confined to the terminal ele- ments, the hyphal portions below points of attach- ment of branches remaining sterile. 3. Two tendencies in the development of fructi- fications were recognized: (a) one leading to an erect dendroidal type, in which successively pro- liferated fertile elements undergo processes of sporogenesis in continuous sequence; (b) the other leading to a prostrate, racemose type, in which sporogenesis 1s delayed in the older branches until the younger branches have also attained their final extension. The majority of species show these tendencies combined in different ways. 4. The sporogenous hyphae of many species are coiled in peculiar spirals. These exhibit pro- nounced specific characteristics in the number, diameter, and obliquity of their turns, and es- pecially in the direction of rotation, which may be dextrorse or sinistrorse. This phenomenon was later found not to be constant, however, but to vary with the composition of the medium. 5. Sporogenesis begins at the tips of the fertile branches and proceeds basipetally. In some species the process involves the insertion of septa, which are, in certain cases, relatively very massive, and in others, so thin as to be barely discernible. 6. Granules which possess the staining prop- erties and uniformity of size characteristic of nuclei are readily differentiated in the spores of many species; they generally occur singly, but in the larger spores of a few forms, two are often found occupying diagonally opposite positions. As in the vegetative thallus, metachromatic granules occur in the aerial mycelium, being very rarely found in spores or sporogeneous hyphae but becoming very abundant in degenerate sterile hyphae. 7. Peculiar spherical structures appear regu- larly in some forms, both in the sterile axial hyphae, where they may contain either a medium septum or a number of peripheral metachromatic granules, and in the sporogenous hyphae, where they are associated with the regularly spaced septa. 8. The spores germinate readily in proper media, producing from one to four germ tubes, the approximate number being more or less char- acteristic of the species. Orskov divided the actinomycetes into three morphological groups: I. Those that produce an undivided substrate mycelium and an aerial mycelium which breaks up into bodies that possess the quality of spores. Il. Those that produce an initially undivided substrate mycelium. After having reached a cer- tain point, it divides by septa into rod-shaped elements; these continue to multiply with a char- acteristic ‘“‘angular’’ growth; aerial mycelium may or may not be formed; in the former case its ele- ments resemble those of the vegetative mycelium. III. Those that produce a substrate mycelium resembling that of group I, but devoid of aerial mycelium and producing spores borne singly at the distal end of short mycelial branches. According to Orskov the angular growth into which the members of group II pass after the formation of an initial mycelium is similar to the process of cell division in the mycobacteria and corynebacteria. This simi- larity makes it impossible to draw a sharp line of demarcation between true bacteria and actinomycetes. Jensen emphasized that the nocardias, or proactinomycetes, as he designated them, represent a heterogeneous collection of types, standing between the mycobacteria and corynebacteria, on the one hand, and the streptomyces, on the other. : The morphological structure of actino- mycetes depends largely upon (a) the nature of the organism, (b) the composition of the medium, (c) conditions of growth, especially aeration, and (d) presence of growth-stimu- lating and growth-inhibiting factors. The thallus consists of a homogeneous cytoplasm which, as it grows older, becomes vacuolated MORPHOLOGY, CYTOLOGY, AND LIFE CYCLES io and shows refringent granules probably con- sisting of volutin (Lieske). Fat granules and occasional vacuoles containing chromotropic granules were observed by Grigorakis (1931) in the thallus of the organisms grown on an agar medium containing glycerol and pep- tone. Our knowledge of the morphology of ac- tinomycetes has recently enlarged greatly by studies of their cytology, espe- cially through the use of the electron micro- scope. Discoveries of chromatic substance (Schaede, von Plotho, 1940), of lipids (Erik- son, 1947) and of the chemical composition of the cells (Romano and Sohler, Cummins and Harris) have further contributed to a better understanding of their structural properties. Electron microscope studies of the mech- anism of spore formation by members of the genus Streptomyces tended to confirm the view of Lachner-Sandoval, presented as far back as 1898. According to Vernon (1955), been Pr A ee ; eae Ficure 33. Germination of a spore, as shown by electron microscopy, 38,000 (Carvajal, F. Mycologia 38: 589, 1946). streptomyces Figure 34. S. griseus: primary mycelium at 40 hours from submerged culture, showing germinating initial cell. Visual light, X 2000 (Reproduced from: Dickenson, P. B. and Macdonald, K. D. J. Gen. Micro- biol. 13: 89, 1955). 74 THE ACTINOMYCETES, Vol. I spore formation takes place within the hy- phal wall. The hyphal contents divide si- multaneously into fragments, separated by less dense partitions, having the appearance of septa. The spores remain in chains, held together by a sheath-like hyphal wall, which undergoes change as the spores mature. In some cultures, the sheath persists, the spores being liberated by means of a longitudinal split; the old wall remains as a ribbon-like sheath, with cross markings indicating the position of the spores; in other cultures, the sheath disintegrates to small fragments. The surface structure appearance of the spores varies for the different organisms and is believed by some to be a species characteriza- tion (Ettlinger et al., 1958). The formation of spines on the surface of the spores, demon- Figure 35. S. griseus: primary mycelium at 53 hours from submerged culture, showing hyphal fusions. Electron micrograph, & 5000 (Reproduced from: Dickenson, P. B. and Macdonald, K. D. J. Gen. Microbiol. 13: 89, 1955). strated by Flaig ef al. (1952), Enghusen (1955), Baldacci and Grein, and others, has also been reported by Vernon for S. flaveolus, for example. Further information on the structure of the mycelium and spores of various actino- mycetes is found in the work of Elisei (1944), Carvajal (1946-1947), Scotti and Gocchi, Webley (1955), and many others. Sexuality among Actinomycetes The fusion of mycelial threads among ac- tinomycetes has been observed first by Lieske, then by Korber (1929), and others. According to Krassilnikov (1938), this can take place either as the confluence of germi- nating spores, through the germination tubes, which give rise to a single hypha de- veloping into a mycelium, or as the anasto- mosis of two hyphal filaments. Krassilnikov suggested that this actually is or resembles the sexual process comparable to that which takes place in many yeasts. He further em- phasized the great biological significance of this phenomenon, which possibly explains the variability of the actinomycetes. According to Kleneberger- Nobel the morphological changes in the growth of a streptomyces on the surface of a medium take place as follows: The spores germinate, giving rise to a substrate or “primary myce- lium,” which undergoes anastomosis, result- ing in the formation of “initial cells,’ or “fusion cells;’ these produce, on germina- tion, aerial or “secondary”? mycelium, which sporulates to give rise to spores. This in- vestigator considered untenable the deserip- tion of spore formation, by Lieske and Orskov, as taking place without any previous segmentation of the protoplasm. During the process of spore formation, the hyphae were believed by Kleneberger-Nobel to be sepa- rated by transverse septa into small cells, ach of which eventually develops into a spore. Erickson (1949) suggested that such “‘ini- De PRIMARY MYCELIUM [ ‘i © SECONDARY MYCELIU “TETRACYTES" Ficure 36. Diagrammatic life cycle of a streptomyces. The primary and secondary mycelia are separated for convenience of illustration though usually the latter originates within the former. At- tention is called to the gradual shrinkage of endofragments of the primary mycelium to produce iso- gametes; the secondary mycelium may be bisporulative (Reproduced from: Roach, A. W. and Silvey, J. K. G. Trans. Am. Microscop. Soc. 77: 36, 1958). 75 76 THE ACTINOMYCETES, Vol. I tial cells” are artifacts; she believed that aerial hyphae may arise by budding from any substrate (vegetative) hyphae. Wilkin and Rhodes observed that the nature of the medium influences the morphological forms produced by a Streptomyces: on synthetic media, the cycle proposed by Klieneberger- Nobel was confirmed; on complex media, the formation of “initial cells’? and ‘‘secondary mycelium”? was suppressed, however. The ‘primary mycelium” proliferates on com- plex media to give ‘‘chlamydospores,”’ which, on germination, give rise to typical “‘pri- mary mycelium.” Further information on the life cycles of actinomycetes is given by Wilkin and Rhodes, and Roach and Silvey. Dickenson and Macdonald made electron microscopic observations on submerged cultures of two species of Streptomyces. The evidence obtained tended to confirm the theory of Kleneberger-Nobel, that at an early stage in the life history of the organ- ism concerned, fusion occurs between por- tions of the same or different hyphae, cul- minating in the formation of ‘‘initial cells.” Further studies on problems of recombi- nation of nuclear material and on the life cycles of actinomycetes in general, are dis- cussed in Chapter 6. Colony Formation The growth of an actinomycete on a solid or in a liquid medium results in the forma- tion of a mass of unicellular mycelium us- ually designated as a “colony.” This is not a colony in a true sense, since it is not an accumulation of many cells, but rather a mass of branching filaments which originated from a spore or from a bit of mycelium. The two types of mycelium making up a colony of a streptomyces often show fundamental differences in appearance, composition, and biological activities. The substrate or vege- tative mycelium grows into the medium, whereas the aerial mycelium grows on the surface; the well-developed sporulating hy- phae and the reproductive spores are pro- duced in the aerial mycelium. Some actino- mycetes form only the substrate mycelium, whereas others produce both types. Some aerial mycelium-forming cultures may lose this property, and may thus be distinguished from nocardias only by certain physiological properties, as pointed out elsewhere. Some nocardias, on the other hand, also produce a typical aerial mycelium, as shown by Gordon and Mihm (1958). According to Henrici (1930), surface colo- nies produced by various actinomycetes are of two general types: (a) One type is char- acteristic of those strains that form a highly developed, extensively branching mycelium, notably members of the genus Streptomyces. Colonies of this type are very firm, almost cartilaginous in consistency, and adhere to the solid substrate, because the mycelium grows into that substrate; when touched with a wire loop, the colony does not break but separates from the substrate as a unit. In cross section, such colonies usually have a shghtly conical form and show marked radial foldings. At first their surface may be glossy or matted, but, if aerial spores are developed, the surface becomes covered with chalk-like powder which, as the colony grows older, may acquire various shades of color. The powdery spores frequently appear in concentric rings. (b) The second type of colony is characteristic of the strains that do not form an extensive mycelium, notably members of the genus Nocardia. Their thal- lus has a tendency to break up into hyphae of variable length, and in certain strains most of the growth may consist of short filaments, resembling in appearance pleomorphic bac- terial strains. Colonies of this type are less tenacious than those of the first; they often have a mealy consistency and tend to crum- ble when touched with a wire loop. Actinomycete colonies are usually round and smooth, or much folded and lichenoid in appearance. When examined under the mi- MORPHOLOGY, FIGURE 37. Sporulation of S. griseus (Reproduced from: Baldacci, E. biol. 1: 34, 1955). croscope, the edge of the colony shows a characteristic picture of radiating hyphae. When grown in liquid media in a stationary condition, the colonies may be formed in- dividually on the bottom of the container, or they may adhere to the surface of the wall, or they may form a ring of growth or a pelli- cle on the surface. The colonies may also erow in the form of flakes, but the medium is never made turbid, as in the case of bac- terial growth, unless the colonies undergo lysis through the action of enzymes or phages. When grown in a submerged or in a shaken condition, actinomycetes produce small, bead-like masses of growth, some of which may be granular in nature. According to Jensen, strains of Nocardia with more persistent mycelium produce firm agar colonies and a discretely granular growth in liquid media, which remain clear; those with small or rapidly dividing myce- lium show a soft growth in solid media and a diffuse, liquid media. Jensen took exception to Lieske’s (1921) statement that ‘‘every form of tur- bidity of a liquid medium is to be looked bacterium-like growth in upon as evidence of contamination.” He also CYTOLOGY, AND LIFE CYCLES id and Grein, A. Giorn. Micro- pointed out that a similar difference exists m cultures of the genus Actinomyces, where most strains show a granular growth but some give a cloudy growth in broth (Erik- son, 1940; Holm, 1948). The colonies of the bovine strains of this anaerobie genus A ctino- myces are smooth and soft in consistency and do not adhere to the medium; the mycelium undergoes fragmentation very rapidly, giv- ing no extensive ramification; such strains show occasional turbidity in the medium. In contrast, human strains give no stable variants and produce no turbidity (Erikson). attempts divide the actinomycetes into groups on the Various have been made to basis of the size of the colonies (IKXrainsky) or the length of hyphae (Lieske). These properties are not recognized now as of pri- mary significance in classification, since the nature of the organisms and the conditions of growth are of prime importance. Staining Properties The mycelium of the actinomycetes can be dried on slides and stained with ordinary aniline dyes. Methyl violet, carbol-fuchsin, and methylene blue can be used. Fresh my- 78 THE ACTINOMYCETES, Vol. I ll formation from double hyphal contact. From 6-day Fiaure 38. S. griseus: first stage of initial ce P. B. and Mac- submerged culture. Electron micrograph, X 10,000 (Reproduced from: Dickenson, donald, K. D. J. Gen. Microbiol. 13: 89, 1955). MORPHOLOGY, CYTOLOGY, AND LIFE CYCLES 79 Figure 39. Mycelium and sporulation of a micromonospora (Reproduced from: Jensen, H. L. Proc. Linnean Soc. N.S.W. 55: 249, 1930). cellum in a nondried condition can also be stained with dyes that readily enter the liv- ing cell. There is little difference in the stain- ing properties of the various types of my- celium and the aerial spores. Practically all actinomycetes are gram- positive. Occasionally, a gram-negative form has been reported. The acid-fast properties of actinomycetes, notably among the no- cardias, have aroused considerable attention. Numerous acid-fast strains have been de- scribed. The microaerophilic and pathogenic species of Actinomyces seem devoid of all acid-fastness; so are many species of No- SO THE ACTINOMYCETES, Vol. I cardia. Others are acid-fast to a variable degree that depends both on the organism and on the cultural conditions. According to Jensen, acid-fastness in pathogenic forms is often stronger im vivo than im vitro. Many species can probably best be described as potentially acid-fast, because this property may be apparent for only a very brief period in the organism’s life history and can be induced by special media, like milk (Jensen, 1931-1934; Um- breit, 1939) or synthetic media containing paraffin or high concentrations of glycerol (Erickson, 1949). Acid-fastness 1s not a permanent property of an organism. On continued cultivation on ordinary media, the acid-fast characteristic may be lost; on the other hand, the property may be strengthened by growing the organ- ism in media containing oil or fat or on animal passage. Acid-fastness cannot be con- sidered as a characteristic for species differ- entiation. Some of the observations of stained prep- arations of actinomycetes have to do with the presence in the thin cells of a nucleus and other particulate constituents. Crystal violet and thionine SOz, as well as crystal violet- tannic acid-congo red cell wall stain, can be used (Webb and Clark). Lieske and others failed to observe any true nucleus in actinomycete cells: nuclear Fiaure 40. Formation of coremia by a strepto- myces (Reproduced from: Krassilnikov, N. A. “Manual of the ray fungi’. Acad. Nauk, USSR, Moscow, 1938, p. 34). substance was found in the form of grains, which, in Lieske’s opinion, pointed to the close relationship of these organisms to the bacteria. The occurrence of fatty particles in the cells of actinomycetes was observed by various investigators. Glycogen and chitin could not be found. Substrate Mycelium Actinomycetes produce a substrate or, as it is often designated, vegetative mycelium that usually varies in size, shape, and thick- ness. The color of the substrate growth ranges from whitish or cream to brownish, yellow, red, pink, orange, green, or black. Water-soluble and water-insoluble pigments may be produced, depending on the organ- ism and the composition of the medium. Some of the pigments, especially the dark or chromogenic pigments, are formed upon complex organic media and are often a result of the action of certain enzymes of the tyrosin- ase type upon proteins and their deriva- tives. Other pigments are synthetic in nature and are formed on simple media. The spores of actinomycetes germinate in the medium with the formation of one or more germ tubes. These grow into long hyphae, finally culminating in a complex mycelium. The length and the diameter of the hyphae differ considerably. Some are straight and reach a length of more than 600 yu; others are only 50 to 100 u in length and are much branched and curved. This frequently suggested the division of acti- nomycetes into long-mycelial and_ short- mycelial groups. The vegetative mycelium varies in diameter from 0.2 to 0.8 uw. The branching of the mycelium is typically mon- opodial. Involution forms which have a greater diameter may also be produced (Miinter, 1916). On continued growth, the vegetative my- celium becomes brittle and breaks into fragments of uneven length. Some cultures, MORPHOLOGY, CYTOLOGY, AND LIFE CYCLES Sl with age, undergo lysis, others are subject to attack by specific phages. When inoculated into fresh medium, the finer or disintegrated particles give rise to a normal mycelium. This suggested to some investigators (IXober, Monal, Grigorakis) the possibility of sym- plasm formation as a stage in the life cycle of the organisms. Further study did not substantiate this concept. Club Formation An interesting morphological phenome- non among certain actinomycetes, deter- mined by the environment, is the formation of clubs. These clubs should not be confused with involution forms. They are the result not of swelling of the hyphal tip, but of the secretion of a sheath of slime around the tips of the hyphae; therefore they are comparable to the capsules of bacteria. The clubs are formed in the animal body by pathogenic organisms like A. bovis. They were also observed by Wright (1905) in cultures grow- ing in the presence of animal serum or whole blood and by Bayne-Jones (1925) in glucose broth. They may be formed in response to the presence of some thermostable substance in animal fluids, or to some other condition such as a reduced oxygen tension. They can be readily induced by the addition of 3 to 7 per cent of NH,Cl, and also in sugar- containing media on aging. These swellings usually have several times the thickness of the normal hyphae. In the animal body the clubs are found in groups, each radiating from a mass of my- celium (granules). They give the impression of a star-like arrangement, which is responsi- ble for the name given to the actinomycetes as a whole (Lieske, 1921). Motility Motility in actinomycetes was reported for N. asteroides by Eppinger (1891) and is characteristic of the genus a ee Pe On P la0 1.8 | 126.0 * The reverse mutants were counted on medium IV, and the number of colonies at 0 time on me- dium II, 2.1 & 107, was taken as the base of cal- culation. + The actual number was 3.0 & 1077. culture is gradually increased. This type of adaptation may be reversed when the cells are again grown in a medium free from the antibacterial agent; sometimes, this type of adaptation may prove to be very persistent. Adaptation of microorganisms to anti- microbial agents has been explained as fol- lows: 1. Adaptation occurs by natural selection from an initially heterogeneous population. This theory has lost much support since variations have been found to occur in strains derived initially from a single cell. 2. Adaptation occurs by actual modifica- tion of the metabolism of individual cells. This may be due to the establishment in the cells of a mechanism alternative to that normally in use, or to the quantitative modi- fication of existing mechanisms. 3. The adaptation is due to a change in some center of organization of the cell. The mechanism of acquired drug resist- ance may thus be due either to direct induc- tion or to mutation with selection (Abra- ham). The ease of development of resistance depends upon the organism and the anti- microbial agent. In some cases the organism 110 may become not only resistant to, but also dependent on, this agent, as was found to be true for bacterial strains requiring strepto- mycin for their growth. Variation and the Action of Phage The lytic properties of actinomycetes, especially under the influence of phage, may also undergo a variety of changes, depending largely upon the development of strains re- sistant to phage action. When an actino- mycete culture is attacked by a phage, the culture will clear up after a few hours as a result of destruction of the sensitive cells. After further incubation, which may some- times require days, the culture will begin to grow again as a result of development of a variant which is resistant to the action of the phage. This variant can be isolated and freed from the phage and will in many cases retain its resistance to the action of the phage even if subcultured through many generations. Though the sensitive strain adsorbed the phage readily, the resistant rariant will generally not show any affinity tO di The variant may differ from the original strain in morphological or metabolic charac- teristics, in serological properties, or in colony type. Most often, however, no such correlated changes are apparent, and the variant may be distinguished from the orig- inal strain by its resistance to the inciting strain of phage. It has been suggested that the resistance to phage is due to a heritable change of the microbial cell, which occurs independently of the action of the phage. The mechanism may be more complex when the resistant culture does not develop until several days after lysis of the sensitive cells. The proportion of mutants in a culture and the mutation rate are detected by changes in the colony type produced by the mutant, either in its pigmentation, or in the character of the surface or the edge of the colony. THE ACTINOMYCETES, Vol. I Often, colonies of intermediate character are produced. This is particularly true of cases where the mutation rate is high and where reverse mutation occurs. Genetic Recombinations Recent genetic studies on Streptomyces have proceeded along two main lines: (a) the radiation genetic studies (Newcombe), for which the uninucleate status of Strepto- myces spores offers an advantage; (b) studies on genetic interaction among Strepto- myces. Anastomosis, or the fusion of hyphae, is frequently encountered, with heterokaryo- sis resulting when both types of parental nuclei persist and multiply in a common cytoplasm. Recombination, based on the exchange of genetic characters between nuclei, is a much rarer phenomenon, re- ported for S. coelicolor, S. griseus, and S. fradiae. Sermonti and Spada-Sermonti demon- strated a parasexual process, leading to genetic recombination in S. coelzcolor. Three types of recombination between strains oc- curred: (a) strains carrying all the ‘‘wild”’ characters of the original organism; (b) some “wild” characters of one strain and some mutant characters of another strain; (c) mutant characters from both strains. Bradley and Lederberg used nutritional and resistance markers to establish two parental types of S. griseus. They demon- strated that fusion occurs between the hy- phae of the two parents, giving rise to hetero- karyotie mycelium in which nuclei from both parents were contained in the same hyphae. The spores produced by the heterokaryon were of only one parental type; they were, therefore, considered as homokaryotic. The recombinations did not prove to be stable, however. According to Bradley, various species of ‘an form heterokaryons, 7.e. associations showing genetic interaction between diverse nuclei in a common cyto- Streptomyces VARIATIONS, MUTATIONS, AND plasm. These heterokaryons can be perpetu- ated by fragments of vegetative mycelium, but not by spores; the latter are uninucleate, being derived from a single nucleus. A strain of S. coelicolor forming stable hetero- karyons and producing spores with at least two sets of genes was studied. Nutritionally wild type, or prototrophic, colonies were obtained from growth-factor-dependent, or auxotrophic, combinations by two methods: (a) strains were plated together on a minimal medium; the prototrophs arose after 8 to 16 days; (b) strains were grown together on complete medium for 3 to 6 days; the result- ing spores were transferred to minimal me- dium to select prototrophs, which were puri- fied by several serial transfers on complete medium. Bradley (1958) exposed a population of a single strain of S. griseus, for several growth cycles, to a sterile culture filtrate of another strain of S. griseus. The first acquired several genetic characteristics of the second strain, namely, streptomycin sensitivity changed to resistance, bacteriophage sensitivity changed to resistance, absence changed to presence of soluble pigment, and presence changed to absence of pigment in the vegetative my- celium. The filtrate Contained a low con- centration of streptomycin, which did not inhibit the growth of the first strain, but streptomycin-resistant mutants were se- lected. The observed morphological changes were coupled with bacteriophage and strep- tomycin susceptibility. The hybridization was said to be the result of selection of mu- tants rather than gene transfer. Alikhanian and Mindlin grew biochemical mutants of S. rimosus on suitable agar media, and observed that at the point of contact of the mutant colonies more abundant growth and more abundant sporulation occurred. Nuclear fusion and reduction occur in S. coelicolor somewhere between hyphal fusion in the substrate mycelium, allowing hetero- ADAPTATIONS 111 FIGURE 53. Formation of heterokaryotic col- onies of S. fradiae on minimal agar. Plates 1 and 3 seeded with 10° spores of strain 6F4-1 (methio nine and isoleucine requiring, and streptomycin sensitive) and 6FS-16 (histidine and arginine re quiring, and streptomycin resistant), respectively. Plate 2 received a mixture of parental spores and shows formation of prototrophic colonies (Re produced from: Braendle, D. H. and Szybalski, W. Proc. Nat. Acad. Sci. 43: 947-955, 1957). karyosis and spore production in the aerial hyphae (Hopwood). According to Braendle and Szybalski, all of the wild-type strains of Streptomyces studied were prototrophic, 7.e. they formed 112 colonies and sporulated on synthetic agar with ammonium sulfate and glucose as the only nitrogen and carbon sources. Nutri- tionally deficient mutants were developed by ultraviolet irradiation of the parental prototrophic culture, concentrating the mu- tants by the filtration technique, and finally employing selective media and the replica- plate principle to detect and identify the mutants. Auxotrophic strains were isolated which required single or multiple supple- ments of various amino acids or alternative requirements of two, three, and even four amino acids, often as the result of a single mutation. Several antibiotic-resistant mu- tants were also isolated with the help of the gradient-plate technique (Szybalski, 1958). The first type of genetic interaction, widely observed in these studies, was the formation of heterokaryotic mycelium con- taining both types of parental nuclei in a common cytoplasm. This phenomenon was easily demonstrated by plating a mixture of two types of nutritionally marked conidia on a selective medium. A cross was _ per- formed between two strains of S. fradiae, one streptomycin-resistant and requiring methionine and leucine, and the other re- THE ACTINOMYCETES, Vol. I quiring histidine and arginine but strepto- mycin-sensitive (Fig. 53). A mixture, con- sisting of approximately 10° spores from each parent, was incubated for 2 to 6 days on minimal agar. This mixture yielded several hundred prototrophic ‘‘recombinant’’ colo- nies, whereas the plates seeded with spore suspensions of only one of the parents showed no growth. The formation of hetero- karyons, nutritionally balanced, 7.e. able to grow in the absence of all the nutritional requirements exhibited by any one of the parents, was demonstrated for S. griseus, S. fradiae, S. venezuelae, and S. albus. Only S. coelicolor produced nutritionally unbalanced heterokaryons, which formed tufts of growth between proximal parental colonies grown on the synthetic medium enriched with a small amount of an amino acid mixture. These unbalanced types did not grow on an unsupplemented medium. Heterokaryon formation only between mutants derived from the same parental culture. The interspecific crosses and a limited number of intraspecific crosses between different strains designated as S. griseus were unsuccessful. was observed CH Wael ial / ER ~1 Physiology Any consideration of the physiology of actinomycetes involves a study of their growth and nutrition, their metabolic proc- esses, and their reaction to environmental conditions. Such important phenomena : saprophytism versus parasitism, aerobiosis versus anaerobiosis, thermophilic versus me- sophilic growth, decomposition of organic residues and nitrogen transformation, well as lytic phenomena and death rate may also be considered here. Some of these proc- esses are sufficiently important to warrant more detailed treatment elsewhere in this volume. The activites of a microbial cell consist of a multiplicity of chemical reactions, which are interlinked in a most amazing and be- wildering fashion. Numerous attempts have been made to base an understanding of the metabolism of the various organisms upon the transformations brought about by rest- ing microbial cells. The capacity of such cells to catalyze the transformation of specific chemical substances has frequently yielded information of considerable biochemical sig- nificance. However, the results obtained from such studies have not always been so fruitful in unraveling the complex reactions of microbial cells. Although our understanding of the phys- iology of the microbial cell is limited chiefly to a knowledge of the behavior of pure cul- tures, it is not to be forgotten that, innature, microbes, especially the actinomycetes, live in constant association with other organisms . « Us as 113 and are subject to continuous influences of these associated organisms. In the soil and in water basins, each of these microbes lives in association with thousands of others, as well as with the root systems of higher plants and with tissues of higher animals. Some in- vestigators have even asserted that actino- mycetes lead only a limited vegetative exist- ence in the soil and occur there largely in the form of spores. The question has, there- fore, frequently been raised: How significant are laboratory studies in interpreting the ac- tivitives of these microbes in nature? Path- ogenic microbes, whether they attack plants or animals, are influenced in their growth and nutrition by the hosts which they in- habit and the tissues which they attack. Physiological reactions based upon pure cul- ture studies and upon the growth of organ- isms in artificial media may thus be quite distinct from corresponding reactions brought about by the same organisms in a natural environment. The actinomycetes represent a_ fairly large group of microorganisms widely dis- tributed in all natural substrates. They rep- resent fairly heterogeneous systems differing greatly in their mode of nutrition, metabolic processes, storage and waste products. Since literally hundreds of antibiotics have been isolated as metabolic products of actinomy- cetes, one can only surmise the variety of metabolic reactions that led to their forma- tion. Whenever the chemical composition of a 114 given organism is discussed, whenever its mode of nutrition and growth characteristics are examined, and its biosynthetic reactions analyzed, it is essential to keep in mind that the conclusions reached hold true for a given environment and for a given set of nutri- tional conditions. Changing the environ- ment, as by raising or lowering the tempera- ture of growth, by modifying the conditions of aeration, or by changing the reaction, or changing the composition of the medium, as by introducing different nutrients and in different concentrations, will change the growth characteristics and metabolic pat- tern of the particular organism. Not the least important among these considerations is the recognition of the strain specificity of an organism, whereby certain reactions are limited not to a genus or even a species, but to a certain race or strain. The metabolism of an organism represents a special phase of its physiology. To com- prehend it, we must understand the food- stuffs necessary for the maintenance of its growth and activities; the manner of obtain- ing the required energy; the products formed asa result of such activities; and the various intermediary reactions through which the nutrients pass when they are used for cell synthesis. Normal metabolism of an_ or- ganism, when it grows under natural condi- tions similar to those it finds in a natural environment, is often differentiated from ab- normal metabolism, when the growth of an organism is made to deviate from the nat- ural path of life to which it has been accus- tomed. Such a deviation occurs in virtually all methods used for growing microorgan- isms on artificial media and under controlled conditions. It is only seldom that a microbe grows In nature in a pure culture. Once it has been isolated and made to grow in an artificial substrate, its metabolism may be considerably modified. Among the factors influencing the metab- olism of microorganisms, the following are THE ACTINOMYCETES, Vol. I most significant: 1. The nature of the energy sources 2. The nature and concentration of the nutrients used for cell synthesis, especially carbon and nitrogen compounds, mineral re- quirements, and the need for certain rare elements. 3. The need for specific growth-promoting substances or vitamins. 4. The particular oxygen tension of the medium. 5. Optimum temperature and reaction. 6. Influence of other organisms, with the resultant associative and antagonistic effects exerted by them and upon them. These factors influence the extent of growth of the microbial cell, its chemical composition, and the nature and concentra- tion of specific metabolic products produced. Metabolism of Actinomycetes Any comprehensive discussion of the met- abolic activities of a group of organisms must consider their utilization of various nu- trients, decomposition of these nutrients into simpler compounds, the various mecha- nisms of transformation of these nutrients, involving those concerned with both break- down and synthesis, formation of waste products, and a variety of other reactions involved in the life of living cells. In the very early studies on the growth of actinomycetes, it was found that these or- ganisms vary greatly in their nutrient re- quirements. Some were found able to con- sume simple elements and compounds; others required complex organic materials. Considerable adaptation to various nutri- ents also was observed. The amount of cell material synthesized depended on the avail- ability of the nutrients and on the effect of the accumulated products. Beijerinck first studied an organism he considered to be a bacillus (B. oligocarbo- philus), later found to be an actinomycete, that was capable of deriving its carbon and PHYSIOLOGY energy needs from some simple compounds present in the atmosphere. Beijerinck and van Delden found that the following ele- ments are essential: N, P, KX, and Mg. When a simple synthetic medium to which no car- bon compounds had been added was inocu- lated with a small quantity of soil and in- cubated at 23 to 25°C, there appeared ‘‘a thin, white or feebly rose-colored, very dry film, difficult to moisten.’ The growth of the film continued for months and resulted in the accumulation of considerable amounts of organic material. Either nitrate or am- monium salt could be used as a source of nitrogen. The carbon was derived from vol- atile carbon compounds of the atmosphere. Lantzsch, who identified this organism as an actinomycete, differentiated between the nutrition of two variants, one a filamentous form which assimilated CO; the other, a coccus-like or bacillary form, which assim- ilated aliphatic hydrocarbons. The organism was considered to be an air purifier (Kober). With the growing recognition of the im- portance of actinomycetes as producers of antibiotics and vitamins, extensive studies have been made of their metabolic processes. Waksman, Schatz, and Reilly (1946) found that the growth of S. griseus reaches a maximum in stationary cultures in 10 days and in submerged cultures in 3 to 5 days, followed by the lysis of the mycelium. Growth of the organism is accompanied by a gradual rise in the pH value of the culture and in the ammonia and amino nitrogen contents. The total nitrogen in the myce- lium tends to be higher during the active stages of growth. The production and ac- cumulation of streptomycin parallels the growth of the organism (Table 18). After maximum activity has been reached, there is a rapid drop, especially in submerged cul- tures. The production of streptomycin re- quires the presence in the medium of a complex organic substance, which either serves as the precursor of the streptomycin 115 O ORY WEIGHT MG PER 100 ML @ SUCCINATE ME. PERL «5 © prxio © LACTATE M.E.PERL«5 @ GLUCOSE MG PER IOML TIME, DAYS Figure 54. Metabolism of S. coelicolor (Repro- duced from: Cochrane, V. W. and Dimmick, I. J. Bacteriol. 58: 727, 1949). TABLE 18 Rate of growth and streptomycin production of 8. griseus in stationary cultures (Waksman, Schatz, and Reilly) Per 250-ml portion of medium. Incubation, Growth, inmyces mycin in beth in broth, 0 Ava) “hanes 45 OsGtN ern? 5° 226 BRS 5 0.437 40.3 8: \3e8 An6oe5 7 \) SOAS), 955.8%, 918 ga5.9) 7a 10 0.695 62.4 128 63.3 66.8 15 05640), 55:24 SAO, (-9256= ees 21 60500 87-6 a5. ene 70.8 molecule as a whole or of an important group in the molecule, or functions as a prosthetic group in the mechanism essential for the synthesis of the antibiotic. This substance was designated as “activity factor’’; it can be gradually synthesized by the organism. When it is provided in the medium in a pre- formed state, however, as in meat extract or in corn steep, the process of antibiotic syn- thesis is greatly facilitated. In a study of the metabolism of S. aureo- faciens in complex media containing sucrose and proteins, Biffi et al. found that myce- lium synthesis is rapid during the first 24 hours then slows down in the next 24 hours. During the first 12 hours, sugar consump- tion is negligible, while the NH; content of 116 THE ACTINOMYCETES, Vol. I TABLE 19 Metabolic changes characterizing the two phases during the submerged growth of S. griseus (Dulaney and Perlman) Phase I Phase II Streptomycin Slight production pH Gradual rise Mycelium Rapid growth Glucose Rapid utilization Soluble carbon Lactic acid Oxygen demand Soluble nitrogen Inorganic phosphorus Maximum Used extensively Gradual utilization Slow production and utilization Used at maximum rate Maximum rate of production Reaches maximum Gradual autolysis Small remaining amount exhausted Concentration reaches maximum and remains constant Slow utilization Decreases to minimum Concentration increases Released into medium the culture increases, pointing to the pref- erential utilization of the proteins as a source of energy. With the advance in growth, sugar utilization and ammonia consumption proceed at a rapid rate, parallel to the in- crease in dry weight of the organism. Dulaney and Perlman divided the meta- bolic processes of actinomycetes into two phases, crescense and senescence. In the first phase, there was uptake of soluble nitrogen, carbon, and phosphate into the mycelium; the oxygen demand was high and the utilization of glucose was rapid, but there was very little antibiotic production. In the second phase, mycelial weight de- clined, phosphate and nitrogen were ex- creted into the medium, oxygen demand fell, and streptomycin was produced. Some O DRY WEIGHT, MG PER 100 ML @ SUCCINATE, M.E.PER Lx5 © pHxi0 © LACTATE ,M.E. PER Lx5 @ GLUCOSE,MG PER IOML TIME’, DAYS Figure 55. Metabolism of S. griseus (Repro- produced from: Cochrane, V. W. and Dimmick, I. J. Bacteriol. 58: 727, 1949). lactate also formed during the early stages, but this disappeared rapidly (Table 19). Van Dyck and DeSomer also recognized two stages in the growth of a streptomyces GS. aureofaciens). The first stage is char- acterized by cell synthesis and nutrient up- take. The second stage is characterized by a slower increase in cell synthesis; protein and ribonucleic acid decrease, and desoxyribo- nucleic acid changes but slightly; the de- crease in nucleoprotein cannot be ascribed to lysis, since it is not accompanied by a de- crease in weight of mycelium or by an in- crease in the nitrogen in the medium. The course of metabolism of S. venezuelae has been studied by Gottheb and Legator (1953). The course of growth and the meta- bolic processes of an actinomycin-producing strain of S. chrysomallus were reported by Dietzel et al. (1950). Schmidt - Kastner found, for example, that the addition of DL- isoleucine and sarcosine resulted in certain new actinomycins differing in their peptide chains. The metabolic processes of neo- mycin-producing S. fradiae were examined by Giolitti and Lugh (1956). Numerous other metabolic processes of actinomycetes, notably of members of the genus Streptomyces, have been described. Sekizawa (1958), for example, found that a culture of Streptomyces produces ethoxy- ethene-1 ,2 dicarboamide, as represented by PHYSIOLOGY the following formula: Leavers 4 —NHz | | OO O CoH; The nutrition of the various organisms and the effects of certain specific environ- mental factors may be considered in fur- ther detail. Inoue (1958) reported a high level oxygen demand for S. griseus grown on soybean medium at 24 and 96 hours. With deficient aeration and in alkaline media, the sec- ondary high level oxygen demand increased; with an excess aeration the latter disap- peared. Streptomycin inhibited the oxygen uptake of young cells at above a certain concentration; this inhibiting action is some- what prevented by the addition of 10° M Mg and 10° M Mn. The carbon dioxide output of S. griseus gave a Maximum rate at 24 hours. The Qo: value and Qco», curve were lower with my- celium than with spore inoculation. The R.Q. value indicated minimum at 48 hours. Both young and old cells were inhibited by streptomycin, but the inhibitory action was influenced by the relative concentra- tions of streptomycin and an unknown fac- tor in the medium. The young cells were more sensitive than the old cells. There was believed to exist a difference in the char- acters (or the enzyme systems) of the young and old cells. The carbon dioxide output was inhibited by streptomycin much more than the oxygen uptake. Fermentation processes of soybean me- dium by two strains of S. griseus fell into four phases: (a) the amount of mycelium increases, glucose consumption is_ slight, oxygen demand is high, and streptomycin production is very low; (b) the amount of mycelium is almost constant or slightly in- creases, glucose consumption is high, strep- tomycin production increases, and oxygen demand decreases markedly; (¢) mycelium 117 increases again and reaches a maximum be- cause of a secondary germination or growth; (d) autolysis occurs, glucose is completely and streptomycin production reaches the maximum. In the casein me- dium, the fermentation process falls into 2 consumed, phases, and the secondary germination or growth is not observed. The Qo» values showed the same tendency both in soybean and casein media. In the latter, the presence or absence of metal salts did not influence the Qo» value, but it played an important role in streptomycin forma- tion. Carbon Nutrition Actinomycetes grow in nature on a wide variety of substrates. The nutrition of ac- tinomycetes can be considered on the basis of the various essential elements required, notably, carbon, nitrogen, and certain min- erals, as well as sources of these elements. These sources range from complex organic environments, such as drained peat bogs, high organic soils, and composts of straw or of stable manures, to fairly simple media, such as poor sandy soils and simple syn- thetic substrates. Actinomycetes are able to utilize a great variety of organic compounds as sources of energy. These compounds include organic acids, sugars, starches, hemicelluloses and cellulose, proteins, polypeptides and amino acids, nitrogenous bases, and numerous other substances. Some actinomycetes can also attack fats, hydrocarbons, benzene ring compounds, and, to a more limited de- gree, lignin, tannin, and rubber. There is considerable selectivity in the utilization of these substances by different kinds of actino- mycetes. Some of the nutrients, like glucose, maltose, dextrin, starch, glycerol, amino acids, and proteins, are consumed very readily; in fact, they are the best sources of sarbon. Sucrose, xylose, raffinose, and cer- tain other sugars, sugar alcohols, and sugar 118 acids are utilized less readily, but more readily by some actinomycetes than by others. Cellulose, chitin, sterols, and poly- uronides can be utilized as sources of energy and for cell synthesis by only certain or- ganisms. Each one of these reactions is of considerable biochemical interest. Some of them have been utilized for identification of specific organisms. The biochemical reac- tions involved are discussed in detail in Chapter 9. Salzmann (1901) found the following car- bon sources most suitable for the growth of actinomycetes (Streptothrix odorifera, prob- ably a streptomyces) : various carbohydrates and succinic, malic, tartaric, and citric acids. Unsuitable sources were formic, ace- TABLE 20 Carbon utilization by different actinomycetes (Lieske) Carb Streptomyces Nocardia Actino- Pa ae eae (No. 12) (No. 74) "Sin Glucose Jocbses Sle h =! Sucrose oe asic = Maltose JLdE Ea SL Lactose oe eet ow: Levulose Wide ee =: Dextrin Sled ALLL = Starch + x ae Inulin ae ak Sew ete a Glycogen +4 JE a etE SEY ee Cellulose = = os Ethyl alcohol = seats = Methyl] alcohol = see a Glycerol Jodie e Se Se Mannitol deco aoe ux Asparagine Gingieete +4 Le Tannin a at = Amygdalin = zs = Caffein = = = Potassium acetate “ oe = Sodium citrate - eee = Blood serum aft eee Ae Control = = wee * — = no growth; + = slight; ++ = mod- erate; +++ = good; ++++ = excellent growth. THE ACTINOMYCETES, Vol. I tic, propionic, butyric, lactic, benzoic, and oxalic acids. Miinter (1913) demonstrated that various carbohydrates, organic acids, and alcohols are readily utilized as carbon sources by a variety of aerobic long-hyphal organisms now known to belong to the genus Strepto- myces. Lieske (1921) compared the carbon uti- lization of representatives of three groups: (a) an aerobic, long-mycelial, sporulating form (No. 12), probably a streptomyces; (b) an aerobic, short-mycelial form (No. 74), probably a nocardia, and (¢) an anaerobic form isolated from human actinomycosis (Si), probably an actinomyces. One per cent urea was used as a nitrogen source (only the nocardia gave a trace of growth with urea as the only carbon source). The carbon sources were used in 2 per cent concentra- tion. The results are shown in Table 20. Lieske used liquid media in his studies. He fully recognized the fact that had other media and additional cultures been used, the results would no doubt have been dif- ferent, especially on a quantitative basis. In agar media, for example, starch utilization undoubtedly would have been different. In general, formic, oxalic, tartaric, ben- zoic, and hippuric acids are unfavorable car- bon sources for actinomycetes; under cer- tain conditions of nutrition, however, some of these can also be utilized by certain or- ganisms. Acetic, lactic, citric, propionic, pyruvic, succinic, and malic are good sources. Ethyl alcohol and ethylene glycol, as well as erythritol and dulcitol, are un- favorable nutrients. Glycerol and mannitol are, on the other hand, highly favorable sources. Starch and certain hemicelluloses, such as mannans, are excellent sources of energy and carbon for a large number of actinomycetes. Utilization of pentose and hexose phosphates has been studied by Cochrane and Hawley. The significance of carbon utilization, in PHYSIOLOGY the form of sugars, organic acids, and aleo- hols, for diagnostic purposes has been em- phasized by Krainsky and Waksman. Arab- inose is not assimilated by most species, sucrose is used by some, and cellulose by only a few (Waksman). Inulin is utilized readily by most species. Gottlieb and Pridham emphasized the selective utilization of some of these com- pounds in the species characterization of actinomycetes. They found that all species are able to utilize d-glucose, d-mannose, starch, dextrin, and glycerol, but not eryth- ritol, phenol, cresol, and the sodium salts of formic, oxalic, and tartaric acids. Certain compounds are utilized by some organisms and not by others. This is true particularly of rhamnose, raffinose, xylose, lactose, man- nose, dulcitol, inositol, and the sodium salts of acetic and succinic acids. Only certain carbohydrates favor the production of streptomycin by S. griseus. These include glucose, starch, and maltose. The addition of inorganic phosphate to S. griseus media results in an increased rate of glucose utilization; this is accompanied by almost complete suppression of streptomy- cin production. Pentoses were found to be poor carbon sources; glucose and mannose were best, especially when combined with proline. Maltose was the best of the disac- charides. The trisaccharides offered inferior nutrients. Inulin was inferior to starch and dextrin. Mannitol was a promising carbon source, but none of the organic acids proved suitable. Numerof et al. reported that, in a medium containing glucose, acetate, and glycine, S. griseus utilized only glucose for the syn- thesis of streptomycin, although the other two compounds also had to be present for efficient production of the antibiotic. All the four carbons of glycine and acetate could account for less than the equivalent of one of the carbon atoms in the streptomycin molecule. More than half of the acetate and 119 ce i=) nd o ~ » un Co oO So — uw Mg. glucose used/mg. mycelium =a oO Y o Lactic acid (mg./ml.) 10 | DS. 4 576 Time (days) Ficure 56. Utilization of glucose and produe- tion of lactic acid by S. griseus (Reproduced from: Hockenhull, D. J. D., et al. J. Gen. Microbiol. 10: 364, 1954). glycine carbon appeared as carbon dioxide. The incorporation of labeled carbon from acetate and glycine into streptomycin was thus highly inefficient, although it was still possible to demonstrate localization in the guanidine carbons of the molecule. Benedict et al. tested a large number of streptomycete cultures for their ability to utilize various sugars. A total of 147 strains of Streptomyces representing 75 species have been tested on 24 carbon compounds for their ability to initiate growth in the syn- thetic medium of Pridham and Gottlieb. Forty-one cultures were tested also on duleitol and on the sodium salts of two or- ganic acids. Of the carbohydrates studied L-sorbose was not attacked by any species of Streptomyces, and erythritol and dulcitol were found to be of limited value in these tests. Relatively poor growth was attained on melezitose, sorbitol, and esculin (Table 21). According to Stapp and Spicher, some 120 TABLE 21 Utilization of various carbohydrates and related compounds by different streptomyces (Benedict et al.) The basal medium of Pridham and Gottlieb was used. Utilization of carbon source Number of Source of carbon strains tested Positive Negative strains strains Erythritol 137 9 128 Adonitol 136 46 90 D-Sorbitol 148 31 117 Dulcitol 41 1 40 i-Inositol 147 71 76 D-Mannitol 146 107 39 D-Xylose 133 102 3 L-Arabinose 137 98 39 L-Sorbose 146 0 146 Melibiose 130 48 82 Melezitose 136 45 9] D-Fructose 140 111 29 L-Rhamnose 137 66 71 Trehalose 138 118 20 Maltose 145 141 4 Sucrose 144 38 106 Lactose 142 O4 48 Raffinose 1438 41 102 Inulin 146 36 110 Salicin 147 106 4] Esculin 145 54 91 Dextran 147 58 89 K-5-ketogluconate 144 32 112 Ja-2-ketogluconate 144 30 114 Na acetate 40 35 5 Na succinate 40 40 0 species of Streptomyces are able to grow in high concentrations of carbon sources, such as 80 per cent dextrin, 10 to 20 per cent glycerol, or 20 to 30 per cent glucose. Kurasawa classified the antibiotic-pro- ducing cultures, on the basis of their sugar utilization, into four groups: 1. Rhamnose- and raffinose-negative, 2. Rhamnose- and raffinose-positive, 3. Rhamnose-positive and raffinose-negative, 4. Rhamnose-negative and raffinose-positive. These were further subdivided on the basis of utilization of xylose, lactose, mannitol, and acetate. The THE ACTINOMYCETES, Vol. I streptomycin-producing cultures fell into group | and were able to utilize all the four compounds according to the secondary char- acterization. The streptothricin-producing organisms also fell into group 1, but they were only acetate-positive and xylose-, lac- tose-, and mannitol-negative. chlorampheni- col fell into group 3, and Producers of acti- nomycins into group 2. Burkholder ef al., found that viomycin- producing strains of S. floridae and S. cal- ifornicus utilized xylose, glucose, galactose, fructose, cellobiose, maltose, mannitol, and starch; they grew poorly on arabinose, rhamnose, lactose, sucrose, raffinose, ducitol, i-inositol, and salicin. The grisein-producing strains of S. griseus, but not the strepto- mycin-producing strains, grew well on arabi- nose and rhamnose (Table 23). MeClung (1954) made a detailed study of the utilization of a large number of carbon compounds by species of Nocardia. He found that carbon compounds having an alpha- glucoside linkage (maltose, starch, dextrin, trehalose) are used more often than those having a beta-glucoside linkage (cellulose, lactose). He came to the conclusion that no relationship exists between carbon com- pound utilization and the morphological groups. Since no two organisms used exactly the same carbon sources, the possibility of using carbon compound utilization as a means of species differentiation was strongly suggested. However, the carbon compounds used by six strains of NV. asteroides were not the same. This suggested that different iso- lates of the same organisms differ in their ability to use carbon compounds. A substance related to vitamin B, is ef- fective in stimulating the growth of JN. corallina (Reader, Peters et al., Lutz). Mar- tin and Batt have shown that this organism requires the addition of thiamine to synthetic media, especially for the utilization of am- monium ions. Various organic acids, such as malice and aUTPIANVAYOJAXO aYT[-ULAWIOUT}OB 10 AoUa}od Moy JO suTBIyg snuab -“OULOL yo -oLyphisa ~y ,OPBUTDONS-B Ny _9]V4O0B-BN snjo -asi1ib ei L0)09 anv) -1a09 "Sf -21909q “6 | 49 BUTOONS-B AT 49} B]IIB-BN | ,[oplsouy —[0F19[NG | [oor Sul[quiasad suIRiys -usydwe Z ON Aouajod MoT -10[TYO DIOIqIUR MON : | | snuab snuab snuab snuab snuab snuab 40]09 saqnd -OWOLYI -OWOLYI -OWOLYI -OWLOLYI -OWOLYI snjoaa -OWOLYI 217209 “§ ‘sg -0aN0 “Sy -oayd “¢ -0980L “SF -O9SOL "Sf -098OL *S -pyf sy -001j0 “5 | | i | 4,9) 8U _oyeu _O}BUTIONS-B NV -19ONS-BN -LOONS-BN 4.9}BY90B-B Ny | | _[Oytsouy _[oysouy _[oysouy ,[o}souy ,[oqsouy _jowuuRyy ,Jopyluueyy Joye yy ,Joprauryy [ou TNC | | | | | | ,asoulyey _osouyyey SOUR YY _asouuleyy SSOUWR YY ,SSOUUIBY YY | | T ON BLig}IBq VALBDU-WIBIZ UOdN VAL}OV Jou dTOIqIyUR ‘RLIDJOBG ISBJ-Plow OF OIYSTUOSRyUYy MON i = rs 7 a a — ae it | snuab snuab snuab snuab snoa1y} -OWOLYO -OWOLYI -OUWLOLYO -OWLOLYD -pjab -¥ snq]D “Ss snap “gy sng “¥ -O98OL “Sf -oayd “o iat “¢ nae ‘S | | 9) eu _ayeu a _OSOJORTBY) ,9SOoN]) -1lONS-BN -100NS-BN 4998 19IB-BN 98098] +[OFUUBIT _aso[Ay | _9}8499B-BN _980JIB'T _[oyrueyy 49SO[AX | OR JOIR-BN ,9S0JIR'T ,[opluuRyy _osoj Ay | 9} BIIOB-BNT a ,980J0B'T ,[oyruuryy _OSO[AY _OYRI}IO-BN | {OP RBIJLO-B NY | 4978 190B-B N 980} ORT _Jopuue yy _OSO[AX -uajood *¥ SUIBIYS sutonpoid- ur xu, daryg sn 9IDQAVYO “J mo | sis | snasiib “oy | 49YBJOIB-BN 49S0JOR'T 4 [opluuRyy 4OSO[AY osoulyed ‘_osouwmeyyy (BMESEINY) UOYDz17n LWbns fo sispq ay) UO sadfimojdaas burvonpoid-ayjorquup fo uornoyfissy),) TG WIAV | 121 TABLE 23 Utilization of carbon compounds by viomycin-producing streptomyces (Burkholder et al.) Carbon source floridae californicus | poaees Lees = fgee i dena | 7 fet Weta L-Arabinose 0 to ++ +to+ —-— 0 Oto + Sai ie eae L-Rhamnose 0 Oto+ 0 = 0 ++++ D-Lactose 0 to ++ Oto + 0 0 0 to ++-+ + to +++ D-Maltose OSSESRSE) SED SFSFS5S= 00 == SESRSPS5 SESPSESR SPSeS= UO) S=SeSF55 Sucrose Oto + Oto + 0 0 Oto + Oto + D-Raffinose 0 0 0 == 0 to + Oto + Inulin Oto + | Oto + 0 —- Oto + Oto + Starch =PSRSRSE SParse SPSESESE) SESESSSF) SRSPSE UO SSSeSese SRSESES= 7-Inositol 0 Oto + 0 + Oto + Oto + D-Mannitol | ++++) +4 to ++++)| +++4++4+) +++4++4+/] +++ to +4+4+4 aiaiainieiaiaia D-Sorbitol Oto + Oto + 0 = Oto + + TABLE 24 The utilization of carbon and nitrogen sources by 8. coelicolor (Cochrane and Conn) = Relative : ae Relative Carbon source* eats a are Nitrogen sourcet en poe Beer None 17 QO None 30 By D-Glucose 100 100 + L-Asparagine 0.50 100 100 D-Mannose 202 200 Glycine 0.29 83 36 D-Galactose 84 97 L-Leucine 1.00 76 36 D-Fructose 79 96 L-Tryptophan 0.78 98 82 D-Xylose 143 121 Urea 0.24 86 51 L-Sorbose 26 0 NaNO; 0.64 18 0 L-Arabinose 65 46 (NH;,)sHPO, 0.50 57 0 Starch 107 87 Ammonium acetate 0.58 18 0 Inulin 32 QO Peptone 1.00 146 118 Trehalose 90 38 Tryptone 1.00 91 106 Cellobiose 81 95 Casitone 1.00 116 100 Maltose 62 43 Pepticase 1.00 175 118 Lactose 105 64 Casamino acids 1.00 116 129 Sucrose 34 0 Sodium caseinate 1.00 72 53 Glycerol 135 170 ~—- Gelatin 1.00 106 29 Mannitol 82 88 Egg albumin 1.00 44 35 Dulcitol 20 0 Sorbitol 27 0 Acetic acid 33 33 Lactic acid 60 0 Fumarie acid 69 0 Suceinic acid 47 0 dl-Malie acid 60 0 Tartaric acid 20 0 Citric acid 24 0 Glueconie acid 82 25 * Basal medium (gm/1]): asparagine, 0.5; yeast extract, 0.5; KoHPO; , 0.5; MgSO,;-7H2O, 0.25; and minor elements. Carbon source 5 or 10 gm per liter. 7+ Dry weight and pigment intensity of glucose control taken as 100. t Basal medium (gm/1): glucose, 10.0; yeast extract, 0.5; K2HPO, , minor elements. § Dry weight and pigment intensity of asparagine control taken at 100. 122 0.5; MgSO,-7H:,O, 0.25; and PHYSIOLOGY citric, are excellent sources of carbon, as shown by Cochrane and Conn (Table 24). In an evaluation of salts of organic acids, Pridham, Hall, and Shekleton (1951) found that the sodium salt of acetic acid appears to be more promising than the sodium salt of succinic acid. They observed that little or no growth was attained in 36 species of Streptomyces on Na formate, Na oxalate, or Na tartrate, but that virtually all strains could utilize Na citrate. Nine strains out of 32 produced H.S in Kligler’s peptone-iron agar. Jagnow reported that even oxalic acid can 123 = aa Pe jn xr o 4 w 3 @ oe ray x PE a o ° = x 22 S ef3 x $s (3 2 =) «x!j2 w < Lat] vu 3 x = ee £ 12 24 36 48 TIME , HOURS Figure 57. Chemical changes during fermenta- tion of S. aureofaciens (Reproduced from: Biffi, G. et al. Appl. Microbiol. 2: 289, 1954). TABLE 25 Utilization of carbon sources by viridogrisein- and griseoviridin-producing and related streptomyces (Anderson et al., 1956) S. griseus S. lavendulae Carbon source S. fragilis S. griseoviridis (strep- (grisein) (streptomycin) | tothricin) None | Oto + | Oto + 0 to + 0 to + 0 to + L-Arabinose |+++ to ++++4 +4++4 to SP SRSP S| ++++ Oto + 0 L-Rhamnose | SRSESES= SeSRqR UG) S=4e5=4= a iealiealaceatg 0 0 to + D-Xylose SSSSS5 00 S555 S55 Sega id Seseqe0 yassss5 ub S=s5q54- SeS5S55= 0 to + Glucose | SRS5SES= ‘Gieeas WO) S=555=5= aR 4R S54 SRSR455F RP Sa SeS= D-Galactose pinta lasoitini arS= WO) SeSeSeS=) Seae 10 Ses=sese SRR SRSF ee eeieaate D-Levulose +--+ OnE S=S5555= | x i a + to ++ D-Mannose SRSES5=5 S=5= 110) S=5=5=5= SESPSESE | SPSP SES SPSSS59= D-Cellobiose SeS=S555 SESPSF OQ S=seSeS= qPSESR55 Searqre WO sesesesq|| qes-qqae D-Lactose 0 to ++ ++ to +++-+ 0 to +++ 0 to ++ | Oto + D-Maltose sip aitpacataeata RPSrSS5 SP Sear 45 SESRSRSe SESE S=45 Melibiose 0 to + 0 0 to + Oto + +++ Sucrose 0 to + | 0 0 to + 0 to + 0to + Trehalose SRS5 Se | SPSeS545 SeS5S55° aRSRSE AF == Melezitose 0 to + 0 0 to + 0 0 D-Raffinose 0 to + | 0 to + 0 0 0 Dextrin t++++ | Ht ee tte +4+++ Inulin 0 to + 0 to + Oto + 0 to + 0 Starch SRSRSRSq +++ to ++++ SESE SRS= | FESSSESq SESESESF Adonitol 0 to + 0 0 | + to ++ 0 Dulcitol 0 0 0 0 0 Glycerol ++ to ++++ +++ to ++++ +++ | fe /++++ i-Inositol 0to + Oto + 0 | 0 0 D-Mannitol | ++4++ SE SRSaS= +++-+ sRarsrar 0 D-Sorbitol 0 to + 0 to + 0 to + | 0 to + 0 to + Aesculin | 0 to + 0 to + 0 to + | 0 to + 0 Salicin /++ to +4+++ 0 to + 0 to + | +to+++ | + tod | | ee 124 Ss) zx = Be o = 8 Bi, Zz = < 46 \( w ants 1 we 2 20 = : > = g See Fi Glan - = = = oe te ~ a = Ee F x me G 12 24 36 48 60 TIME , HOURS Figure 58. Growth and chlortetracycline for- mation by S. aureofaciens (Reproduced from: Biffi, G. et al. Appl. Microbiol. 2: 289, 1954). be broken down by 25 per cent of all actino- mycete strains freshly isolated from soil. The presence of yeast extract and the use of am- monium sulfate in place of nitrate as a source of nitrogen greatly facilitate this re- action. Numerous other studies have been re- ported on the utilization of various carbo- hydrates and their derivatives by different actinomycetes, as exemplified in the work of Anderson et al. (Table 25). Mariat (1958) studied the utilization of 15 carbon compounds in synthetic media by various pathogenic strains of nocardia and streptomyces. Glucose was utilized by all strains and fructose by almost all. Paraffin was utilized by all strains of N. asteroides and N. brasiliensis. Mariat recorded the fol- lowing series for two species of nocardia and three streptomyces: N. asteroides: Glucose > fructose > glycerol > mannitol > the other carbon compounds which were practically not uti- lized. N. brasiliensis: Glycerol > glucose > fructose > galactose > mannitol > xy- lose > arabinose > saccharose > maltose > the other compounds which were practically not utilized. S. madurae: Glucose > glycerol > starch > xylose > mannitol > fructose > saccharose > galactose > maltose > Na THE ACTINOMYCETES, Vol. I acetate > lactose > Na citrate > the other compounds which were not utilized. S. pelletiertz: Glucose > fructose > Na acetate > the other compounds tested which were not utilized. S. somaliensis: Glucose > maltose > fructose > the other compounds tested which were not utilized. As sources of energy and carbon, proteins and their derivatives are frequently pre- ferred to carbohydrates by actinomycetes, especially species of Streptomyces. This is shown by the fact that when a protein or a peptone is present in the same medium with glucose or another available carbohydrate, an actinomycete may attack the protein first, not only as a source of nitrogen but also as a source of energy and carbon; consider- able waste nitrogen is thereby liberated in the form of ammonia. The favorable effect of glucose in increasing the growth of actino- mycetes in the presence of protein is due partly to the neutralizing effect on the am- monia produced from the peptone by the acid formed from the glucose. Tyrosine can be used by certain species of Streptomyces, with the formation of dark-pigmented com- pounds. Some of the amino acids, like leucine, are utilized by actinomycetes only in the presence of an available carbohydrate Urea ‘an serve as a source of nitrogen, but not of carbon. The metabolic changes involved in the utilization of polypeptides and amino acids as sources of carbon have been studied by Woodruff and Foster (Table 26). Decomposition of Cellulose, Chitin, and Agar Among the carbon sources for the nutri- tion of actinomycetes, cellulose occupies a unique place. The capacity of certain actino- mycetes to decompose cellulose is well estab- lished. The cellulose-plate method or a liquid medium containing the necessary inorganic salts, a source of nitrogen, and filter paper PHYSIOLOGY as a source of cellulose can be used to demonstrate this capacity. Krainsky found that certain pigmented cultures are par- ticularly active in decomposing cellulose. Black or red rings are formed on the paper; on agar plate, clear rings are produced by the colony, indicating cellulose decomposi- tion. When filter paper is placed in vessels con- taining a synthetic solution, with ammo- nium salt or nitrate as a source of nitrogen, and some calcium carbonate, and inoculated with various cultures, many of the cultures will be found growing on the paper above the surface of the medium. When the resid- ual cellulose is determined, a definite ratio will be found to exist between the cellulose decomposed and the nitrogen assimilated. Meyer isolated a strong cellulose-de- composing culture of an actinomycete that produced a green pigment and an earthy odor. It is difficult to tell from the descrip- tion whether this organism was a strepto- myces or a micromonospora. Jagnow reported a widespread capacity of soil actinomycetes to utilize chitin, both as a carbon and as a nitrogen source. None was able to utilize keratin, however. Jagnow found that about 50 per cent of all the freshly isolated cultures of streptomycetes, notably members of the S. albus, S. griseus, S. diastaticus, and S. antibioticus groups were able to attack chitin. Humm and Sheppard isolated from marine sources three actino- mycetes: S. marinus, N. flava, and N. atlantica. Hach of them was capable of di- gesting agar. Both nocardias produced or- ganic acids from carbohydrates more ac- tively than did the streptomyces. The latter utilized organic acids more readily than did either of the nocardias, and therefore its failure to produce an acid reaction in carbo- hydrate media may be ascribed tentatively to coincidental utilization of any organic acids formed during decomposition of the ‘arbohydrates. TABLE 26 Metabolic changes and efficiency of carbon utilization by 8S. lavendulae (Woodruff and Foster) Tryptone| Glycine Mycelium, dry weight, mg 101 106 Glucose consumed, mg 488 782 NH;-N liberated, mg | 4 22 Nitrogen compounds deaminated, | 92 | 162 mg | Lactie acid produced, mg | 126 58 Volatile acid as acetic, mg | ete | le Conversion of glucose to laetie | 25.8 7.5 acid, % | Conversion of glycine to acetic | 10.3 acid, % Iefficiency of carbon utilization, %| 24.8 | 14.3 Utilization of Unusual Carbon Compounds Many actinomycetes, notably nocardias, show a predilection for unusual types of ‘arbon compounds as sources of energy. This is true of phenols (Gray and Thornton, 1928), pyridine (von Horvath, 1943; Moore, 1949), pyrimidines (Lara, 1952), glycerides (Perlman and Langlykke), and_ steroids (Turfitt, 1947), chlorine-containing aro- matic compounds such as p-dichlorbenzene (Erikson, 1941) and chlorohemin (Jensen and Thofern), paraffins (Haag, 1927; Jensen, 1951-1934; Krassilnikov, 1938; Umbreit, Y= 4N MEDIUM ---4NP 12 24 36 48 TIME , HOURS Ficure 59. Effect of added KsHPO, on nucleic acid synthesis by S. aureofaciens (Reproduced from: Biffi, G. ef al. Appl. Microbiol. 2: 291, 1954). 126 1939; Erikson, 1949), and other long-chain carbon compounds (Webley and de Kock, 1952). This property is usually associated with oxidative metabolism, potential acid- fastness, production of red or orange pig- ments of the carotenoid type, and lack of diastatic and proteolytic enzymes. The saprophytic strains, with yellow, greenish, or no pigments (Jensen, 1931-1932; Krassil- nikov, 1938; von Plotho, 1948), seem to be devoid of acid-fastness and fail to utilize paraffin, but they are more fermentative and often show diastatic and proteolytic effect. Nitrogen Nutrition Proteins, peptones, and certain amino acids form the best sources of nitrogen for actinomycetes, followed by nitrates, am- monium salts, and urea. Actinomycetes are unable to fix nitrogen and have to depend, like the great majority of fungi and bacteria, upon fixed compounds of nitrogen for their cell synthesis. Miinter (1914) made one of the first de- tailed studies of nitrogen utilization by cer- tain actinomycetes, now recognized as streptomyces. Lieske (1921) used a 2 per cent glucose solution containing a small amount of MgSO, and K,HPO,, and 1 per TABLE 27 Nitrogen utilization by different actinomycetes (Lieske) Strep- = * Actino- Nitrogen source tomyces (eas myces | (No. 12) et (No. Si Potassium nitrate = op — Ammonium sulfate a he = Hippuric acid = = as Uric acid = = Urea 5 ie le = Caffein os a oi Asparagin aie = = Potassium rhodanate a = et Peptone Bee tl gs le one gs eae Blood serum Control +++ +++ - THE ACTINOMYCETES, Vol. I TABLE 28 Utilization of different amino acids by a strepto- myces as compared to that of a fungus (Waksman and Lomanitz) Growth, NH:3-N dry pro- basis, duced, mg mg Glycine Trichoderma 50 24.3 Glycine Streptomyces 59 30:5 Alanine Trichoderma 80 22.0 Alanine Streptomyces 126 39.2 Glutamic acid Trichoderma 218 29.1 Glutamic acid Streptomyces 169 28.4 cent of the various nitrogen sources. Incuba- tion took place at 37°C (Table 27). Lieske recognized that had other species and other conditions of growth been used, different results would no doubt have been obtained. Fedorov and Jlina (1956) have shown that nitrates and nitrites are excellent forms of nitrogen for various actinomycetes. They are reduced down to ammonia and _ as- similated for all syntheses. The reduced forms of nitrogen (ammonia and hydroxyl- amine) are also readily utilized, but in lower concentrations. Organic nitrogen sources (urea, amino acids, peptone) are utilized even more readily. Cell synthesis, however, is low, not exceeding 10 to 12 per cent, when the ratio of C:N is less than 20:1 in the organic substrate, free ammonia accumu- lates. The nitrogen content of the mycelium varies with the ratio of carbon to nitrogen in the substrate. The great majority of actinomycetes be- longing to the genus Streptomyces are able to liquefy gelatin and utilize casein. The nocardias, as a rule, are unable to do so. Many of the actinomycetes are able to co- agulate and later peptonize milk, though peptonization frequently occurs without previous coagulation. Blood serum is lique- fied by many streptomyces. Complex pro- teins, such as hoof meal and horn meal, can also be attacked by certain forms, such as S. fradiae. PHYSIOLOGY 127 A study of the comparative utilization of different amino acids by a streptomyces and a fungus has been made by Waksman and Lomanitz, as shown in Table 28. In some cases, the actinomycetes are even more ef- ficient than the fungi. The comparative de- composition of plant proteins by a strepto- myces and a fungus is shown in Table 29. The utilization of nitrogen sources by S. griseus, from the point of view of strepto- mycin production, has received considerable attention (Dulaney, 1948). The range of utilization of various nitro- gen compounds by certain nocardias and streptomyces grown in synthetic media were reported by Mariat (1958) as follows: N. asteroides: Asparagine > urea > casein hydrolyzate > POsH(NHa)2 > NO;3K > NO;sNH, > SO.(NH,)2 > NO2Na; the last compound was not utilized. N.. brasiliensis: Casein hydrolyzate > PO,H(NHs)2 > NO3:K > asparagine > urea > SO.(NHa)2 > NO3NH,s > NONa which was not utilized. S. madurae: POsH(NHs,)2 > urea > as- paragine > casein hydrolyzate > NO3K > NO3NH, > SOs(NHa)e > NO.Na which was not utilized. S. pelletieri: Urea = asparagine = casein hydrolyzate = PO,H(NH4,)2. The other compounds were not utilized. S. somaliensis: Casein hydrolyzate > asparagine. The other compounds were not utilized. Yagashita and Umezawa (1951) studied the nitrogen utilization of S. phaeochromo- genes, an organism that produces chloram- phenicol in natural media and in synthetic media containing glycerol, sodium nitrate, and different amino acids. They observed that alpha-aminobutyric acid, norvaline, leucine, phenylalanine, thyroxine, methio- nine, lysine, and tryptophan increased the production of the antibiotic over that given by the basal medium; while glycine, alanine, valine, isoleucine, serine, glutamic acid. TABLE 29 Decomposition of plant proteins by different microorganisms (Waksman and Starkey) Protein Cell Protein Glucose Organism decom- growth, posed, mg | mg. | a a : Bas Ps destin — Trichoderma 541 167 aa Trichoderma 375 359 _ Streptomyces | 162 46 } + Streptomyces | 348 | 187 | ‘ ~ ~ } yy he | hd ~ Ghiadin | — Trichoderma | 854 | 151 | + Trichoderma | 495 | 460 = Streptomyces 240 | 78 | cystine, and histidine had little effeet and in some instances gave less than the basal medium. The most effective was phenyl- alanine. Alpha-aminobutyrie acid, methio- nine, and serine, if added to a medium containing phenylalanine, increased the pro- duction of antibiotic; norvaline, leucine, lysine did not have this effect. Corum et al. (1954) showed that S. erythreus produced an antibiotic in synthetic media containing glycine and that the micro- organism synthesized alanine first, then valine, and later several amino acids and small peptides appeared. Sackmann (1956), studying a_ strepto- myces related to S. roseochromogenes, which produces an antibiotic on synthetic media composed of a basal medium containing several amino acids, asparagine, and urea, showed that aspartic acid, glutamic acid, glycine, alanine, asparagine, and urea gave good production, while gamma-amino-bu- tyric acid, leucine, isoleucine, methionine, and cystine gave very poor production of the antibiotic. Dulmage tested the utilization of 23 amino acids and other nitrogenous com- pounds by S. fradiae for growth and neo- mycin production. Best growth was obtained with gelatin and a easein digest, and with arginine, D-glutamice acid, L-glutamic acid. L-histidine, L-lysine, L-proline, and LACTIC ACID STREPTOLIN i) mg /ml NITROGEN HOURS Fiaure 60. Metabolic changes produced by a streptolin-forming streptomyces. Streptolin, units 10° ml; glucose, mg/ml; lactic acid, mg/ml (Repro- duced by special permission from: Rivett, R. W. and Peterson, W. H. J. Am. Chem. Soc. 69: 3007, 1947). DL-threonine; best production of neomycin, with alpha-alanine, L- and DL-aspartie acids, L- and D-glutamie acids, L-histidine, L-proline, DL-threonine, and N-Z amine. Some actinomycetes, notably nocardias, attack proteins to a rather limited degree. Casein may not be hydrolyzed. Even gelatin, which is readily used by the great majority of streptomyces, is attacked by only some nocardias, and frequently not very readily. In general, nocardias are unable to utilize xanthine, tyrosine, and certain other amino acids. The presence of glucose does not have the same depressing effect upon the decom- THE ACTINOMYCETES, Vol. I position of amino acids by actinomycetes, as shown by the amount of ammonia lib- erated, as it does upon fungi (Waksman and Lomanitz). A study has been made of the ratio of carbon to nitrogen consumption by S. lavendulae. This was found to depend upon conditions of growth, nature of organism, and age of culture. With sugar and tryptone in the medium, the ratios increased to about 300 per cent as growth advanced, resulting in greater oxidation of the carbohydrate as compared to the utilization of the nitrogen in tryptone for cell synthesis. This was true especially for submerged cultures: the abun- dance of available oxygen brought about a ereater oxidation of carbohydrate as com- pared to the tryptone consumed (Woodruff and Foster). Romano and Nickerson (1958) studied the utilization of amino acids as sole sources of ‘varbon and nitrogen by S. fradiae. Alanine, histidine, lysine, glutamic acid, proline, and arginine supported growth; aspartic acid, threonine, leucine, isoleucine, and methio- nine did not. A coenzyme I-linked glutamic dehydrogenase was found in a cell free ex- tract of the organism. It was suggested that this was the mechanism by which members of the glutamic acid series are utilized via the tricarboxylic acid cycle. Certain actinomycetes, when growing in a peptone medium without any carbon sources, are able to produce urea (Guitton- neau). Out of 477 cultures of streptomyces isolated by Stapp, 177 were able to use urea readily as a source of nitrogen. Some of the cultures used assources of nitrogen, xanthine, hypoxanthine, and adenine, in concentrations of 0.05 to 0.1 per cent. A large number also used uric acid, but not uracil, pyridine, imidazol, and pyrrhol. According to Moore (1949), certain species of Nocardia are able to utilize pyridine, aniline, nicotinic acid, and nitrobenzene as a source of nitrogen, and phenol plus am- PHYSIOLOGY monium ion as the sole source of carbon, nitrogen, and energy. Changes in reaction as a result of growth of various actinomycetes depend both on the organism and on the composition of the medium (Waksman and Joffe). Protein-rich media give an alkaline reaction even in the presence of sugars, largely because of am- monia accumulation. When actinomycetes are grown on media containing sugars and ammonium sulfate as a source of nitrogen, the media usually turn acid as a result of the consumption of the ammonia and the ac- cumulation of sulfate. Media containing sugars and sodium nitrate may first turn acid, then alkaline, as a result of the con- sumption of the nitrate and the accumula- tion of the sodium ion in the medium. The great majority of actinomycetes prefer a neutral or a slightly alkaline reaction for their growth. Very few prefer an acid réac- tion. Oxygen Consumption by Actinomycetes The oxygen uptake by S. lavendulae, with glucose and glycerol as sources of carbon, was found to be 60 and 45 per cent, respec- tively. The incomplete oxidation was due to assimilation of some of the products for cell synthesis and to the formation of incom- pletely oxidized products, such as lactic acid. Shaking and motion of cultures of actino- mycetes usually do not affect all forms alike. The effect of motion has been compared to that of temperature: in both cases, energy is brought from the outside to the living cells; this exerts a favorable action up to a certain limit; above that limit, the results may become unfavorable. Shaking the cul- ture brings about an exchange in the at- mospheric gases, the organism obtaining a continuously fresh supply of oxygen. This affects favorably its growth and activities. The type of growth produced by an actinomycete in submerged culture varies 129 STREPTOMYCIN Ag-/ ML MYCELIUM PER CULTURE MG/PER 40ML URS Sy oR OSes iS) SS Via 1¢ PROLINE 14 12 f NH,-N MG/IOML PA A 05) le $2703. 4. Sis 16. 7) 0 a tO! ii DAYS OF FERMENTATION Figure 61. Fermentation characteristics of S. griseus (Reproduced from: Woodruff, H. B. and Ruger, M. J. Bacteriol. 56: 316, 1948). greatly from stationary growth. In place of the ordinary mycelial mat of stationary growth, the growth in submerged culture is limited to flakes or to bead-like masses or pellets which may fill the whole container. The physiology of the organism may also be markedly affected, one type of antibiotic being produced, for example, in stationary cultures and another type in submerged. Pine and Howell studied 11 strains of the A. israeli and A. naeslundiit types. These strains were found to require CO» for ana- erobic growth. Some of them were found to be obligate anaerobes to microaerophiles, others were facultative anaerobes. Further studies of the respiration of Streptomyces species have been carried out by Cochrane et al., Kemp and Sayles and many others. 130 Autotrophy among Actinomycetes As pointed out previously (Chapter 3), Beijerinck and van Delden in 1903 isolated from the soil a nonmotile coccus and a rod- shaped organism that grew in pure mineral solution and produced, after 2 or 3 weeks, a snow-white, nonwettable surface pellicle. They suggested that the cultures grew at the expense of the traces of volatile organic compounds found in the laboratory air. Later, Beijerinck (1913, 1914) reported that the cultures could also utilize He in the at- mosphere. The two cultures were described as Actinobacillus (Bacillus) oligocarbophilus and as Actinobacillus (=Streptothrix Cohn) paulotrophus, a thread-forming organism. In 1922, Lantzsch cultivated, from a surface pellicle produced spontaneously on a quartz suspension in water, a culture similar to Beijerinck’s Bac. oligocarbophilus and found it to be closely related to the actinomycetes. He designated it as Actinomyces oligocarbo- philus. CO was used as a source of C. Kober (1929) obtained, from an enriched culture of algae heated for 5 minutes at 71° C, a pure culture of a white actinomycete which was related to the cultures of Bei- jerinck and Lantzsch. Krassilnikov (19388) placed the culture among the proactino- mycetes (Nocardia). 4 z a Ww a °o = = re) « ° 2 ® te) 24 48 72 96 THE ACTINOMYCETES, Vol. I Ware and Painter (1955) isolated from clear sewage, on a mineral medium with KCN as the only C, N, and energy source, a ‘“‘strongly autotrophic” actinomycete. Takamiya and Tubaki (1956) observed an actinomycete growing on a phosphate solution. The culture could be grown in a pure mineral solution. The culture grew chemo-autotrophically, utilizing the process of H» oxidation in order to assimilate CO» . It was named S. autotrophicus. Three other cultures of chemo-autotrophic actinomy- cetes were isolated. Hirsch demonstrated that a culture of Nocardia, N. petroleophila, grew slowly but steadily upon a_ purely mineral medium in contact with laboratory air. No growth took place without CO». By means of tracer technique, it was established that the CO, is assimilated and incorporated in the cell substance. As energy sources for the COs, assimilation, the organism uses volatile, aliphatic hydrocarbons with 9 to 14 carbon atoms. These are incompletely oxidized, with the uptake of oxygen and release of only small amounts of CO». Metabolism of Aerobic Actinomycetes Garner et al. (1950) have shown that high- streptomycin-yielding strains had a lower respiratory activity (slower use of glucose GROWTH 3 x LACTIC ACID MG. PER ML. GLYCEROL of LACTIC ACI a eeee x Oe oe Occcecccccnce 120 144 168 192 HOURS Fiacure 62. Utilization of glycerol and sodium lactate by S. venezuelae (Reproduced from: Gottlieb, D. and Legator, M. Mycologia 45: 512, 1953). PHYSIOLOGY and slower COs, evolution) and a higher cell dry weight in the initial stage of growth than did the low-yielding strains. High-yielding strains showed active production of volatile nitrogen compounds during the first day, followed by a decrease during the next few days when streptomycin production was at its highest, and then by a slow reappearance of the nitrogen compounds during autolysis. In low-streptomycin-yielding strains, release of nitrogen compounds during autolysis was more pronounced. Added phosphate reduced streptomycin yields. Added calcium in- creased the yield, probably because of the formation of insoluble phosphates. Typical fermentation diagrams were pre- pared by Perlman and Wagman. The pH curve for fermentations in glucose-contain- ing media was typical not only of S. griseus, but also of most species of Streptomyces. Addition of extra phosphate to the medium depressed streptomycin production and at the same time increased the rate of sugar consumption. Biffi et al. found that addition of small amounts of phosphate to the medium results in increasing consumption by the strepto- myces of sucrose and the accumulation of pyruvic acid. In the first stage of growth @ fe) N, MG PER ML. 3 fo) fe} Nn > > ° MYCELIUM MG. DRY WEIGHT TOTAL N AND NO a 2or O08 ° 24 131 there is an increase in desoxyribonucleic acid synthesis. There is also a delay in protein decrease on addition of phosphate; this is accompanied by a lower antibiotic (chlortetracyeline) yield. Cochrane and Peck (1952) have shown that whole cells of S. coelicolor oxidized some compounds of the tricarboxylie acid cycle. The cells failed, however, to metabolize citrate and a-ketoglutarate. Cell-free prep- arations oxidized glucose (in presence of adenosine triphosphate), citrate, a-keto- elutarate, succinate, fumarate, and malate; they also decarboxylated oxalacetate. Se- lected reactions or groups of reactions found to be catalyzed by cell-free extracts included the oxidation of citrate to a-ketoglutarate, the conversion of malate to pyruvate, and the condensation of malate and acetate (or pyruvate) to citrate. The effects of diphos- phopyridine nucleotide on malate and fumarate oxidation and of malonate on the oxidation of a-ketoglutarate were consistent with the operation of a tricarboxylic acid cycle. Metabolism of Anaerobic Actinomycetes Anaerobic actinomycetes show only rela- tively limited growth and biochemical ac- 120 168 192 HOURS Ficure 63. Changes in nitrogen-components of medium during growth of S. venezuelae (Reproduced from: Gottlieb, D. and Legator, M. Mycologia 45: 512, 1953). 132 tivity. According to Erikson, they do not attack egg or blood serum; they do not clot or hydrolyze milk; they seldom grow on gelatin; they have little or no hemolytic action on blood agar. Certain strains iso- lated from human infections have been found to show a slight degree of hemolysis on blood-agar plates at different times, but not consistently. They do not produce soluble pigments on protein media or in- soluble pigments in their cells. The growth of A. bovis on sugars is not accompanied by gas formation. Glucose is the most readily available source of energy; acid is formed. Maltose, lactose, and sucrose are also utilized by all strains. Positive or negative reactions with salicin and mannitol have been found of value in differentiating strains, such as human versus bovine. A. bovis was found by Rosebury to have a limited tolerance for oxygen, which varies, however, among strains. 9 a eee eee eee ee eee THE ACTLNOMYCETES, Vol. I The introduction of synthetic media for the growth of actinomyces (Howell and Pine) made possible the study of the ability of these organisms to produce lactic acid. According to Erikson and Porteous (1953), A. israeli has the capacity to convert as much as 30 to 60 per cent of the glucose utilized to lactic acid, under suitable condi- tions of growth. Like aerobic actinomycetes, A. bovis is killed by heating at 62 to 64° C for 3 to 10 minutes, but it apparently survives drying for a long time, particularly when kept at low temperatures. Lieske, however, re- ported that anaerobic forms are highly sensi- tive to drying, being unable to survive even for one day. Influence of Temperature The range of temperature within which many microbes are able to develop is com- paratively wide. Most microbes begin to Ya Lent facie ns a en es en on ee i Pa is : 10) 2 4 de wee © Meee eee eee eee eos vd —— NO GLUCOSE srereeee O19 “ —— 0.5% ” enone 1% " a“ 6 8 10 DAYS Figure 64. Effect of glucose concentration on pH (Reproduced from: Krassilnikov, N. A. 1950, p. 206). PHYSIOLOGY 13 90 ~ °o € ~ Da =a = so O > = Oo - a uJ a 30 2) oo DAYS Figure 65. Effect of glucose on streptomycin production; key same as in Fig. 64. (Reproduced from: N. A. Krassilnikov, 1950, p. 206). grow only above a certain temperature, al- though they may remain alive, without multiplication, at much lower temperatures. When the temperature reaches a point which is specific for each organism, growth begins. The rates of reproduction and of the meta- bolic reactions increase rapidly with a fur- ther rise in temperature up to a certain point, which is again specific for each or- ganism. A still further rise in temperature leads to a drop in the rate of growth, until finally a point is reached at which growth stops. According to Haines (1932), the ordinary saprophytic actinomycetes found in cold stores and in soil fall into two groups: 1. Those organisms that have their optimum temperature for growth at 37° C, their range of growth extending from 40 to 5° C, witha lower limit at just about 0° C. 2. Those that have a less sharply defined optimum tem- perature, growth being rapid at 20 to 30° C; corresponding to a lower optimum tempera- ture is also a lower minimum temperature, growth being slow but good at 0° C, with a minimum between 0 and —5° C. The con- clusion was reached that actinomycetes are probably of greater practical significance in modern trade practice in chilled meat, eggs, and possibly fruit than in well-frozen meat. The presence of active cultures is sufficient, without actual growth, to cause a ‘‘musty”’ taste in the stored product. Three points are thus established in the temperature range for every organism: (a) a minimum or lower limit of growth; (b) a maximum or upper limit; and (¢) an opti- mum at which growth is at its best. The optimum temperature may not be a sharp point, but may cover a comparatively wide 134 THE ACTINOMYCETES, Vol. I range. Bacteria pathogenic to man and to warm-blooded animals develop within a much narrower range of temperature than do saprophytic bacteria. M7. tuberculosis, for example, has its minimum at about 30° C, its maximum at 42° C, thus giving 12° C as a range of growth. On the other hand, many saprophytes have a range of growth of al- most 40° C. According to their temperature relations, microbes are usually divided into three groups: 1. Psychrophilic forms, with a minimum at 0°, an optimum at 15 to 20°, and a maxi- mum at 30° C. 2. Mesophilic types, with a minimum at 9 to 30° C, an optimum at 28 to 38°, and a maximum at 48 to 50° C. 3. Thermophilic organisms, with a mini- mum at 40 to 49°, an optimum at 50 to 65°, and a maximum at 60 to 75° C. The water-inhabiting organisms, particu- larly the marine types, and most lumines- cent bacteria comprise the first group. The pathogenic forms and most of the sapro- phytes belong to the second group. The third group includes certain bacteria which develop in hot springs and in soils of warm climates; many bacteria and actinomycetes are found in high-temperature composts. This classification is purely arbitrary, since there are no sharp lines of demarcation be- tween these groups. The above limits of temperature are not constant, and depend on the composition of the medium, concen- tration of nutrients, and other environmen- tal conditions. When the temperature is reduced below the minimum or raised above the maximum, growth of the organism stops. An injurious effect follows only a considerable change in temperature. This effect is more marked on the upward scale than on the downward scale. Microbes are much more resistant to temperatures below the minimum than to those above the maximum. Lowest possible temperatures are usually not sufficient to kill microbes. Even tem- peratures of liquid air were not sufficient to kill staphylococei and M. tuberculosis. How- ever, some bacteria are rapidly killed at 0°. Repeated freezing and thawing have a more deleterious effect than freezing alone; dif- ferent organisms vary in this respect. Bac- teria and spores in a dry state are preserved by liquid air. The mycelium and spores of certain fungi were not killed even by —110°, whereas the mycelium of other fungi was destroyed readily at low temperatures, the spores being more resistant. Most bacterial cells, except the thermo- philic forms, are killed at a temperature of 56° C for 1 hour; at 60° C, 10 minutes is sufficient, whereas at 80° C only 1 minute will kill these bacteria. The temperature and time necessary to kill bacterial cells vary with the kind of bacteria and with the composition of the medium; a longer period is required for bacteria in organic media than for suspensions of bacteria in water or in salt solution. The optimum temperature for growth of most of the actinomycetes usually falls be- tween 23 and 37° C. Certain actinomycetes are able to grow at temperatures lower than 20° C, whereas some prefer temperatures of 20° to 23° C. The more common forms are readily destroyed at the higher tempera- tures, the resistance of the spores being only slightly greater than that of the mycelium. When a culture is kept for 10 minutes at 70° C, not only the mycelium but also the spores lose their viability. The actinomycetes are able to withstand much higher degrees of dry heat than of moist heat. Knor1 (1933) found that ex- posure of soil to dry heat at 180 to 200° C may not be sufficient to destroy all actinomy- cetes. Some actinomycetes can be adapted to grow at higher temperatures. Lieske, for example, was able, after a few transfers, to grow his culture Nocardia 74 at 48° C, PHYSIOLOGY whereas the original upper temperature limit for this culture was 42° C. Further studies on the effect of tempera- ture and humidity upon the growth and death-rate of spores and mycelium of various streptomyces have been made by Jagnow (1957). Among the actinomycetes, a special group stands out in relation to temperature condi- tions. These are the thermophilic actino- mycetes that are capable of growing at temperatures of 50 to 65° C. These ganisms occur abundantly in manure com- posts, in heaps of hay, and in pasteurized cheese. Gilbert isolated several thermophilic forms from various soils and included them in one species, A. thermophilus. The opti- mum temperature for growth was 55°, with a maximum at 60° C. Most strains ceased to grow at 45°, although some could be adapted to grow on agar media at 37° and even lower temperatures. Gelatin was slowly liquefied. Miehe looked upon the thermophilic actino- mycetes as the characteristic organisms in- habiting the decomposing masses of plant material under high-temperature conditions. The actinomycete spores lost their vitality rapidly, especially on agar media, but sur- vived on hay particles. One organism, desig- nated as A. thermophilus Berestnew, grew well at 40 to 50° C, more slowly at 30°, and not at all at 25 and 60° C. The manner of spore formation of this organism suggests that it was a member of the J/icromono- spora group. Schititze reported the presence in decomposing clover hay of representatives of two types of thermophilic actinomycetes, one of which was designated as A. thermo- philus Beresthew and the other as A. monosporus Lehmann and Schiitze. The latter may also be considered a member of the Micromonospora type. Further studies on the germination of heat-resistant spores of J/. vulgaris led Erik- son (1955a) to conclude that heat activation Oor- 135 at 100°C for 1 minute enhanced the initial germination rate of the spores within the first 38-hour period. When the spores have been subjected to temperatures of 100°C for more than | hour, sectored colonies showing loss of aerial mycelium and impaired viabil- ity frequently developed. Erikson (1955b) further reported that a pathogenic partially acid-fast culture of N. sebivorans closely allied form were capable of with- standing exposure to 90°C for 10 minutes when dispersed in phosphate buffer suspen- sion. Subjection to heat treatment (90°C for 1 minute) of a blood culture belonging to the S. albus group increased the autolytic tendency of the culture and affected the type of sporophore produced during the first generation. Waksman, Gordon, and Hulpoi made a study of the occurrence of actinomycetes in high-temperature composts, as will be shown in Chapter 16. Waksman and Corke examined the classification of the thermo- philic actinomycetes and came to the con- clusion that these microbes represent two distinct groups of the Streptomyces and Thermoactinomyces types. ‘Temperature- growth relationships of J/7icromonospora have been studied in detail by Erikson and Webley. and a Effect of Drying Microbes vary greatly in their sensitivity to drying. Some are highly sensitive and ‘cannot resist drying more than a few min- utes or a few hours; others remain alive for many years. The composition of the me- dium is of great importance in this connec- tion; the presence of protein in the medium greatly increases the period of resistance to drying. Bacteria remain alive in normal air for a much shorter time than in dry air, because of the moisture content of the former. In dry air, many bacteria remain alive for years. Bacterial spores resist drying much more readily and for a longer period 136 than do vegetative cells. The mycelium of most fungi is readily destroyed on drying, while the spores are killed only after pro- longed drying, the period varying with the species. The greater resistance of the spores is due to their lower water content; the mycelium of a Penicillium was found to con- tain 87.6 per cent of water, and that of the spores 38.9 per cent. Actinomycetes are very abundant in dry soils and are, in general, markedly resistant to drying. Acosta kepta culture of an actino- mycete (A. invulnerabilis) alive ina fully dry state for 9 years. Berestneff inoculated a cul- ture of S. violaceus on sterile rye straw and allowed it to grow until sporulated. After being kept in a dry state for 10 years in the laboratory, it was still alive. Lieske prepared dry cultures (on sterile filter paper) of Streptomyces, Nocardia, and Actinomyces in a desiccator, over dry CaCl, and sulfuric acid. The cultures were alive after 18 months. Different organisms differ greatly, however, in their ability to survive drying under ordinary atmospheric condi- tions. Krassilnikov (1938) emphasized the re- markable ability of actinomycetes to survive, under most unfavorable conditions, for very long periods. Influence of Light Actinomycetes do not need light for their activities. In fact, strong light, especially when prolonged, has an injurious effect upon their development. The effect of light de- pends to a great degree upon the medium in which the organisms are grown; the cells are usually more resistant in milk than are those in bouillon. The resistance of the organisms to light is greater in a dried than in a moist condition. The injurious action of light in- creases with the intensity of the source of light. Sunlight acts only on the surface of solid media or in the air to which the or- ganisms are exposed. In liquid media, those organisms which are subjected to the great- THE ACTINOMYCETES, Vol. I est intensity and are only slightly protected, are destroyed. The red and orange rays of the light spectrum, as well as the infrared, or heat ‘ays, have no effect upon the growth and activities of microbes. Blue and violet, and especially the ultraviolet, are the most in- jurious rays of the spectrum. Various ex- planations of the mechanism of the micro- bicidal action of ultraviolet radiations have been offered. It is known that microbes ab- sorb the lethal rays and that the proteins have absorption bands between 2480 A and 2710 A. The effective radiation for steriliza- tion is in the region of wave lengths of 2800 A to 2500 A. The capsulated organisms are most susceptible; the sporulating organisms are most resistant. When a microbial culture is suddenly brought to light, the protoplasm contracts and a partial dehydration of the cell contents takes place under the influence of the rise in temperature. When the culture is returned to darkness, the reverse takes place. Ultra- violet radiation produces an enormous con- traction; the vacuoles are reduced and may even disappear, followed by plasmolysis. The antibacterial action of radiations, comprising both the long ultraviolet (> 3500 A) and short visible (<4900 A) rays, has recently attracted considerable attention from the point of view of destruction of un- desirable organisms and development of mutants from desirable ones. The region of 3500 to 4900 A is particularly effective. It was concluded that the extended nature of the killing curve suggests the production of some toxic substance, or the destruction of some essential compound in the cell, the effect of which, up to a certain limit, does not permanently destroy the ability of the cell to divide and develop further. Wave lengths shorter than 3000 A are most ef- ficient at 2650 A, close to the wave lengths at which nucleic acids act as most active absorbents. The phenomenon of photoreac- tivation greatly influences the killing effect PHYSIOLOGY of ultraviolet light (Kelner, Pittenger and McCoy). Action of Stimulants and Poisons Poisonous substances usually affeet micro- organisms in one of the following ways: 1. At certain low concentrations, which are definite for each substance, there is no ap- preciable action. 2. At somewhat higher con- centrations, these substances may exert a stimulating effect upon growth. 3. A further increase in concentration of the substance produces a bacteriostatic effect upon growth. 4. Still further increases have a microbicidal effect. There is a sharp line of demarcation between stages 2 and 3, but hardly any be- tween 3 and 4, because a concentration which has a bacteriostatic effect may be- come bactericidal on prolonged action. With regard to their antimicrobial effects, chemical substances are looked upon as in- different agents, as stimulants, or as poisons, depending entirely upon the activity of the particular substance. Microbial stimulants have been classified into three groups: (a) chemical stimulants, (b) effective nutrients, including oxygen, and (ec) effective metabolic products. When present in very small doses, toxic substances may act as stimulants; fluoride, for ex- ample, increases the production of zymase by yeasts. The chemical stimuli are often differentiated between those that affect the germination of spores and those that favor growth and reproduction. Kruse proposed the following rules for the toxic action of poisons upon microbes: 1. The bacteriostatic or bactericidal action of a poison increases with the concentration. 2. With an increase in the amount of inoculum, the toxic effect of the medium decreases or disappears. 3. The weaker the nutritive power of the medium, the greater is the toxicity; it is strongest in water and weakest in protein solutions. 4. The poison has a greater bactericidal action at higher tem- peratures. 137 The action of a poison and the resistance of the cell depend upon the rapidity with which the plasma membrane and penetrates into the cell. The fat-soluble substances are, there- the substance passes through fore, more poisonous. The strongest poisons are the metallic salts. AgNOs in a concentra- tion of 1:800,000 will kill most bacteria, and HgCl. is still more toxic; they are most soluble in ether, alcohol, and fats. Iodine and other substances, such as alcohols, chloroform, ether, and CS. behave in a similar manner. Copper salts are not soluble in lipoids, but tend to form complex organic compounds with the cells. Of the various microbial poisons, the fol- lowing groups are found to be particularly effective: salts of certain heavy metals, in- cluding silver, gold, mercury, and copper. The last two have found an extensive ap- plication as fungistatice and fungicidal agents. Iron has little antimicrobial activity, and lead, nickel, and zine are not active as poisons. Mineral acids, particularly those of the halogen group, have a powerful action. Of the soaps, only the salts of saturated fatty acids are poisonous. Potassium per- manganate and peroxides have a_ strong effect. effective. Ethyl alcohol is active only in certain dilutions with water. Of the aromatic compounds, phenol and its derivatives, especially the chlorinated and brominated compounds, are Formaldehyde is most important. A number of other organic compounds, as substituted ammonium salts, salicylanilide, dichlorodihydroxybenzomethane, and many others, have found extensive use as fungi- cides. The dyes may be added to the list of bactericidal agents. In recent years, a new type of antimi- crobial agent has gained universal recogni- tion. It comprises the antibiotics, or com- pounds of microbial origin. Their effect upon actinomycetes are discussed in detail in Chapters 14 and 15. CHAP TER 8 Mineral Metabolism and Effect of Salts on Growth Mineral elements play a highly important role in the growth of microorganisms. They function both as essential nutrients for cell synthesis and as regulatory mechanisms for various transformations that take place in the living systems. The composition of most of the synthetic media used for the growth of actinomycetes bears out this fact. Aside from the required sources of carbon and nitrogen, actinomycetes require phosphorus, sulfur, iron, potassium, magnesium, and cer- tain other inorganic elements. In some cases, as in the production of certain antibiotics, pigments, and vitamins, such elements as potassium, calcium, chlorine, manganese, co- balt, and zine play most interesting parts in the biochemical reactions involved. In most of the earlier studies on the effect of inorganic salts on the growth of actino- mycetes, complex organic media were used. Minter (1916), for example, used a medium containing blood protein, gelatin, and agar. He still observed that potassium and sodium salts, when used in 5 per cent concentrations, are favorable for growth but not for sporu- lation. He further noted that the addition of small amounts of Ca, Ba, and Sr were fa- vorable for growth and sporulation; higher concentrations were injurious. With the recognition of the important role of actinomycetes as producers of antibiotics and vitamins, there has been an ever grow- ing interest in the role of mineral elements in their nutrition. It was soon established that the great majority of actinomycetes, like other microorganisms, grow at rather low concentrations of salts. Some, however, are able to tolerate very high concentrations. Essential Nutrients Among the organisms studied most ex- tensively from the point of view of mineral requirements, S. griseus occupies a leading place. A chemically defined medium is more desirable than a complex organic medium for investigating metal requirements for nu- trition and for antibiotic production. In a study of the mineral requirements of S. griseus by Chesters and Rolinson, a chem- ically defined medium was used. It was made metal-deficient by treatment with chloro- form solutions of diphenylthiocarbazone at pH 7.3 to remove zine and copper and with 8-hydroxyquinoline at pH 5.2 to remove iron and at 7.3 to remove manganese. Media from which the metals were omitted singly were compared to media in which they were included. When zine was omitted, the me- dium supported the synthesis of only 75 mg of cell material per 100 ml of medium. Ad- dition of zine equivalent to 1 part per million of medium permitted maximum growth (550 meg/100 ml) and maximum streptomycin production. Further increases in the con- centrations of resulted in decreased antibiotic production, so that only 50 per ZINC 138 MINERAL METABOLISM AND EFFECT OF SALTS ON GROWTH cent of the maximum antibiotic production took place with 50 parts per million of zine. The addition of 0.05 part per million of cop- per resulted in optimum growth and anti- biotic production. Iron affected growth and formation of the antibiotic at different levels: optimum concentration for growth was 0.3 part per million, whereas optimum antibi- otic production required 1.0 to 2.0 parts of copper. No effect of manganese could be ob- served over a range of 0.005 to 50 parts per million. Thornberry and Anderson developed a synthetic medium for streptomycin produc- tion which contained, in addition to carbon and nitrogen sources, potassium, magne- sium, zine, iron, copper, and manganese. The effect of a number of metals was studied by adding the metals to, or omitting them from, this medium. Growth was estimated by com- parison with that obtained in a complex me- dium. The conclusion was reached that po- UNITS ARBITRARY PARTS 139 tassium, Magnesium, zinc, and iron were needed for good streptomycin production and supported excellent growth. Manganese stimulated antibiotic production but had no effect on growth. Calcium had no effect on either antibiotic production or growth. Temple found that magnesium and potas- sium exerted the most noticeable effect on both growth and antibiotic production. Tron had a lesser effect on growth but was re- quired for high streptomycin yields. A modi- fication of Thornberry’s medium to contain ammonium citrate and inositol required ad- ditional calcium. If tap water were used in place of distilled water, no additional cal- cium was needed. Saunders and Sylvester re- ported that traces of zinc, copper, iron, mag- hesium, and manganese were necessary for optimum streptomycin production. Principe and Thornberry found that the addition of cobalt in a concentration of 0.003 A in- creased streptomycin production by 83 per Blackening of medium opper eHect Surface growth ween gn iganese effect =. - eee, ‘Tee, cy Surface growth Copper effect PER MILLION OF METAL ADDED. Figure 66. Effect of copper and manganese on surface growth and pigment production by S. griseus (Reproduced from: Spilsbury, J. F. Brit. Mycol. Soc. Trans. 31: 215, 1948). 140 cent. Growth was not affected at concentra- tions lower than 0.009 V7 but was inhibited at that and higher concentrations. Chalupka (1957) found that of the various ions tested (K, Na, Mg, Ca, Fe, and Zn), only K (0.05 M) caused a significant in- crease in the mycelial mass of S. griseus and in protease formation. According to Saunders and Sylvester, Zn, Cu, Fe, Mg, and Mn were necessary for optimum streptomy- cin production. Metals had a beneficial effect on growth and streptomycin production also in com- plex media. Spilsbury used a peptone-meat extract medium. The ions of lead, tin, ura- nium, vanadium, cerium, strontium, chlorine, iodine, and fluorine had no appreciable ef- fect at any concentration employed. Slight mg. IN ORY WEIGHT THE ACTINOMYCETES, Vol. I stimulation of growth appeared to result from ions of bromine at 2 parts per million, zirconium at 10 parts per million, and all concentrations of molybdenum employed. Bismuth and lithium were slightly toxie at 2 parts per million; aluminium, cobalt, and nickel were toxic at 10 parts per million; cadmium proved to be extremely toxic at all concentrations employed. The metals most likely to prove worthy of investigation were considered to be copper, iron, manga- nese, zine, and molybdenum, together with the major salt constituents, notably sodium nitrate, potassium phosphate, and magne- sium sulfate. Tryptophan was shown to be essential in the early stages of development. It is of further interest to note that whereas copper and iron caused an increase in growth, MANGANESE SS \LCOPPER \ PARTS PER MILLION OF METAL ADDED, Figure 67. Effect of copper, manganese and molybdenum on dry weight of S. griseus (Reproduced from: Spilsbury, J. F. Brit. Mycol. Soc. Trans. 31: 217, 1948). MINERAL MEPABOLISM only copper yielded an increase in strepto- mycin production. Manganese and zine, however, caused a decrease in antibiotic for- mation (Table 30). Using a basal medium containing glucose in nutrient broth, Woodruff found increased streptomycin production in surface cultures of S. griseus to which sodium chloride, fer- rous sulfate, and zine sulfate were added. Maximum production was affected by the ratio of the supplements and the source of the water. Zine alone resulted in rapid pel- licle formation but decreased streptomycin production, whereas the addition of iron caused a decrease in growth but an increase in antibiotic production. Streptomycin production by S. bikiniensis was affected differently, as shown by Johns- tone and Waksman. The addition of ferrous sulfate had no effect, but the yield of the antibiotic was increased by the addition of zine sulfate to a medium containing meat extract, peptone, glucose, sodium chloride, and tap water. When both zine and iron were added at the same time, no noticeable effect was obtained. Changes in sodium chloride concentration also influenced an- tibiotic production, the concentration being | per cent. By adding most effective sodium chloride to a complex medium con- taining soybean meal, Rake and Donovick obtained increased streptomycin production by S. griseus. Eiser and McFarlane found that complex media with and without so- dium chloride supported the same amount of growth on a dry weight basis, with no difference in the residual glucose, ammonia, or amino nitrogen. In the absence of sodium chloride, streptomycin accumulated in the mycelium, whereas in its presence the strep- tomycin diffused more rapidly into the cul- ture medium. Since the cation and anion could be replaced by other closely related ions in the Hofmeister lyotropic series, still yielding similar results, the authors ascribed AND EFFECT OF SALTS ON GROWTH 141 TABLE 30 Effect of metals on the growth, streptomycin production, and pH of S. griseus (Spilsbury) 7 days 12 days Treatment Dry |Strepto Strepto- | wt, | mycin, | pH | mycin, | pH zm | weg/ml pg/ml Control 2.51] 125 |7.16! 100 |7.70 Copper, 50 ppm 4.64) 80 |7.41) 130 |8.00 Copper, 10 ppm 4.32 150 7.59 100 |8.10 Zine, 50 ppm (0.94) 7.24| 20 7.50 Zine, 10 ppm fen 65: 17.27) 15. \7.70 Manganese, 50 ppm 0.98, 115° 17-20) — 7.00 Manganese, 10 ppm ([2.77| 120 |7.06; — /7.60 Iron, 50 ppm 11.58] 115 |6.88] — 17.05 Iron, 10 ppm 2.29) 110 |7.02| 70 |7.70 Cu X Zn 2.84] 145 |7.06] 150 |8.10 Cu X Mn 3.71, 170 7.35 130 8.00 Cu X Fe 14.10] 130 |7.25| 200 |8.20 Mn X Zn 1.72) 80 |7.24, — |7.00 Zn X Fe P32) 40° 17:10; = 4 Mn X Fe 2.25] 55 |7.20| — {7.30 Cu < Zn xX He |4.26, 90 |7.19} — |8.15 Cu X Fe X Mn 4 06] 125 |7.46| 200 |8.25 Fe X Mn X Zn L 90) 45 7.16) 50 |7.70 3.59) 110 7.25, 110 |8.10 Cu Vine < 2m the results to the effects on membrane per- meability rather than to osmosis. The concentration of iron is an important factor. Rao reported a 3.6-fold increase in streptomycin production as a result of the addition of iron to the medium. Asai et al. studied growth and streptomycin production in a complex, iron-rich medium, and found that both were inhibited at iron concentra- tions greater than 0.015 per cent w/v. They attributed the inhibition to a colloid-chemi- ‘al phenomenon in which an iron gel covered the mycelium, with the result that the organ- ism was unable to take up nutrients or oxy- gen from the medium. The production of other antibiotics is also affected by the presence of metal ions. Le- chevalier found that no neomycin was pro- duced by S. fradiae in a medium containing peptone, beef extract, glucose, sodium chlo- ride, and distilled water. When tap water was 142 substituted neomycin was formed. The addi- tion of zinc, even as low as | part per mil- lion to media, resulted in antibiotic produc- tion even in distilled water media. Iron, manganese, copper, aluminum, calcium, and magnesium, had no such effect. Lechevalier was able to show a requirement for traces of potassium, magnesium, iron, and calcium for optimum neomycin production in media con- taining glutamic acid and glucose. Dulmage also reported that in a synthetic medium containing glutamic acid and glu- cose to which metal salts were added, it was necessary to have potassium, magnesium, iron, zine, and calcium for both growth and neomycin production. When glutamic acid was used as the only source of carbon and nitrogen and the medium treated with 8- hydroxyquinoline or ethylenediaminetetra- acetic acid to render it metal-deficient, a re- quirement for iron, calcium, magnesium, and zine for neomycin production by S. fradiae was reported by Mohan and Nickerson. These investigators were able to show a re- quirement for calcium and magnesium for growth, about a 50 per cent decrease in growth when iron was omitted, and no effect on growth when zine was omitted from the medium. Acker and Lechevalier made a study of some nutritional requirements of S. griseus for growth and ecandicidin production. A synthetic medium was used; it was made metal-deficient by calcium carbonate co- precipitation. Essential requirements for potassium, magnesium, iron, and zinc were demonstrated. No effect of manganese on either growth or antibiotic production was obtained. In a study of the role of iron for the pro- duction of grisein by S. griseus, Reynolds and Waksman demonstrated that as the concentrations of iron were increased, gris- ein activity increased logarithmically. This was due to the fact that iron is a part of the grisein molecule. When iron was added to a THE ACTINOMYCETES, Vol. I solution of grisein in concentrations of 4 gm/] as ferrous sulfate, partial inactivation of the antibiotic resulted. Complete inacti- vation of grisein occurred when 25 gm of ferrous sulfate were added per liter of gris- ein solution. Iron appeared thus to play a quantitative role. Zine had no effect on grisein production. Kelner and Morton reported that iron has a similar effect on the production of actin- orubin. In a tryptone-glucose medium sup- plemented with mineral salts, ferrous sulfate added in concentrations of 5 mg/] resulted in increased antibiotic production, whereas concentrations of 20 mg or more caused a decrease in yield. In various other studies the production of antimicrobial activity by three strains of streptomycetes was found to be influenced by manganese, zine, iron, and copper. Con- centrations lower than 2 mg/l] were ineffec- tive. Copper was inhibitory at a concentra- tion of 10 mg/l. A complex medium was used in these investigations. The addition of different concentrations of sea water to a glucose-peptone-yeast-extract medium resulted in an increase in soil iso- lates (Jann et al.). Two streptomyces cul- tures gave increased antibiotic production on addition of sodium, potassium, calcium, and magnesium as chlorides, sulfates, or nitrates. With a third culture all of the above metals, especially caletum caused an increase in production of antibiotic. In the case of a fourth culture, only calcium and sea salts gave an increase in antibiotic yield. The importance of cobalt in the produc- tion of vitamin By» is due to the presence of this metal in the vitamin molecule. The need for cobalt demonstrated before the structure of this vitamin was known. Hend- lin and Ruger found that cobalt became a limiting factor for vitamin production by S. griseus, even in a complex medium. Cobalt, added as Co(NOs3)2:6H2O, caused a 3-fold increase in vitamin By». production, compared Was MINERAL METABOLISM AND EFFECT OF SALTS ON GROWTH 143 60 ae om Ag € iS “7 a are 3 =) P) pa -4 < 7s Ma : i , RL WJ ‘va oo O : > 20 ; > va ,” A Mae) ? Or o Yipee Ss %% 2 4 6 8 10 DAYS Ficure 69. Effect on streptomyces mycelial growth produced by various soil extracts: 1. field soil. 2. compost soil, 3. field soil, ash of extract, 4. compost soil, ash of extract, 5. control (Reproduced from: Spicher, G. Zentr. Bakteriol., Abt. 2, 108: 580, 1955). MINERAL METABOLISM AND EFFECT OF SALTS ON GROWTH myces cultures was studied. The metals were then added singly and in all possible com- binations at concentrations of 3 parts per million. Growth was used as the criterion of effect, and the results were analyzed statis- tically. Iron and zine were beneficial for the growth of all eight cultures investigated. Calcium was beneficial for six of the eight; S. lavendulae and S. aureofaciens were un- affected. Manganese was effective for only S. coelicolor. An iron-zine interaction was beneficial in all cases. Calcium was found to retard the onset and rate of lysis of S. fradiae in concentra- tions as low as 1.5 parts per million. It also enhanced the utilization of glycine and glu- tamie acid in the medium. Mohan observed that increasing concentrations of calcium chloride in the medium enhanced the utiliza- tion of glutamic acid. Magnesium could not be replaced by manganese in the medium and its omission resulted in almost no growth of S. fradiae. The effect of copper on four streptomyces cultures was investigated by Heim; growth of S. fradiae decreased 55 per cent when copper was omitted, but there was no effect on S. griseus, S. rimosus, or S. lavendulae. Due to the specific effect of iron, Heim et al. analyzed for the presence of cytochromes. In a survey of 13 cultures five were found to contain only a b type cytochrome, and eight contained both b and c¢ types. It was sug- gested that the importance of the cyto- chromes as respiratory pigments and the presence of iron in the molecule may explain, at least in part, the importance of iron in the metabolism of the streptomyces. Potassium, Magnesium, zinc, iron, copper, and calcium are thus shown to be required for most, if not all, of the phases of the metabolism of actinomycetes. Certain met- als, such as cobalt for vitamin By. produc- tion and iron for the grisein molecule, are required for incorporation into specific mole- cules. A study of sodium requirement indi- 145 cated a special effect of this element on the diffusion of streptomycin into the culture medium of S. griseus. The presence of this element does not appear to be a general re- quirement for nutrition of actinomycetes. Manganese is not required generally for growth or antibiotic production; in some cases it was even slightly inhibitory. The inhibiting effect of aluminum ions was also reported. This would appear to be not a qualitative effect but rather a quantitative one, since Heim could not observe any in- hibition due to its presence in a medium treated with chromatographic alumina. The effective concentrations of various metals in the nutrition of most actinomy- cetes vary according to conditions under which the organism is grown or according to the specific effect required, such as anti- biotie or vitamin production. Metals which exert a beneficial effect at some concentra- tions may be inhibitory at higher concentra- tions. This is common with cobalt in vitamin By. production and iron in the formation of some of the antibiotics. In a study of the formation of chlortetra- eycline and bromtetracycline by different mutants of S. aureofaciens, some were found to be independent of the Cl concentration over a range of 0.02 to 10.0 stoichiometric equivalents, whereas the formation of chlor- tetracycline by other mutants depended on the Cl concentration over the above range. Gallicchio and Gottlieb (1958) studied the effects of the microelements Zn, Fe, Mn, Mo, Co, Cu, B, and Ga and the macroelement, Mg, on growth and chloramphenicol produc- tion by S. venezuelae. Elimination of Zn or Fe from the mixture added to a CaCQs- treated synthetic medium resulted in the suppression of chloramphenicol production. No one mineral in the synthetic medium supported production of the antibiotic, but the addition of Zn with Fe had a favorable effect. A larger concentration of Mg was re- quired for growth than for chloramphenicol 146 production. Mn could replace Mg in the growth requirement, but only a Mn con- centration of 10-°? M allowed any chloram- phenicol production. In a medium containing optimum concentrations of Zn, Fe, and Mg, the presence of the other microelements studied, singly or in groups, had little effect on growth or the production of the antibiotic. Metals are common contaminants of most of the media used for the growth of actino- mycetes. Complete removal of some metals, such as calcium, copper, and iron, is virtu- ally impossible. A positive reaction in any medium indicates, however, a qualitative effect of the particular metal on the particu- lar reaction. More refined experiments are required to explain the enzymatic basis for the reactions observed. The effect of minor elements on the growth and nutrition of actinomycetes has been studied further by Perlman (1949), and by numerous other investigators. Their effect on antibiotic production was examined by Sanchez-Marroquin and Arcimega. Effect of Salt Concentration As pointed out elsewhere, actinomycetes have been reported to occur in sea water and in sea bottoms. According to Kober, S. oldgo- carbophilus, grown in a 0.5 to 0.6 MW NaCl so- lution, produced fewer but larger colonies than in NaCl-free solutions. Although MegSO, is not essential for growth, it had a favorable effect even in fairly high concen- trations. Calcium was not essential for growth, but its absence had an injurious effect unless magnesium was present. Potas- sium, in concentrations of 0.5 per cent KCl, was injurious; this effect was neutralized by high concentrations of MgSO, . S. oligocarbo- philus is noticeably hallophilic and could tol- erate 25 percent MgSO,-7H2O, but only 5 per cent NaCl. At 15 per cent MgSO,:7H.O or at 3 per cent NaCl, the colonies grew well. THE ACTINOMYCETES, Vol. I Stapp has also shown that actinomycetes can tolerate a high salt concentration. Many strains were able to grow in a 10 per cent concentration of KNO; ; NaCl was tolerated by some strains in a maximum 8 per cent concentration, 6 per cent by others, and only 1 per cent by still others; some strains tolerated 30 per cent NaSO, and others only 2 per cent. Some strains were able to grow in a 10 per cent concentration of MgSO, , and one grew weakly even in a 50 per cent concentration of the salt. Sodium thiosulfate was tolerated in 10 per cent concentration. KI and KBr were tolerated in 5 per cent concentration; LiCl only up to 0.5 per cent by one strain and CsCl up to 0.1 per cent. SrCl, permitted the growth of some strains in | per cent concentration. Some strains are resistant to salts usually considered as toxic. In earlier studies on the growth of actino- mycetes, Neukirch (1902) observed that S. ochroleucus grew in broth containing 2 per cent NaCl. Lachner-Sandoval (1898) re- ported that good development of S. albzdo- flavus took place in a medium containing 16 per cent NaCl. The fact that actinomycetes are found in salt-rich substrates, such as curative muds (Rubentschik) and in the sea close to shore, speaks for their adaptation to salt-rich environments. Krassilnikov — re- ported on the ability of various actinomy- cetes (Streptomyces, Nocardia) to grow well in the presence of 10 per cent NaCl or 20 per cent NaSO,. He noted that the major- ity of actinomycetes are able to grow in higher salt concentrations than bacteria. This was true particularly of the pigmented organisms. These results tend to suggest that the role of metallic elements as integral parts of cell systems and of catalytic processes of en- zymes is of considerable importance. McEI- roy and Nason presented certain patterns which appear to be evolving from physico- MINERAL METABOLISM AND EFFECT OF SALTS ON GROWTH chemical and nutritional studies of metals in various metabolic reactions. Molybde- num, copper, and iron are said to be most closely associated with electron-transferring systems. These metals are not required spe- cifically for the combination of substrate to protein, but rather function as ‘electron couplers”? from one protein system to an- other. Magnesium and, to some degree, man- ganese function primarily in group transfer reactions, particularly those involving phos- phate. Manganese and, to a lesser degree, zine and magnesium, predominate in general enzymatic decarboxylation and hydrolysis reactions. Some metals form a stable metal-protein complex and in such cases are considered to be specific. In other cases, there are enzymes in which the metal may easily be separated from the protein and be replaced in function by a metal of equivalent valency. Metals may also play an important role as a structural part of a specific molecule. 147 Iixamples of this kind are the iron in the antibiotie grisein, cobalt in vitamin Bys , and iron in a streptomycete pigment known as ferroverdin. Outward manifestations of the effects of metals upon a microorganism can be ob- served as changes in growth, sporulation, and ability to utilize certain substrates as well as the formation of metabolic products such as pigments, antibiotics, and vitamins. The effect of metals on streptomycete nu- trition has been studied largely from the point of view of antibiotic production. It is common practice to use complex organic media to which are added mineral salt solu- tions containing the metals considered to be beneficial for growth and antibiotic forma- tion. The metals usually used are potassium, magnesium, iron, zinc, copper, calcium, and manganese. Streptomycin formation by S. griseus has been studied more than any other reaction in connection with the effects of metals in the actinomycete system. WESID-< A aad Gin A PTE Ryd Biochemical Activities The manifold aspects of the biochemistry of actinomycetes are still far from elucidated. Nevertheless, considerable progress has been made in recent years in our understanding of some of the chemical mechanisms involved in the growth and activities of these organ- isms. By far the greatest number of investi- gations on the activities of actinomycetes have been concerned largely with antibiotic production and the mode of action of anti- bioties. Limited consideration has also been given to certain other fundamental princi- ples of actinomycete biochemistry. Respiration Hockenhull et al. reported that, under highly aerobie conditions, glucose was con- verted by S. griseus mainly to cell material and CO,. Under restricted aeration, lactic acid was also formed. Pyruvic acid was pro- duced during the stages of most rapid growth. The metabolism of glucose by acti- nomycetes was dependent upon the presence of phosphate, the optimal hydrogen-ion con- centration for both glucose oxidation and the rate of disappearance of inorganic phos- phate being about pH 7. At pH 7.3, there was a gradual increase in glucose utilization from 0.62 mg/ml, in the absence of phos- phate, to 1.64 mg/ml, in presence of 0.01 AT potassium phosphate. Further increases in phosphate concentration did not affect the utilization of the sugar. Phosphate esters, tentatively identified as glucose 1-phosphate and glucose 6-phos- phate, were obtained by Hockenhull in fluo- ride-inhibited systems. Glucose oxidation was depressed by 10~* MW sodium iodoacetate and by 10°? M sodium arsenite, but was stimulated by 10° A sodium arsenate; 10-* M 2,4-dinitrophenol and 107? M so- dium azide had no effect. Streptomycin pro- duction was decreased by 3 X 107? M so- dium arsenate but not by 10-? AZ sodium fluoride or 10-? M/Z sodium iodoacetate. S. griseus metabolized members of the tricar- boxyvlie acid cycle, although citrate and a- ketoglutarate gave much lower values of CO, at pH 7.3 than did pyruvate, acetate, succinate, fumarate, or malate. Keto acids were produced, in presence of arsenite, from fumarate, malate, glucose, lactate, acetate, succinate, glutamate, and citrate in descend- ing order of yield. Except from fumarate, which yielded some material behaving like a-ketoglutarate, the product was chiefly pyruvate. According to Cochrane and Hawley, ri- bose 5-phosphate is oxidized by extracts of S. coelicolor almost to completion, in a series of reactions dependent on triphosphopyri- dine nucleotide, stimulated by thiamine pyrophosphate, and insensitive to iodoace- tate. Fructose 1,6-diphosphate is dephos- phorylated by one or more enzymes, with optimal activity at pH 4.5 to 5.0; it also can be oxidized by triphosphopyridine nucleo- tide-dependent reactions, involving glucose 6-phosphate as an intermediate. The con- clusion was reached that S. coelicolor can 148 BLOCHEMICAL ACTIVITIES metabolize glucose aerobically through either or both of two pathways, the ‘hexosemono- phosphate” and the Embden-Meyerhof system. According to Douglas and San Clemente, homogenized suspensions of S. scabies grown under submerged aerated conditions are able to oxidize glucose, succinate, fumarate, cit- rate, and acetate when placed in a Warburg- type manometric respirometer. The high endogenous respiration is due to oxidation of nitrogenous substances; the cells also oxidize glutamate, aspartate, threonine, phenylalanine, glycine, and valine and leu- cine to some extent. It was suggested that tyrosine is oxidized through dihydroxyphen- ylalanine with subsequent formation of melanin. Addition of dinitrophenol increases the oxygen uptake by cells in contact with pyruvate and a-ketoglutarate, and decreases the uptake in cells metabolizing glucose. As pointed out previously, anaerobic acti- nomyces cultures are able to fix CO. . This has been confirmed by Pine, using C'™, as brought out in Table 31. Succinie acid pro- duced by this organism had a high percent- age of its radioactivity in the methylene earbons: ‘‘When grown in the presence of oxygen, 90 per cent of the total activity of the succinic acid was found in the methylene carbons as compared to 62 per cent when it was grown anaerobically.” Butterworth ef al. (1955) have shown that a C;-C; condensation coupled with tricarbox- ylic acid cycle activity is a major pathway of carbon dioxide fixation by S. griseus. The intramolecular distribution of C1! in glutamic acid, produced by the incorporation of la- beled carbon dioxide, confirmed the theory that the tricarboxylic acid cycle operates extensively in the terminal respiration of S. griseus. Gilmour ef al. (1955) demonstrated the oxidation of tricarboxylic acid cycle inter- mediates by nonproliferating cells of S. griseus. Succinate and a-ketoglutarate were 149 oxidized only at substrate concentrations higher than those normally employed. The cells had to be preincubated in order to ob- tain oxidation of fumarate, malate, and citrate. Gilmour et al. have further shown that the labeling pattern suggests that glu- tamic acid arises from acetate through ex- tensive operation of the conventional triear- boxylic acid cycle. The role of this acid cycle in amino acid synthesis is established by the incorporation of radioactivity into most of the amino acids of the cellular pro- tein from proliferating cells utilizing acetate- 1-C14. According to Perlman, most of the studies on the metabolism of carbohydrates by S. griseus tended to indicate that carbon di- oxide is the main metabolic product; no accumulation of significant quantities of intermediates in this oxidation has been re- ported. Some doubt was expressed whether the Krebs tricarboxylie acid cycle operates in S. griseus metabolism of glucose (Garner and Koffler). The report of succinic acid production by other species suggested that at least some of the carbohydrate is metab- olized by these organisms by way of a ecar- boxylic acid eyele (Cochrane et al.). Lactic TABLE 31 Anaerobic fermentation of glucose plus carbon dioxide-C' by Streptomyces sp. (Pine) Substrate or product umoles/ml cpm/umole Glucose utilized 34.4 Carbon dioxide (initial) 33.4 523.4 Carbon dioxide (final) 14.1 492.8 Formic acid 12.6 50.6 Acetic acid Logs 36 —COOH ae —CH; 51S? Lactic acid 43.2 Succinie acid 11.0 520.2 —COOH 143.3 —CH> 120.3 Beta alanine (predicted) 383.9 (found) 377.0 150 acid has been reported in S. griseus and in certain S. lavendulae fermentations, and acetic acid in others. These acids have been looked upon as transitory products accumu- lating under fermentation conditions and inhibiting normal growth of the actinomy- cete. They are further metabolized by these organisms if the fermentation period is ex- THE ACTINOMYCETES, Vol. I S. fradiae cultures where the medium is poorly buffered, sufficient acetic acid accu- mulates to inhibit further growth and me- tabolism (Hubbard and Thornberry). The accumulated acetate is metabolized when the pH of the medium is raised. Wang et al. (1958) carried out time course experiments on the oxidation of C!4-labeled tended or if aeration is increased. In certain glucose and acetate by S. griseus. With 100 ORESTING CELLS “ 4 GROWING CELLS 80 O = = ra) fe) WwW rN = 60 ~ = 2 O° a oser ar aed i Za es OG fe) «40 3 Le (oe) ¥ 10) | 3 TIME 5 7 IN HOURS Ficure 70. Utilization of acetate-1-C™ by growing and resting cells of S. griseus (Reproduced from: Gilmour, C. M. et al. J. Bacteriol. 69: 721, 1955). BIOCHEMICAL ACTIVITIES young cells, considerably more glucose-C! activity was detected in cellular constituents than with older cells. Much larger quantities of fermentation products were found in the medium in the ease of the latter, however. With respiratory COs, , there was a decrease in the individual chemical recoveries with an increase in the age of the culture. The Embden-Meyerhof-Parnas glycolytic scheme was believed to be responsible for the major portion of glucose breakdown, only a minor part going via the phosphogluconate decar- boxylation route. The age of the cells may influence, however, the extent of utilization of either pathway but not alter the over-all pattern of glucose metabolism. Propionic acid has been reported as a metabolite in micromonospora fermentation (Hungate). Media containing a large num- ber of carbohydrates will support the growth of S. griseus; streptomycin production, how- ever, is low or absent unless glucose, starch, or maltose is present (Dulaney and Perl- man). Streptomycin containing C! has been obtained when S. griseus was grown on media containing carbohydrate labeled with Il LEGEND L] PROLINE Py sLucose fl MYceLium Ey carson DIOXIDE Bl stREPTOMYCIN Y | ° 2 15] CM, Addition of inorganic phosphate to S. griseus fermentations results in increased rates of glucose utilization. It results in an almost complete suppression of streptomycin production. Further studies on respiration of S. griseus have been made by Gottlieb and Anderson, Oginsky et al., and Inoue, whose work was reported in Chapter 7. Extensive studies on the respiration of various carbohydrates by various nocardias were made by McClung et al. (1958). War- burg manometric techniques and cell suspen- sions in which endogenous respiration was minimal (twice washed cells shaken over- night in pH 7.0 buffer) were used. The effi- ciency of carbon utilization was: glucose, fructose > sucrose, Mannose, galactose > maltose > sorbose > lactose > arabinose > cellobiose > rhamnose. When one of the less readily utilized sugars was replaced in part by glucose, the oxygen uptake was con- siderably higher than with either sugar alone, showing that the glucose stimulated the utilization of the other sugar. Growth pat- terns paralleled the respiration patterns. ete ee eI a | 5 ees lll ar = Iii a 5 6 7 DAYS OF FERMENTATION Ficgure 71. Carbon balance of S. griseus fermentation (Reproduced from: Woodruff, H. B. and Ruger, M. J. Bacteriol. 56: 318, 1948). 152 THE ACTINOMYCETES, Vol. I Oxidation of Steroids and Related Com- pounds It has long been recognized that actino- mycetes are capable of decomposing fats (Netchaieva). Until very recently, it was commonly assumed that the only actino- mycetes capable of oxidizing steroids are found largely among the species of Nocardia. Turfitt, for example, demonstrated by means of enrichment culture methods, that the breakdown of cholesterol and other steroids in soils is carried out primarily by nocardias, especially N. erythropolis. Among the other active species, he listed N. aquosa, N. glo- berula, N. coeliaca, and N., restricta. More recently, however, different strepto- myces (S. griseus, S. fradiae, S. venezuelae, and S. aureofaciens) were found (Sisler and Zobell, Perlman et al., Hirsch et al., Herzog et al.) capable of oxidizing steroids and lip- ids. Some species oxidized lipids completely to carbon dioxide, without the formation of any intermediate metabolites. Washed cells of S. albus are able to con- vert estradiol to estrone (Welsch and Hens- ghem); pregnenolone is converted to proges- terone by S. griseus, and progesterone to hydroxy-progesterone by various actinomy- eetes (Perlman et al.). The conversion of progesterone-(A*-pregnene-3 , 20-dione) to A!4-androstadiene-3 ,17-dione by Fried et al. suggested that the enzymes which carry out this transformation are adaptive in origin. The metabolism of progesterone by washed S. lavendulae cells grown in media supplemented with progesterone or in un- supplemented media was inhibited by azide, cyanide, or arsenite but not by selenite or fluoride (Perlman et al. 1953). The conver- sion of progesterone by cells grown in un- supplemented media was also inhibited by the addition to the progesterone-cell-suspen- sion of certain antibiotics, such as strepto- mycin. Addition of any of the antibiotics to the steroid-cell-suspension 12 or more hours after the steroid was mixed with the washed cells had no effect on the conversion. The antibiotics had no effect on the progesterone- oxidizing ability of washed cell suspensions of S. lavendulae grown in media supple- mented with progesterone. According to Vischer et al., submerged cultures of various streptomyces are able to convert cortexone to 16a-hydroxycortexone. Saburi et al. isolated several streptomyces which utilize cholic acid as the sole source of carbon. The utilization of these acids de- pends on the nuclear constitution and the length of the side chains of the bile acids. S. celaticus was found capable of converting cholic acid in a C-22 acid (C22H»20.1). When S. gelaticus was cultured in a synthetic me- dium containing cholic acid asthe sole source of carbon, it oxidized cholic acid to an acid with melting point 280 to 282° (decomposes), which was presumed to be 7a-hydroxy-3 , 12- dioxo-A*-bisnorcholenic acid. When it was grown in a medium containing cholic acid and glucose as the carbon sources, various intermediates such as 7a, 12a-dihydroxy-3- oxocholanic acid, 7a-hydroxy-3 , 12-dioxo- cholanic acid, 7a-hydroxy-3, 12-dioxo-A‘- cholenic acid were formed (Hayakawa et al.). Hayakawa et al. (1958) later demonstrated that S. rubescens possesses an alternate path- way for the degradation of cholic acid from that of S. gelaticus. According to Collingsworth et al., S. fradiae is able to convert 11-desoxy-17-hy- droxycortisone (comp. 8) to 17-hydroxy- corticosterone (comp. F). Webley and deKock studied the oxygen uptake by washed suspensions of Nocardia opaca. The uptake was increased by the presence of n-dodecane, n-tetradecane, n- hexadecane, -octadecane, and paraffin wax. Decyl, lauryl (dodecyl), and octadecyl alco- hols also gave increased oxygen uptake, but amyl, zsoamyl, zsohexyl, and heptyl alcohols were toxic. The long-chain fatty acids (C;— Cys) were all metabolized at very low con- centrations (0.0012 J). BIOCHEMICAL ACTIVITIES (1955), the mechanism of breakdown of w-phenyl-sub- stituted fatty acids by NV. follows: Acids with an odd number of carbon According to Webley et al. opaca Was as atoms in the side chain (phenylpropionic, phenylvaleric, and phenylheptylic were converted to benzoic acid, and cinnamic acids) acid was an intermediate; o-hydroxyphenyl- acetic acid was identified as a common prod- uct when acids with an even number of car- bon atoms (phenylacetic, phenylbutyrix, phenyleaproic, and phenyleaprylic) were used, thus supporting the theory of 8-oxidation as a mechanism of breakdown of short-chain fatty acids by N. opaca. Webley et al. (1957) later demonstrated that a strain of NV. opaca will convert 3- and 4-monochlorophenoxy- butyric acids to the corresponding substi- tuted acetic acids. During these conversions an intermediate is formed which proved to be 6-hydroxy-y-(4-chlorophenoxy) butyric acid. Adelson et al. found in soil a strain of S. griseus that had the capacity to bring about demyelination of bovine spinal cord in vitro. Just what mechanisms this process involves are still difficult to tell. Proteolytic Activities of Actinomycetes Actinomycetes are capable of attacking a large number of plant and animal proteins, as pointed out elsewhere. Macé was among the first to demonstrate their marked pro- teolytic properties. The proteins are hydro- lyzed to amino acids, polypeptides, and ammonia. Waksman and Starkey compared the utilization of proteins of animal and plant origin by bacteria, fungi, and actino- mycetes. The actinomycetes were found to occupy an intermediate position between the other two groups of organisms in the ratio of protein decomposed to protein synthe- sized. The presence of a carbohydrate was found to be of great importance in influenc- ing both the amount of protein decomposed and that of cell material synthesized. When 153 proteins were used as a source of both carbon and nitrogen, the reaction of the medium changed rapidly to alkaline. Considerable ammonia, liberated in the decomposition of the proteins, was lost by volatilization. A number of amino acids can be utilized by actinomycetes in synthetic media. This is true of proline, glutamic acid, arginine, aspartic acid, and histidine. The fact that actinomycetes can utilize ammonia and ni- trate as sources of nitrogen, and that amino acids are degraded to ammonia, suggests that the carbon residue of the amino acids may be more important in the actinomycete economy than are the nitrogen-containing groups. Problems of deamination and amino acid degradation by streptomycetes have been discussed by Gottlieb and Ciferri. Actinomycetes are characterized by in- tense activity in breaking down proteins to ammonia. This is true especially when a long period of incubation is employed. These or- ganisms are capable of allowing a large accu- mulation of ammonia even in the presence of available carbohydrates. When washed cell material of S. lavendulae was shaken with different amino acids at pH 6.8, deamination could be measured by ammonia formation. Arginine and histidine were acted upon most readily; B-alanine was deaminated only about one-third as readily as D-alanine; leucine, isoleucine, and certain other amino acids were deaminated not at all or only in mere traces. The formation of proteolytic enzymes are discussed in detail in Chapter 11. Decomposition of Keratins Actinomycetes possess the unique capacity to decompose keratins. This was shown first in connection with the ability of certain pathogenic forms to attack the skin and horny portions of feet (Acton and McGuire). When a soil is enriched with keratinized tis- sues, such as human hair and feathers, or- ganisms belonging to the genus Actinoplanes 154 oe Gg 60 LEGEND oO PROLINE AA MYCELIUM i Pla 30 4 OAYS OF FERMENTATION THE ACTINOMYCETES, Vol. I WS ES SCS Ficure 72. Nitrogen balance of S. griseus fermentation (Reproduced from: Woodruff, H. B. and Ruger, M. J. Bacteriol. 56: 319, 1948). TABLE 32 Ability of various species of actinomycetes to digest wool (Noval) Wool digested after various incubation Species periods, in per cent 6 7. a eS 430 days days days days S. fradiae 3739 10 20 70 90 S. fradiae 3535 0 10 50 90 S. fradiae 3572 0 O 20 90 S. fradiae 3719 0 O WO Go S. rimosus 0 @ @O 30 S. griseus 3475 QO O° Oe S. griseus 3464 0 OM OF st) S. griseus 3492 OPO NOP 30 S. roseochromogenes OP ORO RO S. aureus oY © © @ S. albus On Oe OF 220 N. polychromogenes Wy ) (0) 2X0) The mechanism of keratin destruction by these organisms has not been determined. Jensen (1930) added to moist soil, keratin prepared from horn meal and allowed it to will develop (Karling, Gaertner, Rothwell). decompose. The process of keratin decom- position was slow; it led to a steady accumu- lation of ammonia and nitrate in the soil. After 120 days, 35 to 40 per cent of its nitro- gen was transformed into nitrate. The addi- tion of keratin produced little or no increase in the number of bacterial colonies on agar platings, but markedly increased the number of actinomycete colonies. Two actinomycete strains were isolated and found capable of thriving on keratin in pure culture; they de- composed this substance with the formation of ammonia. One of the strains could be recognized as S. citreus. The other strain was not named, but corresponded closely to the description of Waksman’s Streptomyces 145. The presence of keratinolytic actinomycetes belonging to the genus Streptomyces in the soil has been demonstrated by various other investigators (Piechowska, Hirschmann ef al.). Detailed studies of the decomposition of hoof meal, horn meal, leathers, and similar materials were made by Noval and Nicker- son and Noval (Table 32). These investiga- tors succeeded in isolating an enzyme prep- aration (keratinase) from a culture of S. fradiae, as is shown in Chapter 11. BIOCHEMICAL ACTIVITIES Decomposition of Chitins Chitin-decomposing actinomycetes are widely distributed in nature. As many as 85 per cent of the S. alboflavus group, 84 per cent of the S. albus, 83 per cent of the S. rubrireticult, 82 per cent of the S. griseus, 75 per cent of S. scabies, and only a few (36 per cent) of the S. violaceus groups possess this capacity. Only a few types of chitin- decomposing actinomycetes have been found in forest soils. This may be due possibly to the lack of small arthropoda in such soils. The actinomycetes attacking chitins do so by means of certain enzymes, designated as chitinase. Berger and Reynolds studied these enzyme systems found in the culture filtrates of a streptomyces capable of hydrolyzing chitin. The following compounds were formed during the digestion process: N- acetylglucosamine, a 8-1 ,4-linked disaccha- ride, N,N’-diacetylchitobiose. At least two enzymes were found in the mixture: 1, a heat-labile fraction was able to split the disaccharide to glucosamine, but was unable to hydrolyze the chitin; 2, a heat-stable fraction contained a chitinase which hydro- lyzed the chitin to equimolar concentrations of the two fractions, but did not cleave the disaccharide; the system is specific for poly- mers of N-acetylglucosamine. The chitobiase hydrolyzed the 6-pheny! glucoside of N-ace- tylglucosamine but not the glucosides of glucosamine or glucose. Further information on chitinase is given in Chapter 11. Decomposition of Cellulose As pointed out previously, various actino- mycetes inhabiting soils, high-temperature composts, and sewage sludge are capable of attacking cellulose. Krainsky, Waksman, and Brussoff were among the first to demon- strate the capacity of various actinomycetes to carry out this process. Unfortunately, little is known of the enzymatic systems in- volved in the action of these organisms upon | or cellulose. The only products obtained are usually slimy materials and pigments that range from red and yellow to blue and black. Further information on cellulase is given in Chapter I1. In a study of cellulose decomposition by termites, Hungate isolated a culture of a micromonospora that decomposed cellulose under anaerobic conditions. The presence of complex organic substances in the medium is required. Among the products formed, acetic and propionic acids were identified, in addition to CO». An old culture of micro- monospora contained cellulase that verted the cellulose to glucose. Hungate isolated another strain of the anaerobic micromonospora from a culture of protozoa from the rumen of cattle. The organism is con- referred to as MW. propionici. Decomposition of Starches and Hemi- celluloses Starches are also decomposed by numerous actinomycetes. The problem of isolating di- astatic enzymes is much simpler, since it is very easy to study this process and isolate final products. Hemicelluloses and polyuronides, includ- ing mannans, galactans, glucans, xylans, as well as pectins, agar (Stanier), and others, are decomposed by a large number of actino- mycetes. Some of these actinomycetes are more active than fungi. Further information on amylase and cy- tase is given in Chapter 11. Decomposition of Rubber Sohngen and Fol (1914) reported that actinomycetes are capable of attacking rub- ber, bringing about a reduction in viscosity and transformation into COs. Several spe- cies, notably A. fuscus and A. elastica, were isolated. They were found capable of utiliz- ing various salts of organic acids, including stearate and palmitate, but not formate. 156 Kalininko came to the conclusion that the decomposition of rubber in nature is carried out by molds and, especially, by actinomy- eetes. Among the latter organisms, three forms were found to be particularly active, namely, S. coelicolor, S. aurantiacus, and S. longisporus ruber. Further studies on rubber decomposition have been made by Spence and van Niel. Decomposition of Paraffin Hydrocar- bons Biittner established that various aerobic actinomycetes that appear to include species of both Streptomyces and Nocardia are capa- ble of attacking paraffin. A film is formed upon the paraffin. Most of the lost paraffin is recovered as CO, . Oxidation of petroleum hydrocarbons by marine organisms was studied by Zobell et al. See also Chapter 7, for hydrocarbon oxidation by nocardias. Production of Odors Many actinomycetes, especially species of Streptomyces, are characterized by the pro- duction of a specifie odor, which is typical of freshly plowed soil. It is musty, or earthy, and occasionally fruity in nature. Rullmann believed that the odor is characteristic of certain species. According to Lieske, only those aerobic forms that produce chalky white aerial mycelium with round spores are ‘apable of forming this odor; the nonsporu- lating forms of the Nocardia type and those streptomyces that produce cylindrical spores do not give rise to any odor. The presence of carbohydrates in the medium favors odor production. The thermophilic actinomycetes are responsible for the more fruity scents, which arise particularly from young cultures. The odoriferous substance can be extracted from the culture. It is soluble in ether and partly in alcohol. Thaysen (1935) found that the odor is produced by certain actinomycetes only under certain conditions of growth and on THE ACTINOMYCETES, Vol. I certain media. Gelatin, for example, does not favor the formation of odoriferous sub- stances. . As is shown in Chapter 3, the odor pro- duced by actinomycetes is responsible for a certain type of spoilage of fish that absorb it into their digestive systems, from which it spreads throughout the bodies of the fish. Lime Precipitation Nadson isolated varicus actinomycetes from the bottom of a lake characterized by limestone precipitation. He mentioned 50 <5 | 370 <5 3480 0 0 | all 0 189 } + 10 <5 30 28 3481 0 0 73 0 174 4 50 <é 260 13 4 0 0 43 0) 201 ao 30 <5 160 <5 3475-2PR 0 >0.01 40 40 129 + > 50 16 | 370 75 3478 0 0 <5 0 —<5) + 0 <5 0 <5 3326a 0 -= — | 0 aS + — — >0.2 <5 Sundaes 0s) tO oes 0 30 niensis _ 3 30 7 33 * Each 60-ml flask of culture received at start 0.1 ml of M-1 phage, amounting to 7 X 107 par- ticles per milliliter of medium. than in water. It is also inactivated in acid media. It was suggested, therefore, that a phage-infected culture of an actinomycete be grown in an acid medium to free the culture from the phage. The action of phage takes place best in organic media; very little or no action is observed in synthetic media. Other antibiotic-producing streptomyces are also subject to attack by specific phages. This is true, for example, of the chlortetra- cycline-producing S. awreofaciens (Weindling and Karpos). 178 THE ACTINOMYCETES, Vol. I Figure 76. Phage particles adsorbed on the surface of a germinating streptomyces spore (Repro- duced from: Mach. F. Centr. Bakteriol. Parasitenk. Abt. II, 111: 555, 1958). TABLE 44 Phage multiplication in cultures of actinomycetes and its effect wpon the production of antibiotics (Reilly, Harris, and Waksman) Total incubation, days hase | Phage* added after hours | : 4 6 Organism of incubation — = — Phage per Phage per eel Phage per | percue ml X 10’ ml X 107 \S units/ml ml X 10% ii units/ml Se) 5 5S eS RR ee eee es | ae wa SEIOGUSCUST AS noe tart ate roar | Controlt 0) 0 | 66 0 90 MSL ATIO i ee SR ar Start 22 650 35 930 | 4S SGIUSCUSE Rte en 24 9500 7000 96 4600 135 WALUGUSCILS AP eee oer a 48 - 166 120 90 | 90 S. griseus 3478. ... Bes are Control | 0) ) ;o— | --- 14 S. griseus 3478. . eer oe Start 8 ee — | _- 15 S. bikiniensis. . ae. eee Control 0) 0 29 a 18 S. bikiniensis ain Nel eer th. Start 0.05 Ons 2A» «|| 0 30 SEE LAVEWIOILLGC. «she ee Control - 0 15 — | <0 S. lavendulae : Start - 8.8 <10 — <10 N. asteroides ; Control - — - 0 a N. asteroides... : : Start — — 9.4 = *7 X 107 phage particles added per ml of culture. +t No phage was added to control cultures. LYTIC MECHANISMS 179 Different phages are able to attack the same organism. Cultures of S. griseus made resistant to one type of phage may in time become sensitive to another. The formation of lysogenic strains containing a prophage was demonstrated by Welsch (1954). Welsch (1957) further demonstrated that several ac- tinophages can be found in a single natural substrate. They differ in the morphology of their plaques, their host-range, and their an- tigenic properties. A given actinophage may be present in its natural habitat in a concen- tration of 10° to 10* particles, ml. A search was made for truly lysogenic streptomyces. Thymol-sterilized culture flu- ids of various organisms were tested upon many indicator strains. About 15 per cent of the organisms freshly isolated from a natural substrate or taken from a collection of cul- tures actually carried a phage. The true lysog- eny of this phenomenon was demonstrated by the constancy of the ratio of phage to streptomyces in different cultures, and by the maintenance of phage production after re- peated single colony isolation or serial culti- vation in a medium containing a specific an- tiphage serum. The theoretical and practical significance of lysogenesis among actinomycetes has been discussed by Rautenstein (1957). The phe- nomenon of true lysogeny was found to be widely distributed among the actinomycetes. As many as 53 per cent of the S. olivaceus cultures examined possessed that property. Numerous other species proved to be lyso- genic, including S. diastaticus, S. cacaoi, S. candidus, S. griseus, S. antibioticus, and S. scabies. Many of the lysogenic actinomycetes eas- ily liberate the phage when grown as sub- merged cultures for 48 hours on a shaker. In a study of their mutual influence, the lyso- genic state of a series of actinomycetes was demonstrated by juxtaposing agar blocks bearing certain cultures on the superficial growth layer of others. Some of the lysogenic cultures contained various phages with dif- ferent lytic properties. An indicator culture was used to reveal the development of the phage of the lysogenic culture. It often influenced the lytic proper- ties of a given phage. This influence became particularly significant when the indicator culture was itself lysogenic. Phages which have the faculty to cause lysis of their own culture were often isolated from lysogenic ac- tinomycetes. This suggested that many acti- nophages are able to change their lytic property more or less easily. Some phage-resistant variants obtained experimentally differed from their initial cul- tures in their antibiotic properties. Many of these variants were found to be lysogenic. The possibility was suggested that acquisi- tion of new antibiotic properties may be con- nected with lysogenization. Rautenstein suggested that the broad dis- tribution of lysogeny among actinomycetes and the diverse character of changes caused by actinophages in corresponding sensitive cultures influence the variability and the evolution of the actinomycetes in an impor- tant manner. The distribution of lysogenic actinomycetes in nature has been studied further by Bradley. The isolation of actinophage from soil has been studied by Rautenstein and Kofanova (1957), Khavina and Rautenstein (1958), and others. S. olivaceus actinophage has been isolated from greenhouse soil; it proved spe- cific for cultures of S. olivaceus. It was sug- gested to use this actinophage for the identi- fication of cultures of this species. Out of 17 S. olivaceus cultures tested, 9, or 53 per cent, proved lysogenic. The actinophages isolated from these cultures proved identical to one another and somewhat different in their lytic properties from the phage isolated from the soil. Sveshnikova and Pariskaya made a de- tailed examination of the occurrence of actin- 180 THE ACTINOMYCETES, Vol. I FiGcurE 77. Phage particles adsorbed on a streptomyces spore (Reproduced from: Mach, F. Centr. Bakteriol. Parasitenk. Abt. II; 111: 556, 1958). ophage in greenhouse soils. In four out of fourteen samples of soil, free actinophages were revealed by direct count. In most cases these actinophages proved to be polyvalent. In another ten samples of greenhouse soils and in twenty-five samples of forest and meadow soils, as well as in filtrates of field soils, no free actinophage was found. Many actinomycetes isolated from these soils were susceptible to the eight different actino- phages previously isolated. Mach (1958) iso- lated from composts and forest litter three actinophages of which two were polyvalent and one strongly specific. These three phages were morphologically distinet from one an- other, as shown by the electron microscope. Bradley and Anderson (1958) isolated from soil three streptomyces phages and five nocardia phages. Of the former, one attacked a culture designated as NV. paraguayensis, and of the latter, three attacked streptomyces. All other Actinomycetales were resistant to both types of phage, as shown in Table 45. On the basis of these results, the authors con- cluded that the genera Nocardia and Strepto- myces are closely related and should not be separated into different families. Such gen- eralization is hardly justified. The N. para- quayensis used in these tests is not a no- cardia, but a streptomyces, as will be brought out in Vol. II. The nocardia phages that attacked the have been polyvalent phages, a potentiality streptomyces cultures may indicated in the early studies of Wieringa and Weibols (1936). The sensitivity to phages LYTIC MECHANISMS IS] as a criterion for species and varietal char- acterization of organisms will be discussed in detail in Vol. II. The inhibition of the phage by chemical agents was studied by Perlman et al. (1951). Gause et al. (1957) reported that actinomy- cetes possess the ability to produce antibi- otics which defend themselves against actinophage action. It was said that certain substances may defend actinomycetes against lysis by phages and simultaneously display a protective action toward other ac- tinomycetes. Among 1,000 cultures studied, about one-half displayed the ability to hinder actinophage activity, such ability being found both among cultures hindering bac- terial growth and among cultures lacking an- tibacterial action. Hemolysin Production Waksman (1919) demonstrated that the property of bringing about hemolysis of red blood cells is widespread among actinomy- cetes. This property was found to vary quantitatively for different organisms. A comparative study of the hemolytic proper- ties of certain pathogenic forms (Waksman, 1918) brought out the fact that hemolysis of blood in blood agar, liquefaction of blood serum, clotting and subsequent peptonization of milk, and liquefaction of gelatin, all run parallel. Lieske (1921) made a comprehensive study of hemolysin formation by actinomycetes ob- tained from various sources. There was no correlation between this property and the ability of the organisms to liquefy gelatin or to dissolve coagulated egg-albumin. The he- molytie action of actinomycetes was found to be an extracellular phenomenon, occurring during the very early growth of the organ- isms. An active preparation could be ob- tained by growing the organisms on blood agar plates, extracting the agar with salt so- TABLE 45 Effect of several actinophages upon different Actinomycetales (Bradley and Anderson) Lysis of a host by a standard phage suspension is indicated by a plus sign; no lysis is denoted by a minus sign. Actinophages Hosts -- —— : SP-3 | SP-4 | SP-8 |NP-3 |NP-4 |NP-5 S. griseus S34 —/+;/;—-;+]-]- S. griseus S104 +-)/+i)+/+]/+)+ S. griseus 1945 —-|/—-/|/+/+/4+]-—- S. olivaceus S11 + | +/+/4+]/4+]— S. venezuelae $13 +i+/}/+/]/+/]+/]4 S. cyaneus $45 } — | — Fe Geet eon a Nocardia sp. 3403 (eet Bee (hae ln oan N. paraguayensis* | + | +/+ ]+ prey a! N. madurae | =|] =] =) 4+ y= V. brasiliensis | — | a N. asteroides }— | — Actinoplanes sp. p= fe | es] Streptosporangium _ | —|/-—-/|];-/-]/-— sp. Mycobacterium phlei | — | — | Micromonospora sp. | — | — | * This culture is actually a streptomyces. lution, and filtering. Such extracts were cap- able of rapidly dissolving blood suspended in salt solution. The hemolytic activity of the extract was not destroyed by boiling; on the contrary, it Was increased by such treatment. It was lost, however, on heating at 120°C in the autoclave. In this respect, it was similar to the hemolysis of certain gram-negative bacteria. This phenomenon suggests that the active substance is not truly enzymatic in nature. No antihemolysin could be demon- strated in the normal blood or in a patient suffering from actinomycosis. Lieske reached the conclusion that the he- molysin effect was purely accidental and was caused by certain nonenzymatic normal met- abolic products of the organisms. He also emphasized the lack of correlation between hemolysin production of microorganisms and 182 THE ACTINOMYCETES, Vol. I their pathogenicity. Pathogenic organisms, including freshly isolated anaerobes and N. farcinica, were nonhemolytic, whereas many of the saprophytes were strongly hemolytic. The hemolytic property was changeable in nature; freshly isolated, nonhemolysing cul- tures can be made strongly hemolytic on re- peated transfer upon blood agar media. CH APE ER Production Actinomycetes produce a variety of extra- cellular and endocellular enzymes. Some of these enzymes have been isolated from the culture filtrates or the mycelium, concen- trated, and purified. Others have only been demonstrated in the mycelium of the organ- ism. Lysozyme The production of lysozyme systems by actinomycetes at first aroused considerable attention. These systems were confused, however, with autolytic and _ bacteriolytic mechanisms, on the one hand, and with anti- bioties, on the other. In his classical studies of the lytic agents of B. subtilis, Nicolle first suggested that bacteriolytic substances produced by micro- organisms might have properties in common with enzyme systems. According to Welsch (1947), the bacteriolytic system of certain actinomycetes is lysozyme-like in nature and is able to digest bacterial cell wall substrates. However, according to Ghuysen and Salton and Ghuysen, such enzyme systems, unlike lysozyme, are able to liberate amino acids but not reducing substances. Salton defined the enzymic properties of lysozyme on the basis of the following de- terminations: (a) turbidity reduction of iso- lated cell wall structures or lysis where the wall is 7m situ as with intact bacterial cells; (b) liberation of reducing groups; (c) libera- tion of an acetylamino sugar complex of glucosamine and the acidic hexosamine. Such Ld of Enzymes 183 enzymes as the actinomycetin complex pos- sess only the ability to dissolve isolated bac- terial cell walls; they are wrongly classified, therefore, with lysozyme. The ability of puri- fied enzymes isolated from the actinomycetin complex to form amino acids sets them aside from lysozyme, which forms reducing groups. The assertion of Kriss that actinomycetes produce “‘lysozyme”’ still requires confirma- tion of the ability of these organisms to form enzymes that liberate reducing groups and an amino sugar complex of the type released by lysozyme as well as the peptidase type, in accordance with the requirements laid down by Salton. Proteases Minter, Waksman, and Lieske first estab- lished that various actinomycetes, mostly members of the genus Streptomyces, possess strong proteolytic activities. Some cultures were able to decompose very energetically proteins in gelatin, egg-white, and blood serum. This is true of both saprophytic and greatly, in this and quantita- pathogenic types. They vary respect, both qualitatively tively, as can be simply demonstrated by the process of gelatin liquefaction or casein de- composition in ordinary plates. As a rule, species of Nocardia are poorly proteolytic, whereas certain species of Streptomyces are highly active in this respect. The degree and rapidity of proteolysis also vary with indi- vidual species. Stapp found that out of 477 freshly iso- 184 lated cultures of streptomyces, only one failed to liquefy gelatin. The liquefying ac- tion of the others was characterized by vary- ing degrees of rapidity. Many of the organ- isms produce a soluble brown pigment in gelatin, which, according to Beijerinck, is in the nature of a quinone that tends to harden the liquefied portion of the gelatin. The quantitative ability to secrete pro- teolytic enzymes can also be measured by the degree of gelatin liquefaction and of casein hydrolysis. Many species are also able to decompose complex vegetable and animal proteins. Culture filtrates of certain actino- mycetes were found to contain at least two proteolytic enzymes, one capable of digest- ing casein and the other of attacking the proteins of bacterial cells. According to Chaloupka, cultures of strep- tomyces cultivated under different condi- tions secrete more protease in an environ- ment with a low concentration of nitrogen than in media rich in nitrogen. A decrease in the concentration of sugars in the medium brings about a decrease in the secretion of the enzyme. Secretion of protease depends on the form of nitrogen, and is lowest in a protein medium, higher with lower peptides, and highest in media containing complex peptides and amino acids. Low secretion of the enzyme in protein media is accompanied by vigorous submerged sporulation of strep- tomycetes; high secretion is connected with lysis of the mycelium. Growth of the culture and enzyme production are greatly stimu- lated by potassium ions. The proteolytic enzymes of actinomycetes are more resistant to the effect of higher temperatures than are corresponding animal enzymes; the former enzymes are able to withstand heating at 70°C for 30 minutes though at 80°C they are destroyed. Lieske found that the resistance of the enzymes to temperatures is greater than that of the living cells of the organisms, which are killed at 62 to 65°C. Aceording to Krassilnikov THE ACTINOMYCETES, Vol. I (1938), many cultures are destroyed upon being heated at 40 to 45°C for a long time, but their proteolytic capacity is not affected. The proteolytic activities of the various species of actinomycetes are so marked that Waksman (1919) suggested the use of this property for diagnostic purposes. Lieske, however, stated that proteolysis is not a constant property and cannot be used for characterization of the organisms. Krassil- nikov (1938) tested 200 cultures every 8 to 12 months for 3 to 5 years. Various forms of gelatin were used for the test. The results were always identical. A strain that dissolved gelatin rapidly when first isolated continued to do so after 1, 2, 3, 4, and 5 years. Strains that failed to liquefy gelatin at first failed to do so after 2 to 5 years’ cultivation. The nonpigmented forms were most active. The pigmented forms were least active. Proteolysis may occur only at a late stage in the development of the organism. This may be due to the formation of endoen- zymes, Which are liberated on the death of the cells, as contrasted with the exoenzyme produced at an early stage of the develop- ment of the mycelium. The diagnostic prop- erties of proteolysis must, therefore, be based upon early observations during the stage of the rapid growth of the organisms. MceCon- nell (1950) and Dion (1950) studied the ex- tracellular proteases produced in submerged culture. Species of Nocardia, as a rule, possess much weaker proteolytic systems than do Streptomyces species. Some, like the patho- genic N. asteroides and the saprophytic N. ruber and N. viridis, do not liquefy gelatin at all. Some of the yellow species (NV. flava) are weak liquefiers. There are also reports in the literature that pigmented nocardias did not liquefy gelatin. The white (NV. alba) forms, however, are able to liquefy gelatin. No large scale production of proteolytic enzyme preparations has so far been ob- tained from actinomycetes. Sterile culture PRODUCTION OF ENZYMES filtrates of certain species were found to exert a marked effeet not only upon animal proteins but also upon proteins derived from soybeans, peanut meal, and corn meal. Ac- cording to Simon, S. griseus produced pro- tease in a medium containing 2 per cent soybean meal. An active enzyme preparation with a potency equal to that of pancreatin was obtained; the activity did not decrease on dialysis. Casein, soybean, protein, fibrin, and peptone could be used as substrates. The optimum reaction for the activity of the enzyme was pH 8.2. An aqueous solution of the enzyme was inactivated at 60°C in 3 minutes. Further studies on the production of proteolytic enzymes by various actino- mycetes have been made by Naeslund and Dernby (1923). The formation by different streptomycetes of proteolytic enzymes as well as of amylolytic and inverting systems has been discussed in detail by Jensen (Table 46). See also Tytell et al. (1954). Bechtereva et al. (1958) studied the course of accumulation of active proteolytic en- zymes by S. violaceus and S. lavendulae. The period of intensive accumulation of active proteolytic enzymes in a simple synthetic medium and in a corn-extract medium was found to be related to the decomposition of the cells. Upon submerged fermentation in media containing proteins, the release of active proteolytic enzymes may accompany not only decomposition of mature cells but also vigorous growth of the young healthy hyphae. The concentration of the nitroge- nous components in the medium greatly influences the rate of decomposition of the S. lavendulae mycelium and the accumula- tion of active proteolytic enzymes. Rennet or Lab Coagulation of milk by microorganisms can be brought about either through the action of the lactic acid formed from the lactose or by means of an enzyme, usually designated as lab or rennet. Since the ma- IS5 TABLE 46 Production of diastase, invertase, and protease by different actinomycetes (Jensen, 1930) Organism bedtolvats- (inven mele cae S. griseus, 5 strains 0) 0) +++ S. griseoflavus, 2 0 0 ++ 4 strains S. cellulosae, 5 strains 0) 0 +4 S. fulvissimus, 3 0 +4 +4 strains S. olivaceus, 5 strains | 0 0) jp ees S. violaceus-ruber, 3 ao 0 | +4 strains | S. diastatochromoge- | 0 to + | +++ + nus, 3 strains S. bobiliae, 2 strains + -f- +++ + S. halstedit (?), 2|/0to+ ele ee la strains S. aureus, 2 strains Lararar | 0 + S. pheochromogenus Wale 58 ) _ S. erythrochromogenus + — aa jority of actinomycetes do not form any lactic acid from lactose, the production of lab can easily be established. The addition of some CaCl, is favorable to the coagulation process. While the optimum temperature for growth of the organism may be 28 or 37°C, that for enzyme action is 55 to 65°C. Heat- ing to 70°C has no injurious effect, but ac- tivity is destroyed at 80°C for 30 minutes. Lieske obtained an active preparation of lab by precipitating a liquefied milk culture of the organism with alcohol. The lab enzyme is also produced by active cultures grown in other media, such as blood serum. The fact that certain actinomycetes were capable of clarifying the milk without pre- vious coagulation, whereas others brought about coagulation followed by varying de- grees of decomposition of the coagulum, sug- gests the possibility that lab is an enzyme distinct from the true proteolytic enzymes (Waksman, 1918). Keratinase Noval and Nickerson obtained, from a culture of S. fradiae, a highly potent prepara- 186 tion of keratinase that digested hoofmeal, wool, and feathers. Noval (1957) made a comprehensive study of this preparation. Three strains of S. fradiae were isolated and found capable of rapidly solubilizing 80 to 90 per cent of native keratin. One of these, S. fradiae 3739, was isolated as the most active keratin-digesting strain and was used for the preparation of the enzyme. Signif- icant stimulation in the digestion of wool by this culture was obtained by increasing the Cat+ and/or Mgt* concentration of the media. Approximately two-thirds of the cystine of the digested wool accumulated as soluble sulfhydryl compounds in the culture broth during the digestion. The sulfhydryl material was very stable to aeration, heating, and acidification but was substantially de- stroyed by addition of organic solvents to the acidified broth in the presence of light. Neither cysteine nor sulfide was detectable in the culture broth during or after the ac- tive digestion of wool. Most (75 per cent) of the nitrogen of the solubilized wool was ac- cumulated in the form of ammonia. The cell-free culture broth of S. fradiae 3739 was capable of enzymatically digesting keratins and casein. The enzymes that ‘-aused both of these digestions were similar in their optimal activity at about pH 9 and in their nonsensitivity to sulfhydryl rea- gents. Magnesium appeared to be the metal required for the digestion of wool by the culture broths. The culture broths of S. fradiae were cap- able of solubilizing a maximum of 10 to 20 per cent of several native keratinaceous sub- stances; trypsin and papain could solubilize, at the most, about half as much (5 to 10 per cent) of each of the same keratins. By ammonium sulfate precipitation, a product was obtained that had about 16 times as much wool-digesting activity per milligram of protein as did the culture broth. THE ACTINOMYCETES, Vol. I Urease Various actinomycetes, like S. griseus, were found (Simon) to produce urease. This enzyme was found also in cultures grown in urea-free media; hence it is not adaptive in nature. It was suggested that urea may be produced by the organism from guanidine by the action of guanidase. Deguanidase S. griseus was found by Roche et al. to produce a system of deguanidases that are active at pH 7.5 upon different monosubsti- tuted guanidines. This system comprises a mixture of enzymes different from arginase. Its diffusion in the medium can bring about the destruction of streptomycin. Chitinase Nearly all streptomyces are capable of producing an enzyme that has the capacity to hydrolyze chitin. Jeuniaux reported that this enzyme is formed in a simple synthetic medium containing chitin as the only source of carbon and nitrogen. The enzyme is also produced in the absence of chitin; the pres- ence of chitin in the medium was not es- sential for, although it favored, formation of the enzyme. The presence of glucose tended to repress the formation of chitinase. The enzyme was found to be rather unstable in culture filtrates, but the presence of chitin tended to stabilize it. Bucherer has shown that various species of Streptomyces, notably S. griseolus, S. ex- foliatus, S. fradiae, S. aureus, and S. griseus are able to break down chitin. According to Schmidt-Lange and Bucherer, both patho- genic and saprophytic actinomycetes are capable of producing the enzyme chitinase. Yamaguchi (1957) found that different species of streptomyces, such as S. fradzae, have the capacity to produce a powerful cuticle (of pig Ascaris) digestive substance PRODUCTION OF ENZYMES that could be precipitated from culture fil- trates and by ethyl alcohol, acetone, and other protein-precipitating agents. The en- zyme-like substance was distinct from the sasein-digesting agent. Amylases Numerous actinomycetes are able to hy- drolyze starch rapidly, either to the dextrin stage or directly to maltose and glucose. The production of amylolytic enzymes by actino- mycetes was first recorded by Fermi, who found most of the actinomycetes tested capable of producing such enzymes. These results were later confirmed by various investigators, including Caminiti. Some claimed that the starch is hydrolyzed only partially, not to the sugar stage. Krainsky made a detailed study of a large number of actinomycetes that were found sapable of producing amylase. This phe- nomenon was further studied extensively by Waksman and Lieske, who observed that only very few actinomycetes lack the ability to produce such enzymes. For the screening of a large number of cultures of actinomycetes, agar media con- taining starch as the source of carbon are used. The plates are streaked and allowed to incubate. After 5, 10, 15, and 20 days, the surface of the agar is covered with a solution of I-KI, and the amount of starch hydro- lyzed is measured by the width of the clear zone around the streak. Formation of zones 1.0 to 1.5 em wide after 10 days’ incubation is an index of good amylase production. For the production of amylolytic enzymes, inor- ganic sources of nitrogen, especially nitrates, appear to be preferable to organic com- pounds. Stapp recorded that 83 per cent of all ac- tinomycetes isolated from soil and belonging to the genus Streptomyces produced amylase. Although the amylolytic property is char- acteristic of the species, as noted by Krassil- 187 nikov, the wide distribution of this property tends to reduce its diagnostic value. A single alpha type amylase was found to be produced by five different streptomyces species (Simp- son and MeCoy, 1953). The mechanism of breakdown of starches by actinomycetes is discussed further by Bois and Savary (1945). The amylases of actinomycetes are able to withstand the effect of higher temperatures better than are the cells of the organisms producing them. Surovaya obtained a potent diastatic preparation from a culture of S. diastaticus. The organism was grown on a potato medium, and a satisfactory enzyme preparation, designated as ‘“‘superbiolase,”’ was obtained. This preparation was active at 70 to 100°C and had an optimum pH at 6.6 to 6.7. The starch was converted first into soluble form and then into dextrin. Saccharification of the dextrin proceeded much more slowly than the liquefaction of starch. The hydrolysis of mannosidostreptomycin to streptomycin, by various strains of S. griseus, 1s said to be due to an amylase (Christensen ef al., Langlykke and Perlman). Maruta and Tanaka isolated the enzyme mannosidostreptomycinase by precipitation of the culture broth with 1 to 2 per cent lead acetate. The optimum pH for the action of the enzyme was 6.8 to 7.4 and optimum temperature 37°C. Certain aldohexoses, such as glucose and mannose, inhibited the action of the enzyme; D-sorbitol produced an ac- celerative effect. In the regular streptomycin fermentation by S. griseus, the content of mannosidostreptomycin is high during the arly stages of fermentation and low in the older stages. The presence of glucose in the early stages represses the action of the en- zyme. Many actinomycetes are able to attack dextrins, glycogen, and inulin and to produce the corresponding enzymes. Lieske, who was one of the few to study these enzyme sys- 188 tems, reported that the anaerobic pathogenic forms, or species of Actinomyces, differ from the aerobic forms in that they form such enzymes in mere traces, if at all. No attempt has been made to study these enzyme sys- tems in detail or to utilize them for any practical purposes. Polysaccharidases Various actinomycetes are capable of uti- lizing agar and other polyuronides as sources of energy. The agar is thereby liquefied. This is true particularly of such forms as S. coelicolor (Stanier). A detailed study of the enzymes involved in the decomposition of seaweeds and sea- weed products (laminarin and alginates) by actinomycetes has been made by Chesters et al. (1956). Various nocardias (N. cztrea) have been found to be active in breaking down calcium alginate and laminarin. Cer- tain streptomycetes were particularly active in decomposing laminarin. In this respect, they were much more active than bacteria. The enzymes laminarinase and alginase were isolated from cultures of these organisms and found to be highly active upon the corresponding substrates, as well as upon starch and calcium pectate. These enzymes were obtained either from the culture fil- trates of the organisms or by treating the mycelium with 15 per cent ethyl alcohol. Sorensen (1957) found that both S. albus and M. chalcea, when grown in a xylan- containing medium, have the capacity to produce the enzyme xylanase. This enzyme is extracellular and can attack the xylan chain at random along its length, yielding a mixture of shorter or longer chain fragments. These saccharides, of which xylotriose is the shortest, are attacked further by xylanase, giving the following end products: xylose, arabinose, xylobiose, and uronic acid or uronic acid-xylose oligosaccharides. The xy- lanase produced by the streptomyces con- tained two fractions, of which one traveled THE ACTINOMYCETES, Vol. I toward the anode and the other remained at the starting point. The two fractions were found to represent two proteins with identical enzymatic functions. Invertase Invertase is widely produced by actino- mycetes, as shown by Caminiti, Krainsky, and Waksman. Lieske was unable to demon- strate the production of this enzyme by the cultures he investigated, but he did not deny such capacity. The formation of invertase and other saccharidases by actinomycetes has also been studied by Hofmann and Latzko (1950). The ability of some actinomycetes to uti- lize sucrose as a source of carbon is largely dependent upon the property of the organ- isms to produce invertase. This capacity has not been established for all organisms, how- ever. Krassilnikov says that nocardias are able to utilize sucrose without prior inver- sion. According to Waksman, only those forms that are able to produce invertase make abundant growth on media containing sucrose. In view of the constancy of this property, it has been suggested that invertase produc- tion be utilized for species differentiation. Cellulolytic Enzymes Although the property of decomposing cellulose is widely distributed among micro- organisms, neither the cellulolytic mechan- isms nor the cellulases involved in the proc- esses of decomposition are well understood. As shown previously, many actinomycetes, especially streptomyces, are able to grow on cellulose as the only source of carbon. This was established through the work of Fousek, Krainsky, Waksman, and others. Various actinomycetes capable of decom- posing cellulose have been described under different names, such as MJycococcus cytopha- gus of Bokor, Micrococcus cytophagus of Merker, and the organism described by PRODUCTION OF ENZYMES Krassilnikov as Proactinomyces cytophagus, all of which, especially the first, appear to belong to the nocardia group. There is no question that cellulolytie en- zymes are involved in these processes, al- though they have not yet been demonstrated with any degree of certainty. Lipase and Esterase Actinomycetes are found abundantly on fats, especially on butter. Jensen demon- strated in 1902 that certain chromogenic actinomycetes are able to grow in sterile butter and produce considerable amounts of acid. Lieske reported that actinomycetes, especially the aerobic forms producing long mycelium, are capable of attacking a variety of fats. The fatty acids produced are neutra- lized by the salts in the medium, giving rise to characteristic, mostly needle-shaped, crys- tals. To what extent hydrolytic mechanisms of the lipase and esterase type are involved is still uncertain. Some of these systems ap- pear to play an important part in the spoilage of various fats and cacao and in odor produc- tion. Oxidative Enzymes Actinomycetes possess a number of oxida- tive mechanisms, only a few of which are recognized at the present time (Sano, 1902). The production of phenol oxidases by certain actinomycetes has recently attracted con- siderable attention. Hockenhull et al. (1954) studied a-phenyl mannosidase production by S. griseus. After a suitable manometric method for the determination of this en- zyme had been worked out, a phenol oxidase of the laccase type was found by Sevcik in S. antibioticus. This lacease was an endo- enzyme with a maximum activity at pH 4.0 to 4.5. Hydroquinone was oxidized most apidly, catechol more slowly, and p-phen- ylenediamine very slowly. When growing S. antibioticus in submerged cultures on differ- ent media using a rotating shaker, Sevcik 189 found a direct relation between the produe- tion of the antibiotic actinomycin and that of phenol oxidases. On the basis of these results, he concluded that phenol oxidase of the laccase type participates most probably in the biosynthesis of actinomycin by S. antibioticus. Kiister the presence of phenol oxidases in the autolyzate of various cultures of streptomyces such as S. virido- chromogenes. These enzymes are capable of producing humic acid-like substances; this finding was believed to be evidence of the role of actinomycetes in humus formation. Hirsch and Wallace (1951) studied the octanoxidase system of S. aureofaciens, and Kempf and Sayles (1946) the oxidation- reduction potentials of S. griseus. Cochrane made a comprehensive examination of the enzymes involved in the utilization of car- bohydrate by S. coelicolor. The organism forms the enzymes phosphofructokinase, al- dolase, triose phosphate isomerase, triose phosphate dehydrogenase, phosphoglyceryl kinase, enolase, and ethanol dehydrogenase. Growth of S. coelicolor resembles yeast fer- mentation in its requirements for phosphate, demonstrated w (oe) Op UPTAKE, ul ine) [e) TIME 1/N MINUTES Figure 78. Oxidation processes by cell-free ex- tracts of N. corallina: 1. endogenous; 2, thymine; 3. uracil; 4. barbiturate. (Reproduced from: Lara, F. J.S. J. Bacteriol. 64, 281, 1952). 190 adenosine diphosphate, and diphosphopyri- dine nucleotide, as well as in its susceptibility to iodoacetate and fluoride in the breakdown of fructose 1 ,6-diphosphate. Intact cells or extracts were unable to ferment hexoses under normal conditions. It was suggested that obligate aerobiosis of S. coelicolor results from a biochemical le- sion, the inability to regenerate anaerobi- cally the diphosphopyridine nucleotide re- duced in the oxidation of triose phosphate. According to Sato, aerobic actinomycetes possess the respiratory pigments of the a, ), c, d, and dz cytochromes. On the other hand, the anaerobic actinomyces of the alkali type possesses a, b, d cytochromes, and those of the acid type possess no cytochrome at all. Birk et al. demonstrated the production of a b- type cytochrome by S. fradiae. In the presence of 2 ,6-dichloroindophenol, crude extracts of S. scabies were found by Douglas and San Clemente capable of cat- alyzing the dehydrogenation of succinate, citrate, malate, and glutamate. Transami- nase activity was also demonstrated, since a-ketoglutarate was converted to glutamate in the presence of aspartate, leucine, or valine as amino group donors. According to Inoue (1958), S. griseus pos- sesses the oxidase of the intermediates in the Krebs cycles. Glucose, pyruvate, acetate, oxalsuccinate, and lactate were oxidized readily, but the oxidation rate of citrate was very low in the given condition. Formate was not oxidized at all. Glucose oxidation was inhibited by monoiodoacetate and so- dium fluoride. Succinate oxidation was in- hibited by malonate, the latter being oxi- dized slightly. Citrate appeared to be formed as a result of an oxalacetate-acetate con- densation reaction, a reaction not inhibited by streptomycin. Inoue further reported that S. griseus possesses cytochromes a, b, and ¢; their wave lengths were found at 600 my, 567 my, and 550 my, respectively. Cyanide inhibited oxi- THE ACTINOMYCETES, Vol. I dation of acetate, malate, and lactate; glu- cose, pyruvate, oxalacetate, succinate, and oxalsuccinate were inhibited only slightly. Sodium azide oxalacetate slightly inhibited oxidation of succinate and lactate, but not glucose and pyruvate. It was suggested that the cytochrome system acts as an electron transfer system in S. griseus; the participa- tion of some other systems, such as that of flavoprotein, may also be considered. According to Musilek and Seveik (1958), the addition of sodium arsenite in a final concentration of 4 X 10~* WM to the medium of S. erythreus reduced biosynthesis of eryth- romycin by 87 per cent, with a simultaneous increase in pyruvic acid. Sodium acetate and sodium propionate in final concentrations of 0.5 per cent decreased the inhibitory effect of arsenite on erythromycin biosynthesis. Other salts of organic acids did not reduce the effect of arsenite. The latter completely inhibited oxidative decarboxylation of pyru- vate and oxidation of acetate by the washed mycelium of S. erythreus, but only partly inhibited glucose oxidation. Biosynthesis of erythromycin depends on uninterrupted oxi- dative decarboxylation of pyruvic acid to acetic acid. The authors suggested the prob- ability of the part played by acetic acid as the initial substrate in the biosynthesis of propionic acid, which is assumed to be the precursor of the lactone nucleus in the eryth- romycin molecule. Catalase The relation between the development and catalase activity of S. griseus was stud- ied by Kovacs and Matkovics. A close relation was found between streptomycin ratalase activity. A high ‘atalase activity was not necessarily a pre- production and requisite for streptomycin production, but was always present with high yields of strep- tomycin. With only little catalase activity, there was very little streptomycin produced. PRODUCTION OF ENZYMES Penicillinase The ability of actinomycetes to produce penicillinase, the enzyme capable of oxidizing penicillin, is widely distributed. Streptomy- cetes are in general resistant to penicillin; there is no relation, however, between peni- cillin-resistance and formation of penicilli- nase. This enzyme is heat-labile. It can be concentrated by precipitation with ammo- nium sulfate and acetone, a response that suggests its protein nature (Welsch). Tyrosinase The production of a brown pigment by actinomycetes grown on protein media has usually been associated with the ability of the organisms to form tyrosinase. According to Beijerinck, there is involved in the reac- tion the formation of a quinone, which turns brown at an alkaline reaction and in the presence of oxygen. The action of quinone in the presence of iron was found to be simi- lar to that of the enzyme tyrosinase. Since an excess of oxygen is required for the for- mation of the quinone, only limited amounts are found in deep cultures. The quinone is believed to be formed from the peptone in the medium; although good growth was pro- duced on media containing asparagine, KNO;, and ammonium sulfate as sources of nitrogen, only traces of quinone, if any, were found. The tyrosinase reaction is not involved in the production of all black pig- ments by actinomycetes; some species pro- duce such pigments in purely synthetic media in the complete absence of peptone. It has always been assumed that the po- tato scab organism is a chromogene, and that the formation of the black pigment is due to the tyrosinase reaction. Millard and Burr reported, however, that some of the plant pathogens did not give the tyrosinase reac- tion. Afanassiev could not confirm the patho- genicity of these cultures. Skinner concluded that the production of a dark color by the 191 chromogenic actinomycetes is due to tyro- sine metabolism. Gregory and Vaisey found that natural and x-ray-induced mutants of S. scabies were tyrosinase-deficient and did not produce a brown ring in skim milk. All tyrosinase- positive cultures produced the brown ring. The tyrosinase-deficient cultures were viru- lent for potatoes. This fact demonstrates that there is no connection between patho- genicity and the tyrosinase reaction. Steroid Oxidation The ability of various actinomycetes to oxidize steroid hormones has recently at- tracted considerable attention, as shown in Chapter 9. “Resting cells’’ of certain strepto- mycetes are able to transform steroids. Ac- cording to Turfitt (1944), various species of Nocardia are capable of attacking steroids, with the possible exception of halogen-sub- stituted derivatives. The oxidation of cho- lesterol results in the formation of a choles- terone, followed by molecular fission, the products of which may be utilized by the organisms for their further growth. Perlman et al. (1957) studied the enzymes TABLE 47 Aryl sulfatase activity of rapidly growing myco- bacteria, nocardias, streptomyces and corynebac- teria (Wayne, Juarez, and Nichols) Number Number Species of strains of strains tested positive Mycobacteria M. fortuitum 15 15 M. phlei 5 5) M. smegmatis 4 4 M. rhodochrous 10 0 Miscellaneous species 29 29 Nocardias N. asteroides 56 13 N. brasiliensis 4 1 N. madurae 6 l Miscellaneous species 33 6 Streptomyces 8 0 Corynebacteria 6 0 192 that oxidize progesterone. Schiesser studied cortisone-oxidizing enzymes. Harris et al. (1957) found that S. globisporus, S. virido- chromogenes, and other streptomycetes are ‘rapable of deacetylation and oxidation of dehydroepiandrosterone acetate. A detailed discussion of the literature on steroid oxidation by actinomycetes is found in the reviews of Eppstein et al. and Wett- stein (1955). Other Enzymes Numerous other enzymes and coenzymes have been found to be produced by actino- mycetes. It is sufficient to mention coen- zyme A (Gregory et al., 1952). The abil- ity of various nocardias (N. asteroides, N. pelletiert) to bring about the demyeliniza- tion of bovine spinal cord may be due to an enzyme system. Some of these mechanisms produced by Streptomyces and Nocardia species are thermostable and others are thermolabile (Adelson et al.). Anaerobic ac- tinomycetes are able to produce acid phos- phatase (Howell and Fitzgerald, 1953). Wayne et al. developed aryl] sulfatase tests for differentiating saprophytic mycobacteria from the tuberculosis organisms. This study was of particular interest in differentiating atypical mycobacteria and nocardias. Only 21 per cent of all nocardias tested and none of the streptomycetes or corynebacteria produced the enzyme (Table 47). Almost all mycobacteria hydrolyzed demonstrable amounts of phenolphthalein disulfate if suffi- cient inoculum was used and permeability differences compensated for. The interesting conclusion was reached that if J7. rhodo- chrous is a true mycobacterium, it is the only member tested which does not produce aryl sulfatase. THE ACTINOMYCETES, Vol. I The production of enzymes of the isoci- tritase pathway was demonstrated by Bardi et al. (1958) for N. rugosa. Lara (1952) reported that enzyme prepa- rations of the N. corallina group can be ob- tained by extracting alumina ground cells. The activity of these extracts against thy- mine and uracil was demonstrated only when methylene blue was added. These com- pounds were oxidized to substances having the over-all composition of 5-methyl bar- bituric acid and barbiturie acid, respectively. In one experiment uracil was formed from thymine; the results of many other experi- ments, however, indicated that the demeth- ylation of the latter did not normally pro- ceed under the conditions used, leading to the conclusion that normally uracil is not an intermediate product in the decomposition of thymine. Cell-free extracts of N. corallina with activity towards thymine, uracil, and barbituric acid were obtained only from cells grown in either thymine, uracil, or bar- bituric acid; enzyme preparations from glu- cose grown cells were devoid of such activity. This points to the adaptive nature of the enzyme system (Fig. 78). Barbituric acid was hydrolyzed by the enzyme preparation with the formation of 1 mole of CO. , 2 moles of NH; , and 1 mole of malonic acid, for a pathway of thymine and uracil degradation according to the fol- lowing scheme: Thymine — 5-Methy] barbituric acid Ww Tiracil — Barbiturie acid — Maloniec acid + Urea oe CO. NH3 Production of Vitamins and Other Growth Factors Various actinomycetes and their meta- bolic products exert a favorable effect upon the growth of lower forms of life, including fungi and other microorganisms, and upon higher forms of animal and perhaps plant life. This is due, partly at least, to their ability to synthesize several vitamins. Herrick and Alexopoulos grew S. virido- chromogenes in a liquid medium, then filtered the culture and autoclaved it. The fungus Stereum gausapatum inoculated upon this medium produced a heavier growth than usually obtained on sterile media. Even more striking results were obtained when Phyco- myces blakesleeanus was used as the test organism; since this culture is used in as- saying for the presence of thiamine, the con- clusion was reached that actinomycetes produce this vitamin. In a further study of a number of cultures of actinomycetes, it was demonstrated that all were able to produce thiamine or its intermediate or precursor. Mackinnon observed that various strains of S. albus have a marked stimulating effect upon the growth of Trichophyton discoides. This effect was comparable to that exerted by the presence of thiamine. The same effect was obtained when P. blakesleeanus was inoculated on a synthetic medium in which the streptomyces had previously been grown. Filtered cultures of S. albus added to syn- thetic media had the same effect upon the growth of thiamine-requiring organisms. 193 The formation by S. griseus of a growth factor for Leuconostoc citrovorum was demon- strated by Emery et al. (1950). This factor was not identical with either By. or By. This factor had no activity upon pernicious anaemia, but possessed leucocyte-stimulat- ing activity. The production of carotenoids by actino- mycetes has also been demonstrated. Pro- tiva has shown that certain actinomycetes are able to produce riboflavin and flavopro- tein in synthetic media. Formation of Vitamin B,» The announcement, in 1948, by Rickes et al. that certain actinomycetes are able to produce vitamin By. resulted in much in- terest in the commercial possibilities of these organisms as sources of this vitamin. It has now been established that numerous actino- mycetes are able to produce this vitamin if cobalt salts are added to the media to serve as precursors. The production of By: can be measured by microbiological assays. Var- ious strains of S. griseus, including the streptomycin- and grisein-producing forms, and various Streptomyces species, such as S. fradiae and S. aureofaciens, are able to produce some vitamin By» without affecting the yields of the corresponding antibiotic substance. Certain nonantibiotic-producing actinomycetes, like S. olzvaceus, were also 194. found capable of producing this vitamin (Ganguly et al.). According to Hall et al. (1953), S. olivaceus when grown under submerged aerobic con- ditions is capable of producing large amounts of vitamin By. The yields of the vitamin were influenced by the composition of the medium. In the presence of proteinaceous material such as distillers’ solubles, glucose, CaCO; , and cobaltous ion, about 1.5 ug of vitamin B,. was produced per millilter in deep tank fermentors, but as much as 3 ug of the vitamin was obtained in some media. Appreciable amounts of the B-complex vita- mins, niacin, pantothenic acid, biotin, py- ridoxine, thiamine, and riboflavin were pro- duced. By selective cultivation, including mutant formation by ultraviolet light and x-rays, it is possible to increase considerably the yields of vitamin By» by a given culture. The effect of cobalt as a limiting factor in the biosynthesis of the active vitamin Bye by S. griseus was immediately recognized (Hendlin and Ruger, 1950). Shull and Rou- tien (1951), Pridham et al. (1951), Saunders et al. (1951), and Garey et al. (1951) made a survey of vitamin By». production by different actinomycetes. Particular attention was paid to S. griseus (Rickes et al., 1948) and S. fradiae (Jackson et al., 1951). Chemical investigations have brought out the fact that there are several forms of vitamin By. Possibly not all of them occur naturally but are formed during the isolation process. The incorporation of cobalt into the actinomycete metabolite has been estab- lished by the use of radioactive cobalt in fermentation media and the isolation of the vitamin containing the labeled isotope. Some of the vitamin By». produced by ac- tinomycetes is bound on the cells, and may be released by treatment with acid, alkah, ionizable salts, sonic energy, or heat. Sub- sequent to release, the cell-free liquid may be treated with cyanide to convert the vita- min present to the more stable cyanide form. THE ACTINOMYCETES, Vol. I In addition to vitamin By» , various other biologically active compounds, designated as Bio , Bize ; and Byg, are produced by S. griseus (Anslow et al.). The formation of vitamin By», by S. aureofaciens was demon- strated by Lichtman ef al. (1949). The By», was effective, by parenteral administration, in the treatment of Addisonian pernicious anemia. In a study of the production and purifica- tion of vitamin By». by various actinomycetes, Tarr has shown that Bo, was formed by S. griseus and by S. aureofaciens in aerated herring press water containing 2 mg_ per milliliter of added cobalt (as cobaltous ni- trate). Highest recovery of vitamin Bys, in these products (about 1.1 mg per milliliter with S. aureofaciens and 0.8 mg per milli- liter with S. griseus) was obtained by chro- matography on filter paper strips treated with potassium dihydrogen phosphate, elu- tion of the vitamin, and aseptic addition of the eluates to previously sterilized Lactobacil- lus lecchmannii assay medium. Treatment of the crude fermentation products with po- tassium cyanide (2.5 mg. per milliliter) caused partial resolution of Bis, to Bie . Letunova (1958) made a comprehensive study of cultures isolated from the lime of stagnant reservoirs of the biogeochemical province enriched in cobalt. Nearly 86 per cent of these cultures, as well as 90 per cent of strains isolated from lime deposits of the Co-impoverished province, were able to form vitamin By. Certain nocardias are also capable of pro- Bardi et al. (1958) studied a strain of NV. rugosa that was found to release ducing By. free porphyrins into the medium under suit- able aeration conditions. By ultraviolet ir- radiation, mutants were obtained that gave a much higher yield of porphyrins. The rela- tion between vitamin By). and porphyrin production was demonstrated. The free por- phyrins consisted mainly of coproporphyrin III, and of uroporphyrin, probably III, in PRODUCTION OF VITAMINS AND OTHER GROWTH FACTORS minor amount. Traces of coproporphyrins T and II, of uroporphyrin I, and of other non- identified porphyrins were also released in the medium. The total free porphyrin con- tent in broths may reach, in some strains, 20 to 40 ywe/ml. The mutants producing greater amounts of porphyrins gave lower yields of vitamin By». than the parent strain. Less aerated cultures produced more por- phyrins and less vitamin By. The lack of cobalt lowered porphyrin production. Added d-aminolaevulic acid (100 to 500 ug/ml) in- creased porphyrin production, but did not exert any effect on vitamin By» levels. Small amounts of succinic and glyoxylic acids were detected in suspensions of N. rugosa incu- bated with citrate under anaerobic condi- tions. Since glyoxylic acid can be a precursor of glycine, it was suggested that this reaction might have some meaning in porphyrin bio- synthesis by this strain. A detailed discussion on the effect of co- balt concentration in the medium upon the production of vitamin By is given in Chapter 8. Darken (1953) reviewed the production of By by actinomycetes. Other Vitamins and Vitamin-like Ma- terials Actinomycetes have also been found to produce various other porphyrin-like (Cor- tese) and iron-containing compounds, such as grisein. They also have the capacity to pro- duce other water-soluble vitamins, notably coenzyme A, the pteroylglutamic acid de- rivative that promotes the growth of certain strains of Leuconostoc citrovorum. Little is known of the mechanisms in- volved in the direct symbiosis of actino- mycetes with insects, as in the case of the nymph of the hemipterous insect Rhodnius prolixus, the moulting and reproduction of which depend upon its infection with N. rhodnii. Brecher and Wigglesworth (1944) isolated a culture of a nocardia regularly 195 from this insect reared in the laboratory. The transmitted through the egg but is taken up by the young nymph from the environment, such as the microorganism is not contaminated surface of the egg, and more often perhaps from the dry excreta of other members of the species. Insects were reared free from the actinomycete by sterilizing the surface of the egg and feeding with suitable precautions. They grew and moulted nor- mally until the 4th or 5th instar. Moulting was then delayed or failed entirely in spite of repeated feedings of blood. Very few in- sects without the actinomycetes became adult, and those few were almost certainly incapable of reproduction. Normal growth and moulting and egg production were re- sumed when the insects were reinfected with the organism. Little is also known of the role of the streptomycete that is able to infect nema- todes that grow in the cockroach (Hoffman). Other references in the literature concern the formation by certain actinomycetes of substances that exert a stimulating effect upon the growth of various organisms. It is sufficient to mention the work of Rehm on the presence in the mycelium of cultures of the S. albus group of substances that stimu- late the growth of the fungus Aspergillus niger. Growth Stimulating Effect of Antibi- oties The highly significant practical results ob- tained by the stimulating effects of antibi- otics upon animal growth could not all be explained by the action of known vitamins. Moore et al. first observed, in 1946, this effect for an actinomycete antibiotic, namely streptothricin. At the present time, large quantities of the tetracyclines and strepto- mycin are employed in the feeding of non- herbivorous animals. Some of these growth factors are still unidentified (Fitz et al. 1956). An early review of the effect of antibiotics 196 upon the growth of swine has been presented by Brande et al., and a detailed analysis of the nature of the growth promoting effect of antibiotics upon animals has been made by Porter (1957). Porter summarized the evi- dence that their use results in appreciable benefit to growing livestock. Early fears that their use for breeding stock might lead to the establishment of resistant strains of patho- genic bacteria have been found to be ground- less. The feeding of antibiotics to animals was found to result in a healthier environ- ment. The mechanism by which the antibiotics produce this favorable effect on growth of the animals may be due to their action on the tissues, or primarily upon bacteria. This may depend partly on the state of health of the animals. Conditions of poor health can lead to a state of disease, making it difficult to draw a distinguishing line between the therapeutic effect of antibiotics and their nutritional action. Antibiotics may exert a direct antibacterial action on pathogenic bacteria in the gut or animal tissues. Their presence in small quantities in both the gut and tissues may also act as a prophylactic. Even in the absence of a state of disease there is a definite relationship between the effect of antibiotics on growth and the effect on the microbial flora. There is little or no evidence of consistent changes in the types and numbers of the microorganisms in the gut, possibly because present techniques are not sufficiently developed to detect changes in the gut population. Such changes may not take place and the antibiotics may act by altering the metabolism but not the mor- phology of the microorganisms. Porter postu- lated that such alteration may result either in an increased microbial synthesis of known or unidentified nutrients or in a lessened competition by the microorganisms for nu- trients, the host benefiting from both. THE ACTINOMYCETES, Vol. I Stimulating Effect of Products of Ac- tinomycetes upon Plants and Bac- teria Kkoaze demonstrated that the culture fil- trate of a streptomyces had a strong pro- moting effect on the germination of rice plants, in a dilution of 10~ to 1077. A erys- tallme substance was obtained by ethyl acetate extraction and chromatography on aluminum oxide which was active in con- centration of 0.1 mg/ml. It was a neutral substance, soluble in alcohol, benzene, chloroform, and water. On analysis it gave CyoHygN 202. It was found to be L-prolyl-L- valine anhydride (diketopiperazine). The substance had no antibiotic activity. In a study of the effect of antibiotics upon plant growth Koaze et al. (1957) found that certain antibiotics showed a similar promot- ing effect; neomycin B and sarkomycin had a marked effect on the growth of broad beans. Mention may also be made of the growth- promoting effect of certain actinomycetes upon cellulose-decomposing and nitrogen- fixing bacteria (Sanborn, 1926). The mech- anism of this action is still insufficiently understood. Ciferri and Machado noted, during the isolation of an antibiotic produced by a cul- ture of a streptomyces belonging to the S. griseus group, a considerable yellow-green fluorescence of the metabolic liquids. The riboflavin potency of such liquids was as- sayed with mutant strains of Lactobacillus casei and Leuconostoc mesenteroides, giving an activity corresponding to a riboflavin con- tent of 0.9 to 1.2 ug/ml. The fluorescent pigments could be easily extracted with n- butanol and purified by absorption on char- coal and elution with aqueous pyridine. Paper chromatography of such preparations and of the original broths showed at least four spots characterized by fluorescence under Wood’s light but none could be identi- PRODUCTION OF VITAMINS AND OTHER GROWTH FACTORS fied either with riboflavin or its nucleotides or decomposition products. Only spot 2 reacted with ninhydrin. When the spots were eut off the chromatograms and tested in- dividually, only one spot, characterized by a blue fluorescence, proved capable of sus- taining the growth of L. casez. Rubentschik et al. found that various strep- tomyces, such as S. griseus, S. coelicolor, and S. globisporus, when grown in cultures with other organisms, are capable of forming volatile materials which stimulate the growth of EF. colt, B. subtilis, B. mesentericus, and other bacteria. Every Streptomyces spe- cies was said to exert a characteristic effect, S. griseus stimulated the growth of Azoto- bacter. Grossbard reported that various anti- fungal substances produced by actinomy- cetes exert stimulative effects upon the growth of fungi, within a concentric circle adjoining the zone of inhibition. Pigmenta- tion was intensified in cultures of Verticilium dahliae, Helminthosporium victoriae, and Fusarium oxysporum. The last usually fails to produce a pigment on synthetic media, but in response to the metabolites of certain 197 streptomyces cultures, a pigment was formed in optimal media; a considerable intensifica- tion of the pigment (lycopersin) occurred. Colletotrichum atramentarium responded to the same streptomyces metabolites by an acceleration in the maturing and by a greater density of stromata. Ceratostomella ulmi and Streptomyces scabies rapid sporulation. The favorable effect of certain strepto- myces upon the sporulation of other cultures of the same genus has been demonstrated by Dondero and Scotti. These results led to the conclusion that actinomycete metabolites contain specific stimulatory substances in addition to inhibitory substances, or that the substances may be growth-inhibiting at one concentration and growth-stimulating at an- other. The significance of these phenomena in natural processes in general and in the soil in particular is still to be elucidated. This is also true of the stimulating effect of actino- mycin upon the growth of some strains of rhizobia and its inhibiting effect upon others, notably the slow growing strains (Trussell and Sarles). responded by more CoH AUP TER Vie Production Among the most characteristic properties of actinomycetes is their ability to produce a great variety of pigments, both on organic and on synthetic media. This pigment-pro- ducing capacity is so characteristic as to serve for the naming of a large number of the species, notably those belonging to the genus Streptomyces. Very few of these pig- ments are pure. Most of them are mixed, with gradual transition from one color of the rainbow to another. Conn and Conn emphasized the value of pigmentation in classifying actinomycetes. Stapp isolated from various soils 477 cul- tures of streptomyces. Their growth on elycerol-asparagin agar gave the following pigmentation: 31 per cent of the colonies were gray in appearance; 19 per cent yellow (cream to orange); 18 per cent brown; 17 per cent white; 8.4 per cent red; 4.2 per cent blue; 1.3 per cent green; and 0.8 per cent black. von Plotho (1948) divided the actinomy- cetes into four large groups according to the pigments produced: 1. red-yellow; 2. red- blue; 3. red-brown; 4. colorless. Some of the pigments possess indicator properties: at an acid reaction, the red pigment predominates; at an alkaline reaction, the second pigment prevails. The colorless group have given the greatest number of antibiotic-producing strains. Members of the red-blue group pro- duce strong pigments but weak antibiotics. Members of the red-yellow group produce both strong pigments and highly active anti- of Pigments biotics. The members of the red-brown group produce neither true pigments nor active antibiotics. Only very few of the pigments of actino- mycetes have been studied in detail. Some are soluble in water, others are soluble in alkalies, and still others in organic solvents, such as alcohol or chloroform (Table 48). These pigments have been divided into three groups: anthocyanins, carotenoids, and mela- nins. The red-blue pigments of the actino- mycetes belong to the first group; red-or- ange-yellow pigments belong to the second; the black and brown pigments belong to the third. Some of the pigments are fluorescent, and some change in color with a change in reaction of medium. Some are intracellular; others are exocellular and dissolve readily in the medium. Some of the pigments are pro- duced only on certain specific media, notably synthetic media; others are produced on a variety of media. In many cases, a change in the composition of medium results in a change in the nature of the pigment. The pigments of actinomycetes are usually described in terms of various shades of blue, violet, red, rose, yellow, green, brown, and black. The shades of color are also frequently indicated, as light gray, deep gray, mouse gray. The pigments may be concentrated either in the vegetative mycelium or in the aerial mycelium and spores. The pigment-producing property of ac- tinomycetes is variable, depending upon the nature of the medium, the age of culture, 198 PRODUCTION OF PIGMENTS and its previous cultivation. The insoluble pigments are more constant in nature than the soluble kinds. The formation of water- soluble brown to black pigments on organic media has been used to designate the chro- mogenic streptomyces. The tyrosinase action characteristic of these organisms was_ be- lieved by Beijerinck to explain the mecha- nism of the production of this pigment. Miller first studied the pigment produced by S. coelicolor. It is dark blue and diffuses readily into an alkaline medium. If the reac- tion of the culture changes to acid the pig- ment becomes red. This pigment was found to be produced on synthetic media with starch, sucrose, and other carbon sources. This is the reason for the designation of the culture variously as S. violaceoruber and S., tricolor. Beijerinck (1914) described a cul- ture, A. cyaneus, now classified with the nocardias, which produced a pigment similar in its properties to the anthocyanins. This pigment was recently designated as litmoci- din. Lieske recognized two types of pigments among the actinomycetes: (a) the chromo- phores or pigments which are not excreted from the mycelium into the medium, and (b) the chromopars or pigments which are readily excreted. The first group comprises the pigments found in the vegetative myce- lium grown on synthetic media; these are yellow, orange, red, blue, violet, brown, black, and green. The aerial mycelium of these cultures may be white, rose, lavender, red, yellow, orange, green, or gray. The sol- uble pigments are usually yellow, blue, and red; occasionally they are green, orange, or brown. Kriss established that even the chromo- phore pigments are partly dissolved in the medium, possibly because of the lysis of the mycelium and the spores. Some of these pig- ments are insoluble in water and are bound to the proteins. Others are dissolved in the fats and lipoids of the cell. Some may be 199 TABLE 48 Solubility of actinomycete pigments (von Plotho) Chloro- form Butyl alcoho Group Ether CS2 CCh | Water Red-yellow + + ae alte = = Red-brown _ = = = ab s2 Red-blue = = Ze = es Ss water-soluble, but are unable to pass through the living cell plasma; on the death and lysis of the cell, these pigments may dissolve into the medium. The solubility of the chromopar pigments in water is due to the greater pene- tration of the pigment through the cell wall. Kriss suggested classification of the pig- ments of actinomycetes into four types: I. Pigments soluble in water and in 96 per cent alcohol; these are capable of pass- ing through the living cell plasma. This group has been subdivided into (a) antho- cyanins, soluble only in water, and (b) hy- droactinochromes, soluble in water and in alcohol. II. Lipoactinochromes, insoluble in water but soluble in alcohol and in other organic solvents. III. Pigments insoluble both in water and in organic solvents. IV. A combination of water-soluble and water-insoluble pigments. Lieske isolated a culture of a streptomyces that produced a carmine-red pigment that, when boiled in dilute acid, became soluble in alcohol and in ether. Certain actinomy- cetes produce a red pigment that is made soluble only by the action of concentrated HCl; on treatment with H2SO, it is changed to a blue-green pigment. N. polychromogenes produces a red pigment, soluble in chloro- form, ether, and acid, but not in alcohol, glycerol water, or dilute alkali; this pigment is also changed to blue-green by H.SO, . Certain light yellow pigments produced by actinomycetes are insoluble in organic solvents, but are soluble in dilute KOH so- 200 lution; they are changed, on treatment with concentrated H.SO,, first to green, then to dark brown. The yellow-red pigment of N. corallina was identified as belonging to the lipochrome group of fat-soluble pigments. Nature of the Pigments Anthocyanins These pigments are readily soluble in wa- ter and in aqueous glycerol, alcohol, and other water-containing solvents. They are insoluble in absolute alcohol, chloroform, and other organic solvents. They are red in dilute acid solutions and blue in alkaline so- lutions. The neutral point is about pH 7.5. The blue pigment of S. coelicolor has been extracted with cold and hot water as well as with alcohol. This pigment became red when treated with acid, and green when treated with 25 per cent alkali solution. Addition of lead acetate to the aqueous solution of the pigment brought about the formation of a violet precipitate. Miller (1908) first sug- gested the similarity of this pigment with anthocyanin. Kriss confirmed these results fully. Krassilnikov also confirmed the results and emphasized that the anthocyanins or allied pigments are characteristic of several actinomycetes. He pointed out that all the blue pigment-producing actinomycetes be- long to S. coelicolor Miller. The cultures all produce a blue pigment which diffuses into the medium, conferring upon the medium the corresponding color. If the reaction of the medium changes to acid as a result of the growth of the organism, the color of the culture becomes red; on alkalinization of the medium, the color again turns blue. According to Waksman, the pigment pro- duced by S. violaceoruber behaves as an indi- ‘ator, being red in an acid medium and blue in an alkaline medium; the change in pig- mentation takes place at pH 6.6. According to Frampton and Taylor (1938), the pig- ment produced by S. violaceoruber is an THE ACTINOMYCETES, Vol. I anthocyanin; it was isolated as the crystal- line picrate. These workers considered the pigment as a rhamnose glucoside, the carbo- hydrate groups being rhamnose and glucose. The sugars were believed to be separately attached to the anthocyanidin residue. Jean Conn concluded that the two blue pigments produced by S. coelicolor and S. violaceoruber are not identical; the first is similar but not identical to azolitmin. On the basis of this differentiation, she believed that the two organisms represent distinct species. Oxford and Erikson et al., however, could not accept the phenazine or anthocyanin nature of these pigments. Tonolo et al. (1954) described the isolation, from a culture related to S. coelicolor, of a pigment designated as streptocyanin. The course of pigment production in a glycerol medium is shown in Figure 79. The pigment was soluble in acetone, pyridine, and diox- ane. It colored blue-violet with H.»SO, and decomposed at 290 to 300C°. It gave a band in the visible spectrum. A quinoid structure was postulated for the pigment. It showed antibiotic activity. Green Pigments Actinomycetes were found to produce sol- uble green pigments, which is the reason for such species names as S. viridis, S. virido- chromogenes, and S. verne. Some of these pigments are soluble in glycerol and in al- kaline solution, but not in organic solvents. The color of the pigment in water is green; in glycerol, yellowish green. The composition of the medium influences the nature of this pigment. Green pigments produced by ac- tinomycetes were also reported by other in- vestigators. One of the most interesting of the green pigments was recently studied by Chain and Tonolo. They isolated a culture of a strepto- myces that produced on yeast agar an in- tense green nondiffusible pigment. This pig- also formed in submerged, ment was PRODUCTION OF PIGMENTS 201 8 0.7 5 4 iS a OLS 7 Q71.0 : lie: uw? pa Zz =e = bey Qa > = os ok 0 O ac o 6 ea0°5 2 0.1 0.1 ° rr) 24 48 72 96 120 144 168 TIME IN HOURS FicuRE 79. Course of fermentation by streptomyces in 200-liter tank (Reproduced from: Tonolo, A et al. Rend. ist. sup. sanita 17: 958, 1954). well-aerated cultures, in a glucose-contain- ing medium, at pH 7.0. The pigment was extracted from the mycelium with ethanol, then transferred into butyl acetate, and the solution percolated through a column of alumina. The column was developed with 95 per cent alcohol, followed by absolute alco- hol. Three bands were formed. The main, or middle, band was erystallized, yielding 100 to 200 mg of solid material per kilogram of moist actinomycete growth. This pigment was named ferroverdin. The formula assigned was C3o9H,OgsN2Fe. The pigment was found to be insoluble in water and in benzene, chloroform, and certain other organic sol- vents. It was soluble in methanol, ethanol, acetone, and glacial acetic acid. It was re- duced by hydrogen gas, in the presence of a proper catalyst, to a colorless substance. The iron was closely bound, but, after catalytic reduction, it appeared in ionic form. Hydroactinochromes This group, according to Kriss, comprises pigments that are soluble in water, 96 per cent alcohol, and chloroform, but not in ether, acetone, or CS.. In a natural state, they are violet. In dilute acid solutions, they are orange; in an alkaline solution, dark violet or blue. These pigments are usually found in cultures admixed with other pig- ments, red or orange in color. Krassilnikov included among the cultures producing this type of pigment S. violaceus Gasperini and S. violaceus niger Waksman and Curtis. The first is said to produce only a small amount of the orange pigment, whereas the second also forms a dark melanin pigment. According to Kriss and Krassilnikov, no pigment of this type was found in the nocar- dia cultures, although they reported that one such culture isolated by Berestnew had the sapacity to produce such pigments. 202 Lipoactinochromes These pigments are soluble in alcohol and in other fat solvents. They are red, orange, or yellow in a natural state. Kriss divided them into two subgroups: (a) Bright orange pigments soluble in petrol ether. The color does not change in an acid solution, but be- comes lilae in an alkaline solution. The pig- ment is readily dissolved in CS.. (b) Pig- ments insoluble in petroleum ether. They give a rose-red color in alcoholic solution. In dilute acid solutions, the color is red; and in alkaline, yellow. This group of water-insoluble pigments includes the carotenoids. These are produced by the red, orange, and yellow species. Reader demonstrated two such pigments among actinomycetes, one of which was designated as corallin, an ether solution of which gave two bands of absorption in the spectrum. Rhodomycin, another red pigment, was studied by Brockmann and Bauer (1950). Prodigiosin Pigments A prodigiosin-like pigment, yellow in an alkaline solution and red in an acid solution, was isolated by Dietzel from the mycelium of the organism producing actinorhodin. The dry mycelium was treated with methanol, THE ACTINOMYCETES, Vol. I then shaken with dilute alkali. The yellow pigment remained in the butanol solution, whereas the blue pigment dissolved in the alkali. The butanol was distilled off and the residue was dissolved in benzol and chroma- tographed on an AlsO; column. The deeply red zone was treated with methanol. On shaking with NaHCOs solution, the pigment changed to yellow. The pigment proved to be prodigiosin-like in nature. The chemical formula, based on elementary analysis, was Co5H36-O;N3Cl. Several other streptomycetes produced similar pigments. It was suggested that the lipoactinochromes X and B_ of Kriss belong to this group of pigments. Arecamone ef al. (1957) confirmed Dietzel’s results concerning the production of a pro- digiosin-like pigment by certain strepto- myces, notably S. ruber and S. roseodiastati- cus. This pigment had antibiotic properties against gram-positive bacteria. Brown-black Pigments Various actinomycetes, notably species of Micromonospora and certain streptomycetes, produce a pigment that ranges from orange- brown to brown to black. Beijerinck assumed that the brown substance was a quinone. Waksman, Rubentschik, and others suggested that it is a result of the action of the enzyme TABLE 49 Growth and soluble pigment production of 8S. griseus tn calcium salts of alpha-hydroxy and dibasic acids (Benedict and Lindenfelder) Streptomycin-producing strains of S. griseus Waksman 3496 Calcium salt | Growth Pigment Citrate... «aoe + | Yellow | Malater ss tetoe: ...) +++-+4+ | Green Lactate.... + | Light yellow Rartrates -: _ - Succinate eae | Deep yellow Malonate... + Light yellow | Fumarate... +t | None Waksman 9 Carvajal 2060 Growth | Pigment Growth Pigment am |——_—— | |——— = | + | Yellow | ++-+-+ | Light yellow eset) oneen | ee Light yellow oo | Amber | + | Light yellow a. | Deep) yellaw, | + | None + | None | _- | None + | None + | None PRODUCTION OF PIGMENTS tyrosinase. The majority of the ‘chromo- genic” actinomycetes produce such pigments on organic media. Certain species produce these pigments also on synthetic media. Some of the brown to black pigments re- main in the mycelium (‘‘melanin’’); others dissolve in the medium. The nature of the medium greatly influences the nature and intensity of the pigment produced. Percival and Stewart made a detailed study of the mechanism of melanin forma- tion. They found that in the presence of tyrosinase, the formation of melanin from tyrosine is apparently due to the oxidative formation of the red indole quinone through the action of the enzyme. The subsequent reactions, the formation of 3,4-hydroxyin- dole and its further oxidation to melanin, are able to take place merely in the presence of molecular oxygen, and without the interven- tion of any enzyme. Environmental conditions, notably the degree of oxidation and the temperature of incubation, influence the formation of pig- ments. Aerobic conditions and lower tem- peratures (7—15°C) favor pigment formation in the culture. At 37°C pigment formation is greatly diminished. Role of Pigments in the Life and Me- tabolism of Actinomycetes Various hypotheses have been proposed to explain the role of pigments in the growth of the microbial cell. Their function in the respiratory mechanisms of the cell has been suggested. Some have claimed for them a role in the defense mechanism of the cell against the action of foreign cells or against the effect of sun rays. The recent interest in the subject of antibiotics has tended to concentrate attention upon these substances that are pigmented in nature. Benedict and Lindenfelder (1951) have shown that the different varieties of S. gr7- seus possess characteristic pigment-produc- ing capacities on special media. Some of the 203 streptomycin-producing strains are able to form yellow pigments on synthetic calcium malate media and green pigments on calcium succinate media. Grisein-producing strains are unable to produce such pigments (Table 49). Antibiotic Pigments eS A large number of actinomycetes produce pigmented antibiotics, which recently have received much consideration. Benedict pro- posed a system of classification of pigmented antibiotics produced by actinomycetes (Table 50). Some of these groups deserve more detailed consideration. Actinomycins Actinomycin was the first antibiotic iso- lated in a pure state from a culture of a streptomyces. It crystallizes from ethyl ace- tate or from acetone-ether mixtures as red platelets, m.p. 250°, [a]? (C = 0.25 per cent in ethanol) —320 + 5°. It is soluble in chloroform, acetone, ethanol, hot ethyl ace- tate, carbon disulfide, and benzene, but only slightly soluble in water or ether. It is stable in aqueous alcohol when boiled for 30 min- utes, but unstable in dilute acid or alkali. An alcoholic solution of actinomycin gives no coloration with ferric chloride; it shows char- acteristic light absorption in the visible re- gion (E}~2, = 200 at 450 ») and in the ultra- violet region (E}”,, = 216 at 215 my). It is highly active against certain gram-posi- tive bacteria. Waksman and Tishler found that 10 ug given intraperitoneally or sub- cutaneously, killed 20-gm mice in 24 to 48 hours. Various forms of actinomycin have since been isolated. Interest in this group of anti- biotics has grown especially since it has been demonstrated that they exert a marked effect in the treatment of certain forms of cancer. The chemical studies of Dalgleish et al., of Brockmann and Grubhofer, and of many others added greatly to our understanding 204 THE ACTINOMYCETES, Vol. I TABLE 50 Classification of pigmented antibiotics of actinomycetes (Benedict) I. Yellow to greenish yellow to orange. it Low solubility in water, insoluble in ether, soluble in other organic solvents. a. Golden yellow base, C22H2;N 2OsCl, low toxicity, active against bacteria and larger viruses. Chlortetracycline b. Yellow amphoteric compound, Co2H2sN2O0y, , low toxicity, active against bacteria and viruses. Oxytetracycline ce. Greenish yellow weakly basic, C37H3sNiO4 , strongly antifungal. Fradicin Insoluble in water, slightly soluble in ether, soluble in other organic solvents. a. Yellowish substance, active against yeasts and filamentous fungi. Actinone b. Golden yellow compound, CgHipN 2025. . , Aureothricin ce. Brilliant yellow neutral compound, CsHgsN2O.S>2 , active against various bacteria and fungi. Thiolutin d. Orange yellow, CysHi2NsOuSy . Thioaurin e. Yellowish orange basic substances, C3:H3sNsOs-3HCI (A), highly active and extremely toxic. Xanthomycins A and B f. Saffron-yellow weakly acidic, Co;HisOs , active against gram-positive bacteria. Resistomycin Soluble in water and in organic solvents other than ether. a. Yellowish orange, active against various bacteria. Luteomyecin b. Yellowish green neutral, active against gram-positive bacteria. Actinomyceline ec. Yellowish weakly acidic; active against yeasts and filamentous fungi. Flavacid Insoluble in water and ether, soluble in other organic solvents. a. Yellow acid substance, acid stable, active against gram-positive bacteria. Griseolutein b. Yellow substance; antifungal and trichomonadicidal. Trichomycin II. Yellowish red to dark red, blue and purple. With the exception of grisein and the microcins, all are primarily active against gram-positive bacteria. : i Practically insoluble in water, slightly soluble in ether, and soluble in other organic solvents. a. Red cyclic peptides, C4o-41Hs56-57N7-sOu, very toxic Actinomycins b. Strongly resembling actinomycin, possibly identical. Actinoflavins . Acid-base indicators, changing from red to blue a. Non-nitrogen-containing quinone, CogsH2 Oro , alkali-soluble, ether-insoluble. Actinorhodin b. Anthrocyanin-like pigment, soluble in water and ether. Litmocidin c. Low nitrogen-containing compound; red form, water-insoluble, ether-soluble. Rhodomycetin d. Amphoteric substituted quinones, low water solubility. Rhodomycins A and B e. Reddish purple pigment. Coelicolorin Red color due to a metallic element a. Weak acid, containing iron and amino acids, CyoHNioQ2oSFe, soluble in water and phenol. Grisein PRODUCTION OF PIGMENTS TABLE 50 grisein, ec. Grisein-like substances. 4. Possibly containing pyrrole nuclei a. Red, weak base, C55-67H96-104N 18015 . 5. Violet pigment a. Soluble in ether, water, and alcohols 6. Partially purified compounds 205 Continued b. Impure, grisein-like factor, production influenced markedly by iron, narrower spectrum than Antibiotie 3510 Albomyein Sparingly soluble in water and ether. Noecardianin Rhodocidin a. Neutral, reddish purple powder; antifungal and antibacterial. Microcin A b. Acidie, yellowish red powder; antifungal and antibacterial. of the chemistry and activity of this group of antibiotics (Waksman, Katz and Vining, 1958). Actinorhodin Brockmann and Pini, and von Plotho iso- lated, in 1947, a culture of streptomyces which produced a polyoxyquinone pigment, designated as actinorhodin. It was soluble in acetone, pyridine, and dioxane. It gave a blue color with H.SO,, decomposing at 270°C. This pigment formed two distinct bands in the visible spectrum. It was active antibiot- ically. The antibiotic is believed to possess a quinone structure, having three free hy- droxyl groups, one carboxyl group, and two hydroxyl groups adjacent to a carboxyl group (Brockmann and Hieronymus). Shockman and Waksman found that rhodo- mycetin is similar to actinorhodin. The anti- biotic shows a blue color in alkaline solution, dark blue in sulfuric acid, and becomes red- violet on addition of boric acid. Thiolutin, Aureothricin, and Thioaurin This group of antibiotics is characterized by the presence of sulfur in the molecule. Tanner et al. have shown that thiolutin (CsHsN2O.2S2) crystallizes as brilliant yellow needles which have no definite melting point but darken at about 255°C. It is soluble in alcohol, chloroform, glacial acetic acid, Microcin B pyridine, and dimethylformamide; slightly soluble in ether, petroleum ether, and ben- zene; and sparingly soluble in water. It is very stable in acid solution and withstands heating for 1 hour at 100°C. It is active against a variety of gram-positive, gram- negative, and acid-fast organisms, as well as fungi, Hndamocba histolytica, and certain hemoflagellates. Aureothricin also belongs to this group. Bolhofer ct al. obtained bright orange- yellow crystals of thioaurin (Cy4H)2N,O,8,) which melted with decomposition at 179 to 181°C. Thioaurin is relatively insoluble in water and in many organic solvents. It in- hibits the growth of various bacteria, but unlike thiolutin, it shows little activity against fungi. Coelicolorin This is a purplish red powder, m.p. 142 to 146°C, which was found (Hatsuta) to be red at pH 5.0, purple at pH 6 to 7, and green at pH 8.0 or above. It is soluble in water at pH 8.0 or above. It is soluble in ethanol and other organic solvents, but insoluble in pe- troleum ether. It is active primarily against gram-positive bacteria. Xanthomycins A and B These yellow antibiotics were isolated by Thorne and Peterson. Rao and Peterson con- 206 verted xanthomycin A to the crystalline hydrochloride, the elemental analyses giving Cz,H3.N,Os-3HCl. The antibiotics showed characteristic quinoid properties and con- tained four methoxyl groups and one methyl- imide group. Prolonged reaction shows the release of two primary amino groups. They were soluble in organic solvents. Acid hy- drolysis gave ethanolamine, methylamine, and ammonia in a ratio of 2:1:1. Separation of A and B was accomplished by Craig countercurrent distribution. Xanthomycin strongly inhibits the growth of many gram- positive and gram-negative bacteria, but does not affect M. tuberculosis in dilutions as low as 1:2000. It is very toxic to experi- mental animals. It is important to add further that, in the presence of certain metallic ions, some anti- biotics may form pigmented compounds. The tetracyclines give green compounds with copper and nickel and red compounds with ferrous and ferric iron (Albert). THE ACTINOMYCETES, Vol. I Luminescence of Actinomycetes Certain -actinomycetes have frequently been observed to give off luminescence under given conditions of culture. According to Rudaya (1958), there exists a correlation be- tween the antibiotic activity of S. rémosus and its luminescence intensity in the ultra- violet. This is true especially when the cul- tures are grown on solid media. The rate of luminescence depends on the composition of the medium and on the age of the culture. The character of the luminescence was found to change with cultivation and storage, yel- low luminescence being replaced by a blue one. Certain variants show only bright yel- low luminescence. It has been suggested that luminescence analysis be used as a guide for the primary selection of S. rimosus strains by subjecting young cultures to ultraviolet irradiation on solid media which are favor- able for antibiotic production. Antagonistic Properties Phenomena of Association and Antago- nism Soils and water basins are inhabited by mixed microbiological populations. Among the members of these populations numerous associations and antagonisms occur. Com- plex populations also occur in other sub- strates, as in human and animal digestive tracts, where they may be responsible for certain mixed infections. Pasteur, the biochemist, and DeBary, the botanist, were the first to emphasize the significance of possible antagonistic relations among different microorganisms living in such mixed populations. When two organ- isms were grown on the same substrate, one usually was found, sooner or later, to over- whelm and even bring about the death of the other. Such antagonistic activities em- brace phenomena other than mere competi- tion for or exhaustion of nutrients: they were found to be due largely to the formation of specific chemical substances (antibiotics) responsible for these effects. The terms “‘antagonism”’ and ‘‘antibiosis”’ usually refer to the reduction in growth and activities of organisms living in association. The organisms thus affected may respond by exhibiting temporary or permanent modi- fications in their physiological characteris- tics; their morphology may be changed; a reduction in virulence may also result. Sev- eral types of antagonism are now recognized: 1. Antagonism in vivo versus antagonism 7n vitro. 2. Bacteriostatic or fungistatic, bac- 207 iD. oO. tericidal or fungicidal, and lytic effects. Antagonism of function versus antagonism of growth. 4. Direct and indirect antagonism. 5. One-sided or two-sided antagonism. 6. Iso- antagonism and heteroantagonism, namely, antagonism between strains of the same species and antagonism among different species. Among the various types of antagonism, the most definite and best understood are those that result in the formation of anti- biotic substances, formerly spoken of toxins, lysins, or bacteriolysins. The physi- cal, chemical and biological properties of these substances vary greatly. Some are de- stroyed by boiling, others on exposure to light. Some are resistant to heat and to ultra- violet rays. Some are soluble in water, others in special solvents. Some are highly toxic to animals, others are relatively nontoxie. Numerous early investigators observed the depressive effect of fungi upon bacteria and vice versa. It is sufficient to mention the observations of Tyndall (1876). How- ever, the first well-recognized antibacterial preparation was that of pyocyanase, pro- duced by Ps. aeruginosa, formerly known as B. pyocyaneus. Emmerich and Low (1899) considered pyocyanase to be an enzyme sys- tem. Emmerich and Saida (1900) actually used it to destroy (dissolve) tubercle bacilli. Subsequent to these early studies, an ex- € c US tensive literature accumulated on the pro- duction of antibacterial substances by bac- teria, culminating in the treatise by 208 Papacostas and Gaté (1928) on ‘Microbial associations and their therapeutic applica- tion’? and the more recent systematic work of Dubos on tyrothricin. The fungi as producers of antibacterial substances also received considerable atten- tion (Waksman, 1945). The most spectacular work was that on penicillin, first recognized by Fleming (1929), and finally established by Chain, Florey, e¢ al. (1940) as an impor- tant chemotherapeutic agent. The actinomycetes were first recognized by Gasperini (1890) as potential destroyers of fungi and bacteria. Lieske reported, in 1921, that actinomycetes, especially the aerobic, spore-forming types (streptomyces), are not hindered in their growth by other THE ACTINOMYCETES, Vol. I organisms, but, on the contrary, are able, in spite of their slow development, to suppress the growth of almost all bacteria and fungi. This was found to be true on plates in which actinomycetes were seeded after the growth of fungi and bacteria had already occurred. When a culture of an actinomycete and a staphylococcus were mixed, streaked on agar plates, and incubated at 37°C for 24 hours, the plate became covered exclusively with the growth of staphylococci; on further ineuba- tion, however, the actinomycete assumed the upper hand and gradually replaced the bacteria. The growth of the actinomycetes, under these conditions, may have been bet- ter than in pure culture, thus giving the im- pression that the bacteria actually served as FIGURE 80. Production of clear zones on bacterial plates by antagonistic organisms. ANTAGONISTIC PROPERTIES a growth stimulant. On the other hand, Ps. pyocyaneus repressed the growth of various actinomycetes in mixed culture, and even brought about destruction of the actino- mycetes. The antagonistic effects of actino- mycetes were ascribed by Lieske to their ability to excrete toxic substances that have the capacity to inhibit the growth of other organisms or even to destroy them. Miller (1908) first observed that certain pigment-producing streptomyces (S. coeli- color) are able to suppress the development of yeast-like fungi. In a study of the associ- ative and antagonistic effects of certain actinomycetes and fungi, Porter (1924) dem- onstrated that several organisms now known to belong to the genus Streptomyces, namely S. tricolor, S. albus var. ochraceus and S. nigrificans, produced marked inhibitory ef- fects upon the growth of fungi. He suggested that such inhibitory action may aid in species identification. Gratia (1924) definitely established the potentialities of actinomycetes as powerful antimicrobial agents. He and his associates isolated a preparation, designated ‘‘myco- lysate,” which was actually used in the treat- ment of many clinical cases caused by pathogenic bacteria, notably the typhoid organism. Rosenthal isolated, in 1925, from dust, an actinomycete which he designated as the true biological antagonist of the diphtheria bacillus. He inoculated the surface of an agar plate with an emulsion of the bacillus and then introduced the actinomycete culture into several spots on the plate. After 2 days’ incubation, the actinomycete colonies were surrounded by large transparent zones, whereas the rest of the plate was covered with the growth of the diphtheria organism. When an emulsion of this organism, pre- viously killed by heat, was mixed with agar, and the mixture poured into the plates and inoculated with the actinomycete, the colo- nies of the latter were surrounded by clear 209 zones. This demonstrated the fact that the actinomycete produced a lytic substance which diffused through the agar solved the dead bacterial cells. and dis- Forced Antagonism The nature of the antimicrobial effects of different microbes is greatly influenced by the energy and nitrogen sources in the me- dium. Schiller believed that antagonism could be induced by using microbes as nu- trients: in a dilute glucose solution without nitrogen, yeasts were said to be “forced” to kill and digest bacteria; if the yeasts were added to a fully developed bacterial culture, a bacteriolytic substance produced which was also active outside of the yeast cells. However, when the bacteria were inoc- ulated into cultures of yeasts suspended in distilled water, the yeasts were killed. Vari- ous efforts, however, to adapt cultures of actinomycetes by the process of ‘forced antagonism” to grow on specific bacteria failed to yield antibiotics that the organisms would not produce normally when grown upon proper artificial media. Was The Soil as a Source of Antagonistic Actinomycetes The soil may be considered as the major source of antagonistic organisms, especially actinomycetes. Extensive studies carried out in our laboratories on the enrichment of soil with J/. tuberculosis did not lead to any special development of actinomycetes active upon these organisms. This can be explained by the fact that the production of antibiotics by an organism does not take place in re- sponse to certain specific nutrients. Thus, the activity differs from enzymatic processes that exhibit the phenomena of adaptation, whereby the organism benefits directly from a particular reaction. The activity is differ- ent, also, from that following soil enrichment which results in the development of nitrify- ing and nitrogen-fixing bacteria. The forma- 210 tion of antibiotics does not appear to be correlated, therefore, with the stimulation of reactions influencing the breakdown of nutrients or certain other oxidation and fixa- tion processes. Greig-Smith, in his studies on the occur- rence of toxic substances in soil, gave a clear description of the antagonistic effect of ac- tinomycetes against bacteria and fungi; the fact that actinomycetes grow only slowly in normal soils suggested the possibility that they comprise an important factor limiting bacterial development. It may be of interest to quote from his paper: “In making counts of soil-bacteria, it is not uncommon to find colonies of Bac. mycoides or of races of Bac. vulgatus spreading over the surface of the nutritive agar. Very often it will be noted that, while the majority of the colonies are covered by the spreading growths, there are a few that are untouched. The mycoides-colony may approach to within two, five, or ten millimetres, and then spread out and surround the colony, leaving a ring of clear agar medium. It is evident that there is fo.) oO 16) (e) Ww (e) Nm je} % OF 544 ACTINOMYCETES TESTED 5 .sS (e) TOTAL ACTIVE >20mm ZONE OF FIGURE 81. INHIBITION AGAINST Distribution of antagonistic properties among freshly isolated actinomycetes (Repro- THE ACTINOMYCETES, Vol. I some product secreted by these colonies which is obnoxious to the spreading colony, whether it be Bac. mycoides, Bac. vulgatus, or to spreading moulds such as Penicillium or Aspergillus. An examination of the colonies producing this toxie effect showed that the majority consisted of Ac- tinomyces or Streptothrix as they have been called. Some of these darkened the medium and were apparently Act. chromogenus. Certain of these colonies were selected, and spotted upon fresh plates, in the centre of which bacteria with spread- ing colonies were planted. The white forms were found to be very toxic, while the dark forms were feebly toxic to the spreading Bac. vulgatus.’’ Evidence concerning the antagonistic potentialities of actinomycetes began to ac- cumulate also as a result of a different ap- proach. Millard and Taylor succeeded in con- trolling potato scab, caused by S. scabies, by the use of green manures and grass cuttings. When potatoes were grown in sterilized soil infected with S. scabies, seab was reduced by the simultaneous introduction into the soil of S. praecox, a saprophytic organism. By increasing the proportion of the saprophyte E. coli B. mycoides B. subtilis Staph. aureus Ps. aeruginosa 1-lOmm 11- 20mm TESH ORGANISMS duced from: Rouatt, J. W., Lechevalier, M., and Waksman, 8. A. Antib. Chemoth. 1: 189, 1951). ANTAGONISTIC PROPERTIES to the pathogen, the degree of scabbing on the test potatoes was reduced from 100 per cent to nil. Goss found, however, that the general soil microflora rather than certain specific or- ganisms had a controlling effect upon the development of scab; inoculation of soil with S. praecox alone gave negative results. Sanford and Cormack also were unable to obtain control of potato scab by the simul- taneous inoculation with S. scabies and S. praecox of steam-sterilized soils or of natural soil enriched with green plant materials; these organisms were found to be perfectly compatible on potato-glucose agar as well as in a steam-sterilized soil. It was suggested that the control of scab on potatoes ob- tained by Millard was possibly due not to the direct action of S. praecox but to certain other undetermined microorganisms favored by the presence of the green manure or by other undetermined conditions. In their studies on the decomposition of plant residues by pure and mixed cultures of microorganisms, Waksman and Hutchings 211 showed that actinomycetes exert an antago- nistic effect upon the of other microorganisms. Thus the ground work was laid for the systematic study of the antago- nistic properties of actinomycetes and their ability to produce antibiotic substances. activities Screening Programs In search for microbes that have the ca- pacity to produce antibiotics, two proce- dures have been generally followed. One is based upon the observation of bacterial plates that had become infected with a cul- ture from the outside, usually dust, whereby the growth of the contaminant was sur- rounded by clear zones in which bacterial growth was inhibited. This was true of nu- merous observations made in microbiological laboratories, the most famous of which is that of Fleming on production of penicillin by a mold. The second method consists in isolating various organisms from natural substrates and testing them, by the agar streak method, for their ability to inhibit the growth of other bacteria and fungi. These TABLE 51 Isolation of antagonistic actinomycetes from various substrates (Waksman, Horning, Welsch, and Woodruff) The organisms in group I were the most active antagonists, those in groups II and III had more limited antagonistic properties, and those in group IV showed no antibacterial effects with the meth- ods used. Group I Group II Group III Group IV Source of organiams | Number ot —— al = | | ee: [ees aa |Percentage a eae Cultures | Percentage | Fertile soil ] 20 27.0 5 | 6.8 I |) bes} 48 | 64.9 Infertile soil 75 1 a Say 8 10.7 Teel eae 52 | 69.3 Potted soil 13 1 oll 1 Het || Os Wh Lit $4.6 Soil enriched with E. 21 heave! 4.8 4! 19.0 4 | 19.0 12 eee coli | Soil enriched with bac- 15 jc 2 SOROMa ln ee 138s yo 1 6.7 teria | | Lake mud 9 | 3 | 33.3 4 44.4 | 0 0 2 | 22.2 Compost 37 1 | 2.7 20 HAO wae eel Olas | 12 | 32.4 Total 244 ere On E890) 4 | 44 18.0 (oa) Geass 138 56.6 | | 212 THE ACTINOMYCETES, Vol. I FiGuRE 82. Relative susceptibility of test organisms to antagonistic actinomycetes (H. coli = 100) (Reproduced from: Landerkin, G. B. and Lochhead, A. G. Can. J. Res. 26C: 505, 1948). procedures, with numerous modifications, have found extensive applications in the study of the antagonistic properties of ac- tinomycetes and have come to be known as screening methods. In 1935, the first comprehensive survey for the occurrence of antagonistic forms among actinomycetes, largely soil inhabit- ants, was begun in Russia. Similar surveys of the occurrence of antibiotic-producing microorganisms, especially actinomycetes, were started in the United States in 1939. These surveys influenced greatly the whole subsequent history of antibiotics. General Surveys The first survey of the occurrence in soil of antagonistic actinomycetes was made by Nakhimovskaia. Out of 80 cultures of actino- mycetes isolated and tested, 47 were able to exert antagonistic effects, but only 27 pro- duced active substances. Gram-positive bac- teria were inhibited, but not gram-negative bacteria or fungi. No relation was observed between antagonism and formation of pig- ments, manner of sporulation, or shape of spores. Since the capacity to produce anti- bacterial substances was possessed by only certain cultures, it was suggested that this property could be utilized for the systemati- zation of species of actinomycetes. It was also suggested that various bacteria could be differentiated on the basis of their sensi- tivity to the actinomycetes. Krassilnikov and Koreniako found that many species of actinomycetes, members of the group now considered as the genus Streptomyces, but not of Nocardia, produce substances that are strongly bactericidal against mycobacteria, micro- cocci, and spore-forming bacteria, but not against gram-negative bacteria. Under the influence of these substances, the microbial cells are either entirely lysed or are killed without subsequent lysis. This chemical na- ture of the active substance was believed to be similar to that of lysozyme. nocardias, Kriss isolated a chemical agent from cul- tures of actinomycetes. The substance was found to be insoluble in ether, petroleum ether, benzol, and chloroform, and was re- sistant to the effect of light, air, and high ANTAGONISTIC PROPERTIES 213 TABLE 52 Surveys of antagonistic actinomycetes isolated from soils and other natural substrata (Benedict ) Per cent of total active against ; No. of Bre a ET ices || an, Be positive ive | fast | Pave bacteria lWacterta bacteria} fungi Nakhimov- SO -59. 4] — — skaia Waksman et al. 244 43 =| Welsch 164 46 — — | — Meredith 7642 | | 1 Burkholder 7369 25M) go — | 7 Landerkin et| 660] 48 | 8 | 20 | 2! al. Emerson et al. 772 52 =H o— 47 Rouatt etal. | 544] 39 10 — — Cercos and 54 90 20 60 20 Rodriguez Kuroya et al. | 1223 | 138 TABLE 53 Distribution of antagonistic properties among actinomycetes* (Johnstone) Zone of Per cent of cultures active against inhibition, mm B. subtilis E.coli M.avium M. phlei Nutrient agar 20-35 21 6 6 23 10-19 46 3 35 30) 1-9 3 13 29 16 0 30 78 30 26 Glucose asparagine agar 20-35 15 0) 0 6 10-19 28 6 35 70 1-9 35 14 8 10 0 22 80 57 14 * Cross-streak method used. temperatures. Kriss also believed that the agent was similar to egg-white lysozyme. Waksman ef al. made a detailed sur- vey of actinomycetes possessing antago- nistic properties (Waksman, 1937, 1941, 1945). This led directly to the isolation of the first antibiotic produced by a member of TABLE 54 Distribution of antibiotic activity of actinomycetes according to genus (l8merson et al.) Genus Strepto- Micro- Nocar- myces monospora dia Tested against bacteria: Number active 399 I Number inactive 370 2 Number tested 769 3 Tested against fungal pathogens: Number active 353 i) Number inactive 399 6 8 Number tested 752 6 8 Tested against both bae- teria and fungi: Number active against - bacteria only 149 fungi only 109 both 233 neither 237 number tested 728 this group of organisms, namely, actinomy- cin. Antibiotic-producing actinomycetes were found to be widely distributed in na- ture, especially in soils and in composts. Two hundred and forty-four cultures were iso- lated and tested. Of these, 106 or 43.4 per cent, possessed some antagonistic properties, and 49, or 20 per cent, were highly antago- nistie (Table 51). An examination of a large series of well-identified cultures of actino- mycetes kept for a number of years in a type culture collection showed similar rela- tions (Welsch). The antagonistic forms were found to belong largely in the genus Strepto- myces. Burkholder isolated 7,369 cultures of ac- tinomycetes from soil. Of these; 1,869 inhib- ited growth of Staph. aureus, 261 inhibited L. coli, and 514 showed an antagonistic effect against C. albicans. With the growing interest in antibiotics throughout the world, numerous other sur- veys have since been made, as summarized by Benedict (Table 52). Most of these re- 214 vealed that 20 to 50 per cent of all the cul- tures of actinomycetes tested possessed an- tagonistic properties. In some surveys the percentage was higher, in some lower. Stapp, for example, reported that 233 out of 477 cultures of streptomyces isolated from soil were active against B. fusiformis. The nature of the medium used for testing purposes is of great importance, as shown by Johnstone (Table 53). Most of the cultures were active against gram-positive, including acid-fast, bacteria; fewer were active against gram- negative bacteria and fungi. The majority of antibiotic-producing actinomycetes are found among the streptomycetes (Table 54). Landerkin and Lochhead isolated from different soils 50 actinomycetes antagonistic to E. coli. When tested against different bacteria, those actinomycetes that possessed “the most intense antibiotic activity”? were also active upon “‘the greatest number of bacterial species,” in other words, had the widest antibiotic spectrum (Fig. 82). A de- tailed analysis of the results obtained by Landerkin et al. is presented in Table 55. Here as well, fewer organisms were active upon fungi than upon bacteria; of the latter, the gram-positive forms were most suscep- tible, the gram-negative least. There were ) TABLE 55 Antibiotic activity of 600 actinomycetes isolated from northern Canadian soils (Landerkin, Smith, and Lochhead) | Degree of activity* Number of cultures | active against | i Inac- Total |Per cent | | tive | active| active Staph. aureus | 140 | 84 | 87 | 349 | 311 | 4 ~I — E. coli Paes eg COG.) “seal Soe Ps. aeruginosa | Oo On 8° -657 33 ff, Ws M. tuberculosis | 8 | 28 |) 98 | 531 | 129 | 19.5 H. sativum 35 | 43 | 39 | 543 | 117 | 17.7 PF. lint | 2 | 14 | 46 | 598] 62) 9.4 30 mm or more; mm; inactive = * 111 = diameter of zone, ++ = 20-29 mm; + = 10-19 less than 10 mm. THE ACTINOMYCETES, Vol. I also marked differences in the degree of sen- sitivity within each group; many more or- ganisms were active upon F. coli than upon Ps. aeruginosa. Of 1,117 cultures of actinomycetes tested by Aleshina and Makanovskaia, 44 per cent were active against staphylococci and 19.4 per cent against the plague organism. Of 170 cultures isolated and tested by Mukherje and Nandi, 40 per cent were active against gram- positive bacteria, 21.1 per cent against gram- negative bacteria, and 31.7 per cent against fungi. Different isolates belonging to the same species varied in their antagonistic properties. Some of the cultures that exhib- ited antagonistic effects when tested by the agar streak method, did not form, in liquid media, substances with the same kind of ac- tivity. In a study of 70 strains of actinomycetes for their antagonistic effect upon root-nodule bacteria, Fogle and Allen found that 25 LEGEND \" ree" ————is Activity Index Figure 83. ‘‘Antibiotie index’’ of actinomy- cetes in relation to depth of soil (Reproduced from: Landerkin, G. B. et al. Can. J. Res. 28C, 696, 1950). ANTAGONISTIC PROPERTIES 215 strains were antagonistic to Rh. lupini. Other species of Rhizobium were inhibited by fewer strains: Rh. trifold was inhibited by only 9 strains, and the cowpea rhizobia by only 1 strain. In 1952, Poppe and Strutz isolated, from soil and other matter, 220 cultures of actino- mycetes. Of these, 62 possessed antibacterial properties. Ten of the active cultures were found to exert a strong effect upon various gram-positive bacteria. Glycerol-glycocoll solution proved to be especially useful in the production of the antibiotic substances. Lindner and Wallhdusser (1955) isolated 2,500 antagonistically active cultures. of streptomyces from 40,000 soil samples. Their distribution in different soils is shown in Table 56. Of these, 77 per cent were active upon gram-positive bacteria (Staph. aureus), 40 per cent upon gram-negative bacteria (L. colt), 32 per cent upon mycobacteria (J. tuberculosis 607), and 18 per cent upon fungi (A. niger). Wallhausser (1951) proposed a series of charts for representing the mode of growth inhibition of one organism by an- other. The results obtained in these surveys were found to depend upon the test organisms used, the composition of the media upon which the organisms were grown, and various experimental conditions. Most of the active cultures belonged to the genus Streptomyces. However, Endo, who tested the activity of 116 strains of Nocardia upon 10 bacteria and 6 fungi and yeasts, found that 27 per cent of all strains were active upon at least one of the test organisms. The conclusion was reached that the Nocardia group has as high percentage of active strains as Streptomyces. The genus MWicromonospora as well was found to be capable of exerting antagonistic effects against certain bacteria. Craveri et al. (1957) isolated 500 cultures of streptomyces from various soils in Italy. Just about a half had the capacity to inhibit microbial growth on solid media. Among the TABLE 56 Distribution of antagonistic streptomyces (Lindner and Wallhiusser) Nature of soil and vegetation Per cent of active strains Composts 8.6 Garden soils 1.4 Field soils 14.8 Pastures 18.6 Forest soils fee Brown soil 74 River muds 3.8 Virgin soils 22.2 latter, about one-third produced an antibi- otic substance when grown in liquid media under submerged conditions. The percentage of the cultures active upon gram-positive bacteria only was higher than that of strains active upon both gram-positive and gram- negative bacteria. No culture was found to be active only upon gram-negative bacteria. Of the cultures active only upon gram-posi- tive bacteria, about 20 per cent were active upon B. subtilis, 20 per cent upon Staph. aureus only, and about 60 per cent upon both. Jarikova et al. (1958) isolated 1,879 actino- mycete cultures from soils of different re- gions of the Soviet Union; of these, 1,262 cultures, or 67.2 per cent, proved active un- der the conditions of their experiments. The largest number were found in the greenhouse soil of the Botanical Garden, in meadow- granular soils, and in steppe soils, as well as in light chestnut soils. The greatest number of cultures having activity were found in soils of eastern and southern regions. Attention must be directed to the fact that generalizations concerning the charac- terization of certain types of soil by the oc- currence of specific antagonists are hardly justified unless they are based upon detailed and oft-repeated investigations. Antifungal Surveys The ability of various actinomycetes to antagonize the growth of fungi has long 60 ll a Y ge 3 el ro) ® 40 | ro) 30 w ro) = 5 Z 20 ° « Ww a ro) B. subtilis P. vulgaris THE ACTINOMYCETES, Vol. I 60 % CAUSING SLIGHT INHIBITION % CAUSING STRONG INHIBITION 50 % CAUSING VERY STRONG INHIBITION TOTAL PERCENTAGE CAUSING INHIBITION 20 m P. aeruginosa . coli BACTERIA AS TEST ORGANISMS Figure 84. Degree of antibiotic activity with bacteria as test organisms (Reproduced from: Emerson R. L. et al. J. Bacteriol. 52: 362, 1946). been known. Major attention was directed toward their activity upon plant pathogenic fungi (Tius, 1932; McCormack, 1935). Alexopoulos was the first to study, in 1941, the distribution of antagonistic activities among actinomycetes against fungi. Of 80 cultures tested against Colletotrichum gloeos- portoides, 17.5 per cent were strong inhibi- tors, 38.8 per cent were weak inhibitors, and 43.7 per cent had no effect at all. Meredith made a survey of the distribution of organ- isms antagonistic to Fusarium oxysporum cubense in Jamaica soils. The antagonists were not evenly distributed, 10 of the 66 soil samples giving 44 per cent of the antagonistic organisms. Those actinomycetes that were antagonistic to Fusarium when grown in their own soil-solution agar were not always antagonistic when tested in soil-solution agar prepared from another soil. A culture of an actinomycete isolated from a compost pro- duced lysis of the Fusarzum. When spores of both organisms were mixed in an agar medium, the fungus developed normally for 2 days but began to undergo lysis on the fifth day, large sections of the mycelium disappearing. On the seventh day only chlamydospores were observed. In 9 days the fungus completely disappeared, whereas the actinomycete was making a normal growth. Leben and Keitt isolated a streptomyces that was antagonistic to various phytopath- ogenic fungi, but not to most bacteria. The active material was heat-labile, soluble in various organic solvents, and in water at pH 9.3. It inhibited the growth of fungi and of only very few bacteria. Of 3,788 actinomycete cultures isolated from soil by Cooper and Chilton and tested against Pythiwm, 896, or 23.6 per cent, showed some antagonistic ef- fect upon the fungus. Certain actinomycetes were found to be responsible for the destrue- tion, in soil, especially in partly sterilized soils, of the mycelium of Ophiobolus graminis, the cause of the take-all disease of wheat. The parasitizing and antibiotic effects of actinomycetes and other soil organisms were ANTAGONISTIC PROPERTIES 217 believed to be responsible for the check in the development of the Ophiobolus in natural soils. Stessel studied the distribution in soil of pathogenic active against fungi. Dilutions of soil samples were added actinomycetes to suitable agar media, so as to permit 80 to 50 colonies to develop on each plate. The plates were incubated for 4 days at 26°C. Suspensions of spores of four mutually non- antagonistic phytopathogenic fungi were then sprayed on the plates. After 2 days’ further incubation, 170 cultures of actino- mycetes producing marked inhibition zones against all the test fungi were isolated. Plate cultures of the isolates were sprayed again but with separate suspensions of eight fungi and two bacteria. Twenty-one cultures, mostly actinomycetes, were selected on the basis of comparative inhibition. These or- ganisms were grown in five liquid media in shaken flasks. The culture filtrates were tested for activity against Glomerella cingu- lata, by the use of a modified cylinder plate method. Sixteen cultures produced an anti- biotic in one or more media. Of the 170 organisms originally isolated, five appeared to produce desirable substances, as deter- mined by the above screening tests. Further studies on the antagonistic effects of actino- mycetes upon plant pathogenic fungi have been made by Mukherjee and Nandi (1955) and others. Some actinomycetes are particu- larly active against yeasts (Takahashi, 1952). The activities of soil actinomycetes upon fungi pathogenic to man were examined by Schatz and Hazen. Pridham et al. (1956) made an extensive survey of actinomycetes, largely strepto- myces strains, for their growth-inhibiting effect against phytopathogenic bacteria and fungi. About 500 pure cultures were sub- jected to primary screening by three meth- ods against a minimum of 12 test organisms. Based on their antimicrobial spectra, some 200 strains were selected as warranting fur- ther study and were grown in a variety of media in shake-flasks. The culture filtrates were tested for antibiotic activity by paper- dise assay against nine organisms. Fifty-two of the 200 strains showed promise. Culture filtrates from eight strains as well as two substances isolated from the mycelium of different then the greenhouse against certain plant diseases. Nine of the 10 preparations showed broad antifungal spectra in laboratory tests and activity against at least one disease in green- house tests. Phytotoxic effects were observed with some of the test materials. The details of the test are brought out in Table 57. strains were tested in Correlation of Antimicrobial Activity and Pigmentation von Plotho correlated pigment production by actinomycetes with their antagonistic properties. Two hundred and ninety-one cul- tures grown in colorless media were classi- fied into four groups on the basis of pigments produced either in the mycelium or in the medium. Activity was determined by testing against Mycobacterium eos. Of the 61 cultures (21 per cent) showing activity, 21 were in the colorless group, 20 were in the red-yellow, 12 in the red-blue, and 8 in the red-brown group. By using media in which pigments could be seen readily, investigators might well try such a correlation to learn whether particular groups could be eliminated with- out further testing. A detailed analysis of the antibiotic pigments of actinomycetes was presented in Chapter 13. Screening for Specific Antibiotic- producing Organisms By using the technique reported by Waks- man, Reilly, and Johnstone (1946) of adding a particular antibiotic to the medium before plating out soil samples, Umezawa ef al. (1949) were able to demonstrate the presence 218 THE ACTINOMYCETES Vol. I TABLE 57 Inhibitory activity of streptomyces against selected phytopathogenic bacteria and fungi (Pridham et al., 1956) Test organism No. of strepto- myces tested No. of strains that produced inhibition zones with indicated diameter 0 mm 0-10 mm 10-20 mm 20-30 mm _ 30-50 mm Agrobacterium tumefaciens 482 389 65 24 4 0 Bacterium stewartii 479 337 81 28 20 13 Corynebacterium fascians 479 170 106 121 52 30 Erwinia carotovora 381 337 32 11 1 0 Pseudomonas phaseolicola 477 327 59 56 21 14 Xanthomonas phaseoli 680 490 90 72 Wi 11 Ceratostomella ulmi 320 284 14 2) 2 0 Cladosporium herbarum 319 257 31 21 | 0 Gibberella fujikurot 66 21 26 1s 1 0 Helminthosporium sp. 23 1 i 14 1 0 Mucor rammannianus 444 295 24 64 38 23 Trichoderma viride 320 300 17 1 2 0 Ustilago zeae 318 283 12 14 9 0 Fusarium oxysporum f. lycopersict + CL. 246 88 1 93 4] 23 herbarum Helminthosporium sp. + G. fugikurot 66 27 22 15 2 0 Helminthosporium sp. + T. viride 297 162 6 70 29 30 T. viride + G. fujikurot 164 84 14 27 29 10 Ustilago zeae + Ceratostomella ulmi 158 25 0 23 49 61 of a rather large number of specific anti- Routien and Finlay emphasized that biotic-producing strains. They found such strains to be present in four of the five soils tested. This would seem to indicate a wide distribution of such organisms. In the process of screening thousands of soil samples from widely scattered geograph- ical areas, Routien and Finlay found or- ganisms producing certain antibiotics to be extremely common. Actinomycetes elaborat- ing streptomycin, streptothricin, chloram- phenicol, actinomycin, and xanthomycin- like antibiotics apparently have a world-wide distribution. The tetracycline-producing cul- tures have been isolated only a few times. One antibiotic was observed from only one culture isolated from one particular’ soil. These investigators found that certain anti- biotics are produced by cultures of actino- mycetes common in soils from somewhat localized areas. On the other hand, soils col- lected within a restricted area were found to yield a number of different antibiotics. variations in the degree of activity of dif- ferent cultures are frequent. Some cultures may decline in potency. Some cultures may produce only small quantities of an anti- biotic, which, on concentration, may be found to possess new and useful properties. The wide distribution of antagonistic properties among actinomycetes has thus been definitely established. Members of the genus Streptomyces are most active. They oecur abundantly in soil (Rouatt ef al., 1951). Even the plant pathogenic S. scabies pos- antagonistic properties (Ark and Oswald), although this organism, as well, is subject to the antagonistic action of certain fungi (Daines, 1937). Various nocardias are ‘sapable of exerting an antagonistic action, as shown by Uesaka and by others. In summarizing the results of evaluation of antibiotic activities of 10,000 streptomyces cultures isolated from various substrates, Woodruff and MeDaniel stated that, on the sesses ANTAGONISTIC PROPERTIES average, 25 per cent of the isolates were found to produce an antibiotic. Approxi- mately 90 per cent of all the antibiotic-pro- ducing cultures formed streptothricin or closely related compounds. A half of the remainder produced streptomycin, one-third (of the other half) formed tetracyclines. Finally, most of those that remained could be identified with one of the various anti- biotics which have been isolated from cul- tures of streptomycetes. In other words, 2,250 cultures gave streptothricin-like anti- bioties, about 125 streptomycin, 40 yielded tetracyclines, 55 produced other previously described antibiotics, and only 30 yielded new antibiotics. Test Organisms A wide selection of organisms is commonly used in testing cultures for antibiotic ac- tivity. Frequently, a highly sensitive or- ganism like B. subtilis or Staph. aureus is used. In a search for a specific antibiotic against a particular parasite, such as J. tuberculosis, the latter itself or a form closely related to it is used as the test organism. In most cases, however, various organisms are used, including gram-positive and gram- negative bacteria, saprophytic and parasitic fungi. Plate method techniques have been developed by Waksman and Reilly (1945), Waksman et al. (1947), and Williston e¢ al. (1947). Recently, actinomycetes possessing ac- tivity against viruses and experimental cancers have been investigated. For this purpose, special procedures are used, such as those involving bacteriophages (Jones and Schatz; Schatz and Plager, 1948). Out of 527 cultures of actinomycetes screened by Landerkin et al. (1957) for antiphage ac- tivity, eight inhibited the development of bacteriophages; of these eight, only one had an effect upon phages of two species of bac- teria. None of the cultures tested had any effect upon the Newcastle virus. 219 Krassilnikov and Kofanova reported that nearly all actinomycetes possess antiphage properties. Some inhibit many phages and others are effective only upon specific phages. Actinomycetes show a characteristic anti- phage spectrum. There is no relation between antivirus and antiphage antibiotics; there- fore, the latter cannot be used as models for study of antivirus agents. An interesting 77 vitro technique utilizing a modified agar plate diffusion method for the detection of antitumor activity was described by Miyamura. Ehrlich’s ascites tumor cells are incorporated into a_ basal medium. The materials to be tested are placed in cups and allowed to diffuse through the medium; the cups are then removed and the plate is flooded with methylene blue; the agar is covered with a glass plate and incu- bated; the diameters of the blue zones are measured. Positive results were obtained with various anticancer agents including the antibiotics trichomycin and sarkomycin and 8-azaguanine. Other antibiotics of actino- mycetes (tetracyclines, streptomycin, and neomycin) gave negative results. Arai and Suzuki published a modification of the above method. Serial dilutions of the materials to be tested are placed in tubes. The tumor cells are added, the suspension is mixed thoroughly, and then buffered glucose agar containing methylene blue is added. The results are read after 3 hours’ incubation. All antitumor substances tested, except 8-azaguanine, gave positive results. These substances included sarkomycin, carzinoph- ilin, the gancidin complex, and others. Rombouts (1953), Stevenson (1954), and Links et al. (1957) isolated certain cultures of streptomyces from soil that produced an antibiotic possessing the capacity of causing the swelling of hyphae of several fungi. The substance responsible for this swelling was designated as the ‘‘bulging factor.’’ This sub- stance, isolated from the medium, proved to be a streptothricin-like base readily solu- 220 ble in water, but not in organic solvents. It was stable in an acid reaction, but was rapidly destroyed in an alkaline reaction and at 100°C. The only report of a upon actinomycetes and not upon true bacteria or fungi was made by Szabo and Marton (1955), who isolated from a culture of an actinomycete an ‘‘anti-actinomycete factor’ that was highly active against ac- tinomycetes, without any activity upon gram-positive and gram-negative bacteria. substance active Effect of Actinomycetes upon Sapro- phytic Soil Bacteria The effect of actinomycetes upon the growth of soil saprophytes and upon plant pathogens has also received considerable attention. Nikolaieva first observed, in 1914, that actinomycetes may exert a repressive effect upon the growth of Azotobacter. Nickell and Burkholder (1947) studied the inhibition of growth of Az. vinelandii by various ac- tinomycetes isolated from soil. When cul- tures of actinomycetes were mixed with soil samples and plated out, the nitrogen-fixing bacteria were either greatly reduced in num- ber or completely eliminated. It was sug- gested that this serves to illustrate the importance of microbial antagonism in eco- logical investigations. Another antagonistic effect of actinomy- cetes upon nitrogen-fixing bacteria may prove to be of even greater economic im- portance. Konishi and Fukuchi have shown that certain actinomycetes are able to in- hibit the growth of root-nodule bacteria; some of the cultures, like S. flavus, were par- ticularly effective. Babak (1958) tested the sensitivity of 60 Azotobacter cultures to various species of Streptomyces and to various antibiotics, such as penicillin, streptomycin, gramicidin, and chlortetracycline. They were all found to be susceptible to the antibiotics. Their sensitivity to the S. coelicolor group was THE ACTINOMYCETES, Vol. I dissimilar; some strains proved inhibited and others indifferent. When the antimicrobial properties of ac- tinomycetes became well recognized, it was only natural that attempts should be made to encourage the development of these or- ganisms in the soil, in order to depress the growth of various organisms capable of causing plant diseases. Activity of Actinomycetes upon Plant Pathogenic Fungi An extensive literature has accumulated upon the antagonistic effects of actinomy- cetes upon fungi, especially upon plant pathogens. References to such effects are reported earlier in this chapter. Winter pre- sented (1949) further evidence concerning, the ability of various actinomycetes to at- tack Ophiobolus graminus, an important parasite that attacks wheat. Sanford and Cormack tested the effect of eight cultures of actinomycetes upon the disease-producing fungus Helminthosporrum sativum. In com- parison with a disease rating of 66 per cent for the untreated pathogen, four actino- mycetes depressed the virulence of the pathogen to 33, 22, 22, and 1 per cent, re- spectively; two had no marked effect; and the other two appeared to increase the viru- lence by 12 and 16 per cent, respectively. Perrault demonstrated that the growth of Colletotrichum sepedonicum in agar media was impeded by several microorganisms 1so- lated from potato tubers affected with ring rot. Four of these organisms were actino- mycetes and were able to produce antibiotic substances that diffused readily through the medium and prevented all growth of the pathogen. One culture produced a lysis of the plant pathogen. MeGahen treated soils in the sugar-cane belt of Louisiana with bagasse compost, with cowpea and soybean trash, and with blood- meal, tankage, bonemeal, and ammonium sulfate. The actinomycete populations were ANTAGONISTIC PROPERTIES 221 measured by the dilution methods; the anti- biotic activities were determined by the de- gree of inhibition of Pythium arrhenomanes in culture. Nitrogen-rich materials, such as cowpea and soybean trash, bloodmeal, and tankage, gave marked increases in the ac- tinomycete populations. The antibiotic index was increased by the use of bagasse compost and cowpea-soybean trash; a decrease of the antibiotic index occurred, however, when the soil was treated with bonemeal, bloodmeal, or tankage. Ammonium sulfate did not ma- terially affect either the population or the antibiotic index. It was suggested that nitro- gen-rich materials, like bloodmeal and tank- age, cause an increase of the nonantagonistic and /or weakly antagonistic actinomycetes in favor of the moderately and highly antago- nistic forms. The cellulosic materials cause an inerease of the antagonistic over the nonantagonistic actinomycetes. Antagonistic Effects of Fungi and Bac- teria upon Actinomycetes In a natural environment, such as soil, the antagonistic properties of actinomycetes, if they develop at all, will be exerted largely in an aerobic environment. Under anaerobic or microaerophilic conditions, the actinomy- cetes themselves may be antagonized; bac- teria, like Ps. fluorescens, have been shown to exert a marked antagonistic action upon actinomycetes, causing their lysis. B. mega- terium can also be antagonistic to certain species of actinomycetes, but also can be antagonized by others. The effect of bacteria upon the potato scab organism was studied in detail by Kieszling, who was able to pre- vent scab development in the soil by the use of such bacterial cultures. Numerous fungi are also capable of exert- ing a marked destructive effect upon actino- mycetes. This is true, for example, of the ef- fect of the fungus antibiotic penicillin upon human and animal diseases caused by actino- mycetes. Are Antibiotics Produced in the Soil? In 1945, Waksman presented evidence that it is highly doubtful that antibiotics are produced in the soil itself or that this phe- significance in nomenon is of any modifying the microbiological population of great the soil. The evidence was based upon the following observations: (1) The fact that an organism produces an antibiotic in artificial culture is no evidence that is is capable of doing so in soil, particu- larly since relatively small changes in a nutrient medium may fundamentally affect the production of antibiotics in pure culture. (2) Many known antibiotics are extremely unstable and could not be expected to re- main unchanged in soil for sufficient time to have any effect. (3) There is no evidence that production of antibiotics affects in any way the survival of organisms producing them. (4) The fact that the organisms found in the soil possess no greater resistance to particular antibiotics than comparable strains found in other substrates add further weight to the non-existence of such antibi- otics in soil, in concentrations sufficient to exert an effect. Brian, Krassilnikov, and others argued against these assumptions. Brian emphasized that some antibiotics are very stable, that they can be produced locally, where they can have a maximum effect. Siminoff and Gott- heb (1951, 1952) could demonstrate the for- mation of antibiotics, notably streptomycin, in sterile soil but not in fresh soil. Pramer and Starkey established that streptomycin is rapidly destroyed in the soil. Some recent evidence that antibotics are produced in soil has been submitted by Stevenson (1956). In most cases, however, the assumption that the occurrence of streptomyces capable of producing anti- biotics will lead to the formation of such antibioties in soil, and that this will lead to the elimination of pathogenic bacteria is far- 222 fetched. Such assumptions have been made, for example, for the elimination of typhus abdominalis bacteria in irrigated _ soils, merely because several streptomyces have been isolated from such soils. Soil Enrichment with Pathogenic Or- ganisms Soil enrichment with various chemical compounds results in the isolation of organ- isms capable of bringing about certain spe- cific reactions. This is true, for example, of the development of organisms capable of de- composing cellulose or pectin by the addi- tion of these substances to the soil; it is also true for the enrichment of soil with sub- stances like sulfur and ammonia to en- courage the development in the soil of sulfur- oxidizing and nitrifying bacteria. It was at THE ACTINOMYCETES, Vol. I first believed that the same principle would apply to the isolation of antibiotic-producing organisms. Claims were actually made that favorable results were obtained. But the in- troduction into the soil of cultures of micro- organisms led to their destruction (Katznel- son, 1940). Waksman and Woodruff (1940) attempted to encourage by soil enrichment the develop- ment of antibiotic-producing actinomycetes. They at first believed this had succeeded. They said that the survival of bacteria added to the soil, but not indigenous to it, resulted in the rapid dying out of such added bac- teria. This was believed to have been accom- panied by an increase in the numbers of soil bacteria and actinomycetes capable of de- veloping on the plate. On further additions of cultures of bacteria to the soil, more rapid TABLE 58 Growth inhibition of streptomyces by their respective antibiotics (Waksman, Reilly, and Johnstone) Nature _ of antibiotic Organism producing antibiotic a. Dilution units per mg, Activity of prepar- expressed as activity against ation per 1 gm S. antibioticus S. lavendulae S. griseus Actinomycin S. antibioticus 100 ,000* 100 5,000 100 Streptothricin S. lavendulae 1007 1,000 0.4 10 Streptomycin S. griseus 1257 1,000 100 1.2 * §. lutea units; crystalline material. + £. coli units; crude preparations. TABLE 59 Cross-resistance among different streptomyces (Krassilnikov) Antagonist = 5 fivats Bl Test organisms Ss Ss Es 3 2 3| 2 but, iz | 4 n 2p) vy | n n S. violaceus —|/+ {c+ | Se Ny Es eet |p S. coelicolor SS | ano aga | a S. ruber | Bigs SE et align eevee S. griseus +/+})4+]/—-|/+]-/- S. globisporus SEP | Pea PAC) Se eee |e ee er S.longisporus |} +/+]/+/+]/+]-—]+ S. roseus | eae ee | | Se S. albus host 4 = Prt ean Reha) A destruction of the added organisms resulted. The nature of the antagonists developing in the soil depended upon the bacteria added, the soil treatment, and the temperature of incubation. Several antagonists were isolated from the soil. Sterile culture filtrates of these antagonists were capable of reducing con- siderably the numbers of bacteria and ac- tinomycetes in the soil. A highly active sub- stance was obtained from a soil actinomycete. Further studies (Waksman and Schatz, 1946), however, on the enrichment of soil with suspensions of living gram-negative bac- teria and with both living and dead tubercu- losis organisms resulted in complete failure ANTAGONISTIC PROPERTIES to isolate cultures of actinomycetes that might possess specific activity against the organisms added to the soil. The writer received numerous suggestions that he use soils from cemeteries in which sufferers from fectious diseases or cancer victims were re- cently buried. He can do no better than to quote from Routien and Finlay, who fol- lowed up such suggestions. They attempted on two oceasions to isolate antagonistic or- tuberculosis and other in- ganisms from such sources. Soil from an orthodox Jewish cemetery, where bodies were interred soon after death without embalming, Was examined for antagonists against the pathogens introduced into the soil with the diseased body. TABLE 60 Cross-resistance of antibiotic-producing actinomycetes (Teillon) Secondary streaks Theoretical results Experimental results EX sy (Coe 1b) IX 1s (1D) Original streak* A O++ + 00 0 + B + 0 + 4+ 0 0+ + C ++ 0+ 4 + 0 + D ++ + 0+ 4+ 4+ 4+ Ae— S. 72mosus; Bb —)S. griseus; ©) — |S. aure- ofaciens, D = S. fradiae. 223 Soil samples and earthworms were dug from the graves, but the cultures obtained “were disappointingly devoid of interesting antagonists.”’ Soil samples were also taken along the route of a sewage effluent from a sanatorium for tuberculous patients. ‘“Sam- ples of the soil taken along this channel of treatment from the raw sewage to a point 50 yards downhill yielded no organism of high antagonistic powers.” Cross-resistance of Microorganisms as Diagnostic Criteria Waksman, Reilly, and Johnstone (1946) first demonstrated that actinomycetes are, as arule, resistant to theantibiotics produced by them (Table 58). However, some or- ganisms tended to be less sensitive to certain antibiotics than to others. These investi- gators suggested taking advantage of this phenomenon for the isolation from the soil of specific organisms producing a particular antibiotic, and for isolation of more potent antibiotic-producing strains from a mixed mother culture. It was later shown (Waks- man and Lechevalier) that certain organisms, like S. fradiae, are sensitive to their own antibiotics. Krassilnikov accepted the phe- nomenon of cross-resistance within the species of actinomycetes as offering a de- TABLE 61 Cross-streak tests with 12 strains of 8S. fradiae on yeast-glucose agar (Waksman et al., 1958) Zones of inhibition, mm Original streak 3535. 3554. 3556a_=3556b 03572, 3594. 3719 = 3566) 3595 3598 93596 ©3597 3535 on0 leone 0 0) alae (Olea) Abe t0) shay ae ayets), lel) (0) 3504 0) ase UO say De LEO! eG aI) AAO) Ws Oey 39068 Mee) Icy pill) Pasty ase) iss) G0) lg) By UL Oe. eo) 3556b ety Paolo ORG) Sica) ha). 0) A). ash ea ZEA) 3072 Wa) Weer IO yay Ye P30) ANA) Page UGG PO) Waly) (ete 3094 eas lla Oe kN PEGS they) PR) al “Waa C)o8} 3719 4.0 PON Ou nom .0) OS 300) on0e 40) G20 3:05 Led 3966 256) 14s Ube OF 14O ALG 4.0) 120) 223) 320) IN6s Fans B95 1D: On ORGAO 223162 6el626 = 70) 0-6) 073 0F6 0 0 3998 LOOM it OU Oe SOLO 222608 180i 720006 30 2.0 0 0 3996 AO Ao, od enol a Oso0 16:0). 20) 10 0.6 0 0 0 3097 O23) LOL G reo 1ORG OG 9) OF aOR sna LOns0 0 0 224 sirable property for species differentiation (Table 59). Umezawa, Oki and Hata, Kuroya, and others also accepted this con- cept for classifying actinomycetes. Teillon (1952), who made a detailed study of the cross-resistance of actinomycetes pro- ducing antibiotics, found various important exceptions to the principle expostulated by Kuroya and Krassilnikov (Table 59). On the basis of these results, he expressed doubt concerning the validity of the claim of the Japanese investigators that “the cross-inhibi- tion test is a useful and easy method of dif- ferentiation.””’ The method of cross-inhibi- tion did not appear to Teillon as sufficiently reliable for differentiating streptomyces cul- tures and their antibiotics. The organisms were shown to form a variety of metabolic products that could readily mask the results. Further, some organisms produced certain other antibiotics that, although in them- selves possibly not quantitatively significant, THE ACTINOMYCETES, Vol. I modified the effects of the major antibiotics produced by the organisms. Some strains related to S. griseus were able, for example, to produce streptothricin as well as strepto- mycin. Phenomena of autoinhibition are also not uncommon among actinomycetes, such as S. aureofaciens and S. fradiae. Finally, the quantitative variations in the production of antibiotics do not permit the laying down of hard and fast rules concern- ing the possible effect of cross-inhibition un- der natural conditions. This is brought out in Table 61 on the auto- and cross-inhibition of various neomycin-producing and other strains of S. fradiae. It is possible to observe occasionally certain self-inhibition of growth by various actinomycetes, especially by pig- ment-forming types. This has been ascribed by Krassilnikov et al. (1958) to special sub- stances of an enzymatic nature designated ce as “neerohormones.”’ Cu AP OR Production of Antibioties Isolation of Antibiotic-producing Cul- tures The actinomycetes occupy a leading place among the antibiotic-producing groups of microorganisms. Already, they have yielded nearly 500 compounds and preparations, in- cluding some of the most important chemo- therapeutic agents now known. Some _ of these antibiotics are active only on bac- teria, others only on fungi; some are active on viruses and phages, and others are ac- tive on tumors. Some of the antibiotics are said to be of a broad spectrum type, able to repress the growth of both bacteria and fungi, or of bacteria, rickettsiae, and the larger viruses. Some have a narrow antibi- otic spectrum, such as those that are largely active against gram-positive bacteria or my- cobacteria, or yeast-like organisms (Waks- man, 1955). Among the antibiotic-producing actino- mycetes, the genus Streptomyces occupies a leading place. Certain groups of this genus, like S. griseus and S. lavendulae, are char- acterized by the formation of a large number of different antibiotics. Some antibiotics or closely related groups of antibiotics (anti- biotic complexes) are produced by different organisms. This is true, for example, of the streptomycin and neomycin complexes. There are also marked quantitative vari- ations in the production of antibiotics by different strains of the same organism. Com- position of medium and environmental con- 225 ditions play an important part in this con- nection. A number of the antibiotics produced by actinomycetes have been isolated in a pure state, their chemical structures established, and their antimicrobial properties studied in detail. Other antibiotics have been described only as preparations, or in such a preliminary manner that it is not certain whether one is dealing with a single entity or with a group of closely related chemical compounds. Only one of the actinomycete antibiotics, chloram- phenicol, has been synthesized. Others have been modified chemically to give compounds with slightly different types of activity. This is true of the formation of dihydrostreptomy- cin from streptomycin and of tetracycline from chlortetracycline. Definite chemical structures have been established for a num- ber of antibiotics, notably the streptomycins, cycloheximide, the actinomycins, the tetra- cyclines, cycloserine, azoserine, erythromy- cins, and sarkomycin. These were found to comprise a variety of highly interesting or- ganic structures, some of which have never been known before. Principles of Antibiotic Production Certain general principles concerning the production of antibiotics by actinomycetes have been established: 1. Different strains of an antibiotic-pro- ducing species may form several antibiotics that are not related chemically: S. griseus 175 THE ACTINOMYCETES, Vol. I ACTINOMYCIN G/ML I50 = = oO fe) 125 ie O = 100p = 2 = uJ 5 et =) > = 50 X “x RESIDUAL GALACTOSE & lay oct 25 0) (0) 2 4 6 8 10 DAYS Ficure 85. Utilization of galactose by S. antibioticus for growth and actinomycin production: A——A = dry weight of mycelium in mg per 100 ml of medium; X——X = percentage of residual galactose; O——O = actinomycin produced, ug/ml of medium (Reproduced by special permission from: Katz, E., Pienta, P., and Sivak, A. Appl. Microbiol. 6: 238, 1958). strains produce streptomycin, streptocin, cy- cloheximide, and grisein; S. fradiae strains produce neomycins and fradicin; S. rimosus produces oxytetracycline and rimocidin. 2. A single strain of an antibiotic-produc- ing organism may form several chemically related antibiotic substances. This is true of the production of mannosidostreptomycin and streptomycin by S. griseus; of neomycin B, and C by S. fradiae; of viomycin A, B, and C by S. vinaceus. 3. Organisms producing the same antibi- otic or closely related compounds are found in different soil regions throughout the world. This is true, for example, of cultures of strep- tomyces producing actinomycin, streptomy- cin, chloramphenicol, the tetracyclines, the erythromycins, and others. Since the strains PRODUCTION OF ANTIBIOTICS 227 producing the same antibiotic may vary, the morphological criteria alone cannot be used as a basis for identifying antibiotics, or the formation of a particular antibiotic for iden- tification of species. 4. A change in the nutrition of the organ- ism may result in a change in the nature of the antibiotic produced. This is true, for ex- ample, of the formation of the various actino- mycins. 5. As a rule, antibiotic-forming organisms are resistant to the antibiotics they produce, as shown in Chapter 14. This phenomenon is taken advantage of in the isolation of fresh cultures capable of forming a given antibi- otic, and in the selection, from a given cul- ture, of more potent strains. Not all anti- biotie-forming organisms, however, behave in the same manner. Methods for Isolating and Testing Antibiotic- producing Organisms In a search for antibiotic-producing cul- tures of actinomycetes, certain steps are usually followed: 1. A sample of soil is plated out on suit- able agar media. Usually, simple synthetic or poor organic media are selected to en- courage the maximum development of colo- nies but prevent these from making heavy growth, thus avoiding overcrowding. 2. After a given period of incubation, which may vary from 3 to 15 days, the colo- nies of the actinomycetes developing on the plates are picked and transferred to fresh agar slants. If care is taken in making the transfer, pure cultures are usually obtained. Otherwise, the cultures may have to be puri- fied by replating and reisolating. 3. The cultures thus obtained are tested, by the agar-streak method, for their ability to inhibit the growth of microorganisms. Various bacteria, comprising gram-positive and gram-negative forms, and fungi are com- monly used as test organisms. In some cases, especially in search for antiviral or antitu- mor agents, more complicated procedures are employed. Such cultures of actinomycetes as are found to possess the desirable anti- microbial properties are selected for further study. 4. The cultures are various liquid media, in stationary or under submerged conditions, for varying periods (usually 3 to 10 days), and the antibiotic spectra of the broths determined. 5. Suitable media and proper growth con- ditions are established for each individual culture. The nature of the medium for the production of the antibiotic is of great im- portance; each culture may require special media for optimum production of the anti- biotic. 6. An effort is then made to isolate from the medium the active substance produced by the culture, concentrate it, and purify it. 7. The isolated antibiotic is studied for its chemical, physical, and antimicrobial prop- erties. A comparison is also made of its anti- biotic spectrum, which should correspond to that of the culture broth from which it was isolated. A lack of proper correlation may be due either to the presence of more than one antibiotic in the broth, or to a chemical mod- ification of the antibiotic in the process of purification. 8. The isolated antibiotic is tested for tox- icity and activity in experimental animals. 9. Before actual production of the antibi- otic is undertaken, the culture is irradiated or treated by other suitable procedures, and the isolated colonies retested, since a freshly iso- lated culture may not be a very potent one. A search for the natural occurrence of more potent strains is often made. 10. The clinical evaluation of the isolated antibiotic is the final step in the group of procedures. This permits one to come to a conclusion concerning the therapeutic po- tentialities of the freshly isolated antibiotic. selected grown in 228 THE ACTINOMYCETES, Vol. I 200 50 150 = 7 = 5 125 125 > ~ O = 5 z 100/X 100 Y z S = Oo z LJ = UO 75 75 6 = 6) = RESIDUAL ) (Je) = = bo (se) S — .63 | 8.53 | 5.53 ~I berculosis: H387Rv 2.00 2.20 5.50 6.50 Ravenel 0.58 0.62 2.50 2.20 BCG 0.52 OF55 1.90 Le 7A0) Nt 0.54 0.56 2.50 2.10 Athy 0.55 0.54 2.20 2.00 Pt 0.62 0.85 2.30 P2720) ODt 0.63 0.75 2.30 2.60 Kt | 1.00 a7) 3.90 3.90 * All figures are given in terms of weight of the trihydrochlorides. On the basis of assays with Kk. pneumoniae, the streptomycin and dihydrostrep- tomycin would have an activity of 820 units per mg, the mannosidostreptomycin an activity of 236 units per mg, and the dihydromannosidostrepto- mycin 228 units per mg. + Strains of M. tuberculosis freshly isolated from human cases. THE ACTINOMYCETES, Vol. I raffnose-negative, but are xylose-, manni- tol-, and lactose-positive. The various strep- tomycin- and streptothricin - producing strains belong to this group. II. Those organisms that show the same type of carbon utilization. These include strains of S. griseus, S. olivaceus, and S. poolensis. Groups III-VI comprised mostly strep- tothricin-, chloramphenicol-, and tetra-cy- cline-forming groups. A combination of chemical, physical, and biological properties of the antibiotics pro- duced by actinomycetes can be utilized in developing a proper system of classification, as will be shown in Chapter 32 (Volume ITT). At present, some 20 different categories of antibiotics may be recognized. Some of these are the following: 1. The actinomycins and other polypep- tides. 2. The glucosidie antibiotics. 3. The macrolides. 4. The pigmented antibiotics. 5. Chloramphenicol. 6. The tetracyclines. Ficure 87. Antibiotic spectrum of S. fradiae (top to bottom: Saccharomyces cerevisiae, Aspergil- lus niger, Serr. marcescens, Micrococcus lysodetkti- Cus). PRODUCTION OF ANTIBIOTICS 7. Grisein and other iron-containing anti- bioties. 8. Sulfur-containing 9. The polyenes. antibioties. 10. The nonnitrogenous substances. 11. Antitumor and antiviral agents. 12. Various unidentified antibioties. their historical significance, may now be discussed Some of these groups, because of in greater detail. The Actinomycins The first representative of the actinomy- cins was isolated and erystallized in 1940 by Waksman and Woodruff. It was produced by an organism described as S. antibioticus. Its chemical nature was studied by Waksman and Tishler, and it was designated as actino- mycin A. Although highly effective against different microorganisms, it proved to be too toxic to experimental animals (Robinson and Waksman). Another representative of this group was isolated by Lehr and Berger; it was studied by Dalgliesh and Todd (1949- 1952), who designated it as actinomycin B. In 1949, Brockmann and Grubhofer (1949- 1953) isolated a third form of actinomycin (C) from a culture of S. chrysomallus. (See also Brockmann, 1954; Brockmann and Grone, 1954; Brockmann and Muxfeldt, 1954-1956). Still other actinomycins were isolated later, in various parts of the world, notably actinomycin D by Waksman and Gregory from a culture of S. parvullus (see Vining ef al., 1954; Johnson, 1956; Bullock and Johnson, 1957), actinomycin I and X (Brockmann et al.), J (Hirata and Naka- nishi), M, and others. Some related com- pounds, like actinoleucin, have also been iso- lated (Ueda et al.). It appeared that in every new screening program of actinomycetes, actinomycin was the first antibiotic obtained (Waksman et al., 1946; Welsch et al., 1946). Although nearly all actinomycins are pro- duced by streptomyces, some are also formed by certain micromonosporas (Fisher ef al.). 201 TABLE 64 Effect of carbohydrates as carbon sources on actino- mycin production by S. antibioticus (Katz, Pienta, and Sivak) Maximum actinomycin Carbohydrate, 1.0 per cent assay, ug/ml L(+) Arabinose 86 D(—) Arabinose 0 D(+) Xylose 79 D-Ribose 0 D-Glucose 20 D-Galactose 124 D-Fructose 25 D-Mannose 10 L-Rhamnose 49 Lactose 24 Maltose 4] Sucrose 0 Cellobiose 14 Sorbose 0 Raffinose i) Dextrin 43 Starch 11 Glycogen 16 Inulin 0 The composition of the medium was found to exert a marked effect upon the composi- tion of the actinomycin molecule (Schmidt- Karsten, Goss and Katz). Seveik (1957) has shown that the production of actinomycin by S. antibioticus depends on the amount of phenol oxidase (laccase type) present in the mycelium and decreases with the pH of the medium, the optimum being pH 5.0. Certain soil bacteria are capable of producing an en- zyme which has the capacity of inactivating actinomycin preparations (Katz and Pienta, 1957). Kach form of actinomycin, produced by different organisms or under different con- ditions of culture, was found to be a heter- ogeneous compound, made up of several in- dividual chemical entities. The molecule consists of a chromophore nucleus to which are attached several amino acids. The nature of these actinomycins and their individual components depend on the makeup of the polypeptide part of the molecule. Waksman, Fiaure 88. Antifungal activity of a strepto- myces. The two filamentous fungi are resistant and the two yeasts are sensitive. Katz, and Vining (1958) proposed a system for classifying the various actinomycins, based on their chemical structure. The paper chromatographic methods are very conven- ient for their differentiation. Among the interesting developments in connection with the study of the actinomy- cins is their effect upon neoplastic growths. The work of Hackmann, begun in 1952, on the effect of actinomycin C on experimental tumors and the clinical observations of Schulte and Lings (1953) opened a new field for the potential utilization of this group of antibiotics as chemotherapeutic agents. These results were soon confirmed by a num- ber of clinical investigators. It is sufficient to mention the studies of Trounce ef al., Sigiura and Schmidt, Nitta et al., Yamashita et al., and Farber and Burchinal (1958). These studies brought out the fact that although different actinomycins may vary somewhat in their toxicity, they are similar in their cytostatic activity (Pugh et al., 1956). Foley reported his observations on the mechanism of the action of actinomycin in bacterial systems (For a review of the literature, see THE ACTINOMYCETES, Vol. I Reilly, 1953; Waksman, 1954; and Hack- mann, 1955). The Basic Antibiotics Streptothricin The first basic antibiotic was isolated from a culture of S. lavendulae by Waksman and Woodruff in 1942. They named it strepto- thricin, thus honoring F. Cohn’s first desig- nation of an actinomycete culture. It was found to possess highly desirable chemical and biological properties and it offered prom- ise of becoming an important chemothera- peutic agent. It was water-soluble and ther- mostable. It was active against various gram-positive and gram-negative bacteria and fungi. It was less toxic than actinomy- cin, but more toxic than penicillin, an anti- biotic that had come to occupy an important place in chemotherapy. Its delayed toxicity prevented its immediate clinical use (Waks- man, 1943). The zn vivo activity of streptothricin was first established by Metzger et al. (1942); its action upon the tuberculosis organism was later demonstrated by Woodruff and Foster (1944). It was later crystallized by Fried and Wintersteiner (1945), and by Kuehl et al. (1945-1946). The search for streptothricin-like substances continued for a long time, partly because of their anti- tuberculosis properties (Weiser et al.) and partly because of their intriguing chemistry. Numerous related compounds were described under a variety of different names. Numerous other preparations were later isolated from cultures of S. lavendulae (Bo- honos ef al.). Some were found to be the same as streptothricin, and others were either closely related or comprised mixtures of dif- ferent antibiotics. Soon after the isolation of streptothricin, another antibiotic, designated as proactino- mycin, was isolated (Gardner and Chain, PRODUCTION OF ANTIBIOTICS 1942) from a culture of an organism believed to be a nocardia (proactinomyces) but now recognized as a streptomyces. Its antibac- terial and other biological properties were later established by Florey et al. (1945) and by Marston and Florey. Streptomycin The experience gained in the study of the formation and isolation of streptothricin from cultures of actinomycetes proved to be highly important in planning a search for other antibiotic agents that would possess similar or even more desirable biological and chemical properties, such as a broad anti- biotic spectrum and a lesser toxicity to the animal body. After further extensive studies of many actinomycetes representing a great variety of species and varieties, two cultures were found to yield an important antibiotic. These cultures were isolated from the soil and from the throat of a chicken. They both belonged to a species described in 1916 as Actinomyces griseus, the first representative of which was isolated by Waksman and Cur- tis in 1916 from the soil. The generic name of the organism was changed by Waksman and Henrici in 1943 from Actinomyces to Streptomyces. To honor this new generic name, the new antibiotic was designated as streptomycin. As previously noted, the two cultures of the streptomycin-producing organism were first isolated in September 1943. Because of the similarity of the new antibiotic to strep- tothricin, both in isolation procedures and in its antibiotic spectrum, rapid progress was made in the development of suitable methods for the growth of the organism S. griseus, for the isolation of streptomycin, and for the evaluation of its antimicrobial properties. In January 1944, four months after S. griseus was isolated, the isolation of streptomycin was announced by Schatz, Bugie, and Waks- man. 233 Special methods were soon developed for the isolation of other streptomycin-produc- ing cultures, as well as for obtaining more potent strains from the mother culture, by using media containing varying concentra- tions of streptomycin (Waksman, Bugie, and Schatz; Waksman, Reilly, Schatz). Streptomycin was the first antibiotic pro- duced by actinomycetes that took a promi- nent place in the treatment of numerous in- fectious diseases In man, animals, and plants. It also proved to be the first drug effective against the Great White Plague of man, tu- berculosis, as first shown by Schatz and Waksman (1944). The activity of streptomycin upon bac- terial infections in experimental animals was first established by Jones et al. (1944), and in experimental tuberculosis by Feldman and Hinshaw (1944), and later by Youmans and McCarter (1945), and Smith and Me- Closkey (1945). The effectiveness of strepto- mycin in clinical tuberculosis was first es- tablished by Hinshaw and Feldman (1946), followed by Cooke et al. (1946), who treated the first case of tubercular meningitis with streptomycin, and soon by numerous others (Keefer et al.). Streptomycin was soon isolated in ecrys- talline form (Carter et al., 1945; Peck et al., 1945) and its chemical nature determined. In standardizing streptomycin (Waksman, 1945), it was found that 1 unit of the anti- biotic, as determined by its activity upon a standard strain of EL. coli, under standard conditions of culture, was equivalent to | ug of the pure base. Gottleb and Anderson have shown that the production of streptomycin by S. griseus is dependent on many factors. Among these are the constitution of the medium, hydro- gen-lon concentration, temperature, oxygen supply, and agitation of the medium. The process of antibiotic formation followed the same general pattern in all the media. No and 234 THE ACTINOMYCETES, Vol. I Figure &9. First experiment in which the effect of streptomycin upon the growth of MW. tuberculosis was demonstrated (Reproduced from: Schatz, A. and Waksman, S. A. Proe. Soc. Exptl. Biol. Med. 57: 246, 1944). streptomycin was detected in the solution imum was reached between 48 and 60 hours during the first 12 hours of growth of the of growth. After this, a decline in the strep- organism. The formation of the antibiotic tomycin content of the solution occurred. increased steadily from then on until a max- This decrease continued until after about 96 PRODUCTION OF ANTIBIOTICS hours, when the slope of the curve decreased with a tendency to level off. In all cases the peak of the streptomycin curve lagged be- hind the peak of the growth curve by about 24 hours. The conclusion was reached that streptomycin production in the medium is not primarily a function of the active growth phase of S. griseus. Only about 25 to 50 per cent of the total streptomycin had been re- leased into the solution by the time the growth peak was reached. On continued growth in artificial media, S. griseus may become contaminated with a phage or virus, designated as actinophage. The growth of the streptomycin-producing organism and the production of streptomy- cin are rapidly diminished. By the use of the plaque method, it is possible to measure the actual concentrations of actinophage in the culture. The number of particles per mil- liliter can reach as high as 10". Phage-resist- ant strains can be readily obtained from in- fected cultures. Streptomycin is a strong base, belonging to the glucosides, in which a diguanido-group is linked to a nitrogen-containing disaccha- ride-like compound. A molecular weight de- termination on the trihydrochloride in water gave about 800 for the free base after the necessary corrections for the chloride ion. Investigations carried out by Hunter et al. on S. griseus with the aid of CO, have shown that the carbons of the guanidine side chains in streptomycin are derived very largely, and possibly entirely, from carbon dioxide. The maximum incorporation of CO, into streptomycin was between 0.4 and 0.5 per cent. A much lower degree of fixation of C™ was obtained when no CO, was passed for the first 72 hours of the fermentation. L- arginine possibly functions as an intermedi- ate in the biosynthesis of the guanidine side chains of streptomycin. A variety of com- pounds, either containing guanidine groups or readily changed into such compounds, are converted by S. griseus into a further sub- stance containing at least one guanidine group. This compound has not yet been iden- tified but may be involved in the biosynthe- sis of streptomycin by the organism. On catalytic hydrogenation of streptomy- cin, two hydrogen atoms are added to the molecule, giving dihydrostreptomycin. Recently it was reported that certain or- ganisms (S. humidus) can produce dihydro- streptomycin directly in the medium. This product is similar to streptomycin in its anti- bacterial and pharmacological properties, ex- cept that in many cases it exerts a less severe effect upon vestibular dysfunction, although it may give greater autotoxicity. These re- sults so far have not been confirmed. Certain organisms belonging to the Strep- tomyces group also produce desoxystrepto- mycin, which was found to be more toxic than streptomycin. Streptomycin is active against a large number of bacteria found among the gram- negative, gram-positive, acid-fast, and spi- rochaetal groups. Many of the bacteria af- fected by streptomycin are able to cause a great variety of human, animal, and plant diseases. Streptomycin is also active against certain plant-disease-producing fungi be- longing to the phycomycetes. It is not active against anaerobic bacteria, protozoa, vi- ruses, and the majority of fungi. The sensi- tivity of a given organism to streptomycin depends not only upon the species, but also upon the strain, and upon the composition of the medium in which it is tested. The bac- teriostatic and bactericidal action of strepto- mycin upon MM. tuberculosis, the causative agent of tuberculosis, is particularly signifi- vant. It is active in a concentration of 0.05 to 0.4 ug/ml. The bactericidal action also varies with concentration and the length of contact with streptomycin, 0.3 ug exerting an effect in 48 hours, and 20 ug/ml in 6 hours. Sensitive bacteria become more resistant to streptomycin upon prolonged contact with the antibiotic. This is of considerable theo- 236 retical and practical importance. Freshly isolated cultures of tubercle bacilli from pa- tients with pulmonary tuberculosis are uni- formly sensitive to streptomycin. When a culture is exposed to streptomycin in rela- tively low concentrations, growth of the mul- ‘iplying cells is inhibited but not that of the nonmultiplying cells. This resistance persists for a considerable time and is not accom- panied by a diminution in virulence. The principal effects of streptomycin on the mor- phology of this organism were a loss of acid- fastness, an increase in granulation, and, in highly bacteriostatic concentrations, a short- ening of the rods. The development of resist- ance of bacteria to streptomycin does not usually result in increased resistance to syn- thetic agents, such as INH and PAS, or to other antibiotics, such as tetracyclines. Among the other problems bearing upon the effect of streptomycin upon bacteria is the development, among certain strains, of dependence upon this antibiotic. This phe- nomenon has highly interesting biochemical and clinical potentialities. No attempt can be made here to review the extensive literature that has accumulated on the effeetiveness and utilization of strep- tomyein. Suffice to say that up to 1952 (Waksman) nearly 6,000 references had ac- cumulated. A comprehensive summary was presented in various publications (Waksman, 1949, 1951). Neomycin Waksman and Lechevalier first isolated neomycin, in 1949, from a culture of S. fra- diae. Neomycin or closely related compounds were also found to be produced by a number of different other organisms, such as S. al- bogriseolus (Benedict et al.) and S. kanamy- ceticus (Takeuchi et al.). They are basic compounds, with a broad antibiotic spec- trum (Table 65). They are active against streptomycin-resistant bacteria, including the tuberculosis organism. They were later THE ACTINOMYCETES, Vol. I found to be a mixture of several closely re- lated compounds, all exerting antimicrobial effects, thus suggesting the term ‘neomycin complex” (Dutcher and Donin, Ford et al.). One fraction isolated from the complex was a basic, water-soluble, nitrogenous substance, designated as neomycin A. It gave 1,700 di- lution units/mg against B. subtilis, by plate assay, but only 50 units/mg against L. colt, by turbidimetric assay. Neomycin B and ne- omycin C, two isomeric fractions, were found to be the major constituents of neomycin (Lechevalier, 1951; Dulmage, 1953; Prieto, 1955; Waksman, 1958). Viomycin and Other Polypeptides Viomycin was isolated simultaneously, in 1951, in several laboratories from cultures described as S. puniceus, S. floridae, and S. californicus (Finlay et al., Bartz et al.). All of these cultures were later relegated by Burkholder et al. to be S. griseus var. pur- pureus. Viomycin was found to be a strong basic polypeptide which, upon acid hydroly- sis, yields carbon dioxide, ammonia, urea, L-serine, a-, 8-diaminopropionic acid, an un- identified guanidine compound, and a basic amino acid that has also been found in strep- tothricin and streptolin (Haskell et al.). Viomycin is active mainly against acid-fast organisms but shows some activity against gram-positive and gram-negative bacteria. Chloramphenicol Among the antibiotics that have found ex- tensive therapeutic applications is chloram- phenicol, isolated in 1947 by Ehrlich and by Gottheb ef al., from a culture of an actino- myces (S. venezuelae). Umezawa et al. iso- lated another chloramphenicol-producing culture which differed from S. venezuelae and to which the name S. omiyaensis was as- signed. Chloramphenicol was the first anti- biotic of anactinomycete to be synthesized. It contains nitrogen and chlorine. It is active PRODUCTION OF ANTIBIOTICS 237 TABLE 65 Antimicrobial spectra of different strains of S. fradiae grown on different media (Waksman ef al., 1958) ee eM rise os Tutt eR, con ee cccons Scans pipes | eRe boi Yeast-glucose agar* 3535 10.0 12.5 12.0 17.2 11.4 8.3 8.0 5.5 5.0 1135 3554 7.5 8.5 9.0 11.0 6.5 4.0 Shot 0.5 3.0 10.0 3556a 15.5 15.5 11.0 20.0 13.0 6.0 7.0 5.0 8-0) $13.0 3556b 10.5 10.0 13.0 16.0 10.5 11.5 6.5 6.0 S.0F 513.5 3572 10.0 11.0 14.0 12.0 12.0 8.5 6.0 2.0 5.0 13.5 3594 9.5 11.5 10.0 14.5 8.3 7.0 4.5 4.0 6.5 12.5 3719 8.5 9.5 9.0 10.5 7.0 0) 6.0 4.5 6.0 4.5 3566 10.5 6.0 9.5 ILL iss 8.5 8.5 eo 0 1.5 8.0 3595 12.0 10.0 10.0 14.5 15.0 10.0 6.0 0 0 5.5 3598 13.0 13.0 13e5 15.0 12.5 11.5 als 0 0 4.5 3596 3.5 5.0 7.0 7.5 4.5 2e5 0 0 0 2.5 3597 8.0 oe 12.0 LZR 10.0 Tod 8.0 0 0.5 4.5 Nutrient agar* 3535 10.0 25.0 20.0 20.0 16.0 20.0 3.0 2.0 6.0 0 3554 13.0 — 24.0 23.0 — 15.0 2.0 33515 0 20.0 3556a — 10.0 = 26.0 27.0 20.0 0.5 0) 0 12.0 3556b = — = = 25.0 — 0 0 0 0 3572 19.0 0 = 27.5 1725 7.0) 0 0 0 0 3594 17.0 17.0 15.0 22.9 15.0 11.0 3.0 iL) 5.0 9.5 3719 18.0 19.4 26.5 PIB a5) 18.0 20.1 0 0 14 0) 3566 20.0 0 — 22.0 21.0 UG 3B 0 0 0 0 3595 20.5 21.5 — 136 12.5 8.5 0 0 0 0 3598 17.5 23 .0 21.0 23 .0 21.0 19.0 0 0 0 1225 3596 4.0 4.0 8.5 4.0 3.0 9.0 0 0 0 0 3597 0 20.0 22.5 13.0 20.5 ZED 0 0 0 Mil * Zones of inhibition in mm. against gram-positive and gram-negative bacteria, rickettsiae, and the larger viruses. Tetracyclines The tetracyclines comprise a group of compounds that have found an important place in the therapy of numerous infectious diseases. They are amphoteric substances, forming crystalline hydrochlorides and_ so- dium salts. The first of the tetracyclines, chlortetra- eycline, was isolated in 1948 by Duggar et al. The culture producing this antibiotic was designated as S. aureofaciens. Like chloram- phenicol, it inhibits the growth of gram-posi- tive and gram-negative bacteria, rickettsiae, and certain of the so-called larger viruses. Oxytetracycline was isolated, in 1950, by Findlay et al. from a culture of S. rimosus. Tetracycline can be obtained either by direct fermentation of media low in chlorides by S. aureofaciens or by the chemical modification of chlortetracycline. In some respects, it ap- pears to have more desirable properties than the other tetracyclines. The anhydro derivative of chlortetracy- cline was found to be particularly effective against actinomycetes (Goodman ef al.). 238 ie ¢) CELLS 7m & uo ro.) ~J VIABLE LOG nm 2 4 6 8 Te) UNITS OF GRISEIN / ml Fraure 90. Effect of antibiotic concentration on development of resistance by EH. coli in agar media: O, Grisein alone; A, Grisein + strepto- mycin | we/ml; O Grisein + streptomycin 2 ug/ml. (Reproduced from: Reynolds, D. M. and Waks- man. S. A. J. Bacteriol. 55: 750, 1948). The Macrolides The first of these antibiotics (erythromy- cin) was isolated by MeGuire et al. (1952) from a culture of S. erythreus. It is a basic compound, soluble in water, alcohol, and other organic solvents. It has significant ac- tivity against gram-positive organisms and some of the more important gram-negative bacteria, such as the Neisseria, Hemophilus, and Brucella groups; it is also active upon rickettsiae, the larger viruses, and some pro- tozoa. A number of other erythromycin-like com- THE ACTINOMYCETES, Vol. I pounds have been isolated and described un- der a variety of names, such as picromycin, magnamycin (carbomycin), oleandomycin, methymycin, and spiromycin. Novobiocin This antibiotic is produced by S. niveus and by S. spheroides. It has been isolated simultaneously in different laboratories and described under different names (strepto- nivicin, cathomycin, cardelmycin, vuleano- mycin, ete.). It is highly active against Staph. aureus, as Well as against a variety of other gram-positive and some gram-negative bac- teria, but it causes allergic dermatitis. Streptovaricin This orange-red group of antibiotics, which contains at least five closely related components, was isolated from the culture of an organism described as S. spectabilis. It is active against various gram-positive, gram- negative, and especially acid-fast bacteria. The Polyenes Most of the previously listed antibiotics are active primarily on bacteria and have only a limited activity on true fungi. There are, however, a number of antibiotics pro- duced by actinomycetes that have a highly selective activity upon fungi. As pointed out previously, the first survey of the distribu- tion of antifungal properties among actino- mycetes was made by Alexopoulos. A large number of compounds have now been isolated and described under the names of actidione, fradicin, thiolutin, nystatin, candi- cidin, ascosin, candidin, trichomycin, anti- mycoin, filipin, amphotericin, and others. Most of them belong to the polyenes. Only a few of them, notably nystatin and tricho- mycin, have found practical application. tecently, Ball et al. made a comprehensive study of the production of polyene antibiot- ics by species of Streptomyces. These polyenes were grouped together on the basis of cer- PRODUCTION OF ANTIBIOTICS 239 2 = 6 8 10 UNITS l2 14 16 20 40 60 OF ANTIBIOTIC / ml Ficure 91. Effect of antibiotic concentration on development of resistance by EF. cold in agar media: O, Streptomycin; O, Grisein; A, Streptomycin + 1 unit grisein/ml; X, Streptomycin + 5 units gri- sein/ml. (Reproduced from: Reynolds, D. M. and Waksman, 8S. A. J. Bacteriol. 55: 749, 1948). tain general properties that may be summa- rized as follows: 1. They inhibit the growth of a wide range of fungi, including yeasts, but are inactive against bacteria. 2. They show a relatively high toxicity when injected into animals, but are much less toxic when given by mouth. 3. They are of low solubility in water, dis- solve more readily in aqueous solutions of the lower alcohols, and are easily soluble in aque- ous pyridine. 4. They exhibit characteristic ultraviolet absorption spectra typical of polyenic chro- mophores (Table 66). Vanék et al. (1958) isolated from soil sam- ples obtained from China a total of 739 ac- tinomycetes; of these, 515 (69.7 per cent) were antibiotically active. A total of 386 cul- TABLE 66 Classification of polyene antibiotics according to their ultraviolet absorptions (Ball et al.) == as Group no. Absorption ( N antihnoiee maxima, mp amed antibiotics | Polyene type | 1 | Tetra- | 290, 305, ene 318 anti- rimoci- din, chromin, am- photericin A Fungichromatin, Fungicidin, mycoin, 2A Penta- SIS eos ene | 351 eurocidin 2B | Penta- | 325, 340, | Fungichromin, fi- ene 358 lipin 3 | Hexaene | 340, 356, | Mediocidin, flava- aegis cid, fradicin 4 | Hepta- | 360,378, | Ascosin, candicidin, ene 405 candidin, tricho- mycin, candimy- | cin, amphotericin | |B, antibiotic 1968 240 tures were characterized by paper chroma- tography using agar blocks, by behavior on agar plates incorporating ion-exchange res- ins, and by ultraviolet absorption spectrum (to detect polyene substances). About half, or 195 cultures, produced a mixture of anti- bioties of both polyene and nonpolyene type. A detailed treatment of these and various other antibiotics produced by actinomycetes will be presented in Volume ITI. Antitumor Agents Various actinomycetes are able to exert a repressive effect against certain forms of cancer and to produce cytologically active substances. Attention has already been di- rected to the effect of the actinomycins, a group of antibiotics with marked cytologic properties. Azaserine is another group of compounds that are highly active against various sarcomas. A similarity in this activ- ity and that against the yeast Klockera brevis was found; this made it possible to use ac- tivity against the latter in studying the puri- fication of the antibiotic. Sarkomycin, iso- lated by Umezawa et al. in 1953, has since been studied extensively by Hooper and others. It was found to be active against ex- perimental tumors, but did not have a great THE ACTINOMYCETES, Vol. I effect in human tumors. The same may be said of another antitumor agent designated as carzinophilin. A number of other sub- stances (DON, mitomycin, sulfocidin) were found to exert marked antitumor activity, but none of these has as yet found any prac- tical application in the control of tumors in man (Osato ef al.). et al. found a high fre- quency of antimitotic activity in the meta- bole products of soil microorganisms. With the use of Allium cepa root tips, they ob- served active antimitotic strains among fungi and actinomycetes. There was no defi- nite correlation, however, between antibiotic and antimitotic activity in the prepara- tions thus obtained. Cavalli-Sforza Antiprotozoan, Antiviral, and Anti- phage Agents Various actinomycetes are able to form substances that exert antiprotozoan effects. This is true particularly of such antibiotics as streptocin, trichomycin, and other sub- stances active upon trichomonads; of puro- mycin and other agents active upon trypan- osomes; and of anisomycin active upon Endamoeba. This field has not yet been suffi ciently explored, but it has marked poten Fiaure 92. The use of bacterial strains sensitive and resistant to a given antibiotic for the identifi- cation of the particular antibiotic. #. coli strains (from right to left): streptomycin-resistant, strepto- mycin-dependent, and streptomycin-sensitive. The plates were streaked from top to bottom: original streptomycin-producing S. griseus; culture producing an unknown antibiotic not of the streptomycin type; an unknown streptomycin-producing culture. PRODUCTION OF ANTIBIOTICS tialities. Other antiprotozoan antibiotics pro- duced by actinomycetes include congocidin, eurocidin, fermicidin, and valinomycin. A number of antiviral substances are also produced by actinomycetes. Some of them, like the tetracyclines, are active upon the larger viruses, such as psittacosis, and have already found extensive practical applica- tion. Others are active upon the smaller vi- ruses but are either insufficiently active or are too toxic to have found practical applica- tion. Among these, it is sufficient to mention ehrlichin, abikoviromycin, achroviromycin, noformicin, hygrosporin, and primycin. A number of actinomycetes are also able to produce antiphage agents (Jones and Schatz). Some of these substances have been given names like chrysomycin. Miscellaneous Antibiotics A large number of other antibiotics pro- duced by actinomycetes, chiefly streptomy- ces, have been described. Some have a wide spectrum. Others have a narrower spectrum. Still others have a very narrow spectrum. A few have found practical applications or of- fer promise. Screening programs for antibiotics are be- ing continued on a large scale. Particular at- tention is being directed at present to agents active against viruses and tumors. Although a number of active substances have already been isolated, none has so far given very promising practical results in the treatment of disease. It is important to note that various antibi- otics of actinomycetes are also active against plant pathogenic bacteria and fungi (Waks- man et al., 1944). The great majority of antibiotics are pro- duced by members of the genus Streptomyces. A few are formed by species of Necardia and Micromonospora. Some of the thermophilic actinomycetes have been found to possess an- tibiotic properties (Kosmacher). None of the remaining genera (Actinoplanes, etc.) were 241 TABLE 67 Release of antibiotic activity from mycelium of 8. griseus by chemical and physical treatments (Perlman and Langlykke) Antibiotic potency released by treatment, Treatment of mycelium suspension* pg/ml None 19 Heated in boiling water bath for 10 min- 38 utes Exposure to sonic energy for 15 minutes — 107 Addition of sufficient concentrated HCl to give: pH 5.1 37 pH 4.2 67 pH 3.0 109 pH 2.5 188 ply ies 175 Addition of sufficient 10 V NaOH to give: pH 8.0 45 pH 9.1 109 pH 9.9 161 pH 10.8 143 Addition of sodium chloride to give con- centration: 0.003 M 19 0.01 M 19 0.03 M 33 0.1M 53 0.3 M 97 Addition of sodium sulfate to give con- centration: 0.003 M 19 0.01 M 43 0.03 M 97 0.1 M 159 0.3 M 147 Addition of sodium citrate to give con- centration: 0.003 M 25 0.01 M 64 0.03 M 142 0.1M 129 0.3 M 130 * Samples from 4-day-old fermentation were centrifuged and the supernatant liquid assayed. The supernatant liquid contained 108 ug/ml of streptomycin. The collected solids were resus- pended in distilled water to original volume, and acid, alkali, or salt was added as indicated to 10-ml aliquots. After 15 minutes of shaking on a mechan- ical shaker the solids were collected by centrifuga- tion and the supernatant liquid submitted for assay. 242 found to exert any antagonistic effects upon other microorganisms. Numerous other compounds were isolated from various cultures of actinomycetes. They are described in detail in Chapters 31—40 (Volume ITI). The Role of Antibiotic Biosynthesis in the Metabolism of Actinomycetes According to Perlman, the role of antibi- otic biosynthesis in the metabolism of actino- mycetes is of considerable significance. An analysis of the cells of S. griseus suggests that the amino acids do not differ in nature from those in other actinomycetes. The fact that a substantial quantity of streptomycin (usually more than half of that produced) occurs bound to the mycelium suggests that the antibiotic may form a part of the cell wall of the organism. This bound streptomycin may be released (Table 67) by treatment of the cells with acid, alkali, or ionizable salts, but not by disintegration of the cells by sonic energy, bacteriophage, or enzymatic treat- ment. Considerable amounts of other anti- bioties, including streptothricin, the neomy- cins, chloramphenicol, and chlortetracycline, have been found to occur bound to the my- celium of the respective actinomycetes and may be released by treatment with acid, alkali, or ionizable salts. This binding does not appear to be a simple ion-exchange phe- THE ACTINOMYCETES, Vol. I nomenon, since addition of streptomycin to the mycelium of the organism does not re- sult in its absorption, and the “binding” power of the mycelium is apparently not a function of its weight (Perlman and Lan- elykke). Chemical Structure and Antimicrobial Activities of Actinomycete Antibi- oties The effect of chemical structure upon the biological activities of antibiotics in general and of actinomycete antibiotics in particular is discussed in detail in Chapter 31 (Volume III). It is sufficient to say here that any shght modification of the molecule may cause a profound change in the activities of the antibiotic. As a rule, the chemical or enzymatic deg- radation of an antibiotic results in its loss of activity. This is not always the case, how- ever, as shown for neamine, a degradation product of the neomycins; it retains its anti- biotic properties, although the spectrum is changed. Nakamura has shown that an anti- biotic of the luteomycin type (CogsH3;NOy) gave, on acid hydrolysis, a greenish black substance, designated teomycie acid (C4;- H,3;NOj 1), which retained its antibiotic prop- erties. However, actinomycin treated with an enzyme preparation completely loses its activity, as shown previously. CH A Pot. E R | 6 Decomposition of Complex Plant and Animal Residues Actinomycetes are capable of attacking a great variety of plant and animal products, notably crop residues; they thus bring about the partial or complete decomposition of these products. Actinomycetes may, there- fore, be considered, on a par with the fungi and the true bacteria, as one of the leading groups of microorganisms concerned in the destruction of organic materials and in trans- formation and mineralization of organic mat- ter in nature. The literature on the soil ac- tinomycetes, beginning with the work of Beijerinck, in the early days of the century, and continuing through recent studies, abounds in data on the abundance and ac- tivities of actinomycetes in composts and in soils rich in organic matter. The essential role of these organisms in the formation and de- composition of humus was early recognized. These processes result in bringing about the liberation of plant nutrients in available forms and are thus of great importance in plant nutrition and soil fertility. Decomposition of Plant Materials In a study of the decomposition of al- falfa by different groups of microorganisms, Waksman and Hutchings found that pure cultures of actinomycetes were able to de- compose, in 39 to 74 days, 33 to 43 per cent of the hemicelluloses and 23.2 to 25.3 per cent of the cellulose, as well as a part of the lignin. Nearly 20 per cent of the total nitro- 243 gen in the plant residues was liberated as ammonia, thus pointing to considerable pro- tein decomposition; much of the nitrogen must also have been used by the organisms for the synthesis of their own cell material. In the decomposition of oat straw, 24.5 per cent of the hemicelluloses were destroyed in 50 days; only little cellulose and some lig- nin were attacked. Cornstalks were only slightly attacked when no lime and phos- phate were added but underwent rapid de- composition when these were introduced. Of the total dry plant material, the actinomy- cetes brought about, on an average, 20 per cent decomposition. They attacked the wa- ter-soluble substances most readily (30.5 per cent), then the hemicelluloses (16.7 per cent), and the cellulose least readily (5.4 per cent). The most striking point was the fact that the actinomycetes decomposed not only the cellulose and hemicelluloses, but the lignin in these materials as well, even to a greater extent than did the fungi, as il- lustrated in Table 68. In a comparative study of the decomposi- tion of cornstalks by several species of Strep- tomyces, alone or in the presence of a fungus or a bacterium, the streptomycetes were highly effective in decomposing a consider- able amount of the cellulose and the hemi- celluloses. Although their decomposing ca- pacity was less than that of the fungus Hw- micola, especially in the absence of added 244 TABLE 68 Decompostion of alfalfa by pure and mixed cultures of microorganisms (Waksman and Hutchings) | | Total Hemi- | Cellu- Inoculum Peerdies co lace | Lignin Control 9.120 0.760, 2.060) 1.170 Soil infusion 6.5297| 0.449] 1.013) — Rhizopus 8.241 | 0.663] 2.000) 1.073 Trichoderma 8.270 | 0.724) 2.079) 1.041 Trichoderma + Strep-7.983 | 0.649) 1.962) 1.023 tomyces 3065 Streptomyces 3065 7.608 | 0.433) 1.582} 0.942 Streptomyces 3065 6.570 | 0.508} 1.538) 0.906 Streptomyces +. soil |5.064 | 0.384) 0.925) 0.821 infusion * Values are given in grams on basis of dry ma- terial. + The amount of the constituents decomposed by each organism or combination of organisms can easily be calculated by subtracting the ma- terial left from that of the control. lime, their activities were highly significant, and particularly when one remembers that the fungus used in this experiment was one of the most effective in the decomposition of plant materials. A number of factors, such as reaction, aeration, moisture, and temperature, exert a considerable influence on the decomposi- tion of plant materials by actinomycetes. This is illustrated in the effect of added lime upon their activities, as shown in Table 69. The maximum decomposition of dried blood by actinomycetes, as measured by am- monia formation, was found to take place at pH 5.8 to 7.7; some organisms showed some activity at pH 5.0, but very little de- composition of this material took place at pH 4.0 and at pH 88. Decomposition of Cellulose and Hemicelluloses As has been pointed out, various actino- mycetes are capable of decomposing different hemicelluloses. Waksman and Diehm made an extensive study (Tables 70-72) of the de- THE ACTINOMYCETES, Vol. I composition by actinomycetes of a variety of hemicelluloses in sand, soil, and liquid media. They found them capable of bringing about considerable decomposition of these carbohy- drates both in an isolated and chemically TABLE 69 Influence of lime and associative organisms upon the growth of actinomycetes on cornstalks (Waksman and Hutchings) Decomposition in per cent | Treat- Water- : Organism | MERE | com | soluble | ellas | Cela CaCOs posed matter lose Control — 0 0 0 0 Streptomyces -- SO; cal 2.7 3065 Humicola - 26.3 | 42.0 4 3.8 Humicola a4. = coil Real | as7(ethal| Zab hh (idk Streptomyces 3065 Streptomyces + ZIAOs E28 om elo ee 6.6 3065 Streptomyces > | 232.0 29.7, \ClGeom ORG 3018 Streptomyces + 14.7 | 33.4 | 14.3 0 3310 Streptomyces + 24.2) | 3523: |) 2286 (eal 3065 + Ps. fluorescens Humicola os PAN ll. || 44o}.25) 9 12.0 Humicola + 24.3 | 41.7 | 18.5 6.4 Streptomyces 3065 TABLE 70 Decomposition of different polysaccharides by var- tous streptomyces (Waksman and Diehm) Milligrams per flask of sand medium Mannan Xylan Galactan See — a myces No. | a : ‘ | Found /Pecom- | Found |Pecom | Found | Decor Control 305.5 -- (152.0 — {170.2 i 26 S39 27452) SSer | S373) TSS ie eou ees 40 15.1 | 290.4) 25.2 | 126.8) 97.2 | 73.0 48 | 25.9 | 279.6] 29.7 | 122.3) — — 50 | 17.8 | 287.7; — — | 69.3 100.9 53 | 23.9 | 281.6) — — | 99.0 | 71.2 DECOMPOSITION OF COMPLEX PLANT AND ANIMAL RESIDUES 245 purified state and in a natural condition in the plant materials. Mannans and xylans were attacked particularly. Decomposition of laminarin by actinomycetes was studied by Chesters et al. (1955). The formation of the enzyme xylanase by shown in Chapter 11. Numerous other in- vestigators have demonstrated the ability of actinomycetes to decompose cellulose and various hemicelluloses. In the degradation of cellulose in the intestinal canal, certain ac- tinomycetes probably play a part, as shown by Hungate (1946) for a species of MWicro- monospora. The active part played by ac- tinomycetes in cellulose decomposition under high salt concentration, with the result that black muds are formed, has been established by Rubentschik (1928, 1932). Cellulose decomposition in composts, un- der thermophilic conditions, was first dem- onstrated by Tsiklinsky (1899) and by Schiitze (1908); later, extensive studies were made by Waksman and Cordon (1939), Waksman et al. (1939), Waksman and Corke, and more recently by Henssen. Other stud- ies on cellulose decomposition by actinomy- cetes were made by Bokor (1930), Meyer (1934), and others. actinomycetes was Decomposition of Proteins The ability of actinomycetes to take an active part in protein decomposition is also highly significant. In a study of the effect of dried blood versus rye straw upon the de- velopment of fungi and actinomycetes in dif- ferently treated soils (Tables 73 and 74), it was found that the addition of protein-rich materials greatly stimulates the develop- ment of actinomycetes as compared to other groups of microorganisms. The ability of actinomycetes to decompose proteins into amino acids and ammonia was first shown by Macé. In view of the fact that actinomycetes synthesize considerably less mycelium than do fungi, only small quanti- ties of nitrogen are assimilated into complex TABLE 71 Comparative decomposition of xylan in corncobs by fungi and actinomycetes (Waksman and Diehm) Milligrams per flask Organism Found Decomposed \Cronaltolly Aaa ee 405.5 TGWUZQDuss 2... scart ae » altaya 237.6 Penicillium... 6... ae PPaarels) 181.9 Trichoderma...... PRA fe 3 331.0 74.5 ANS DYahTULG Close treme thane, aicks 302.9 102.6 Streptomyces 26........... 372.1 33.4 Sereplomuces OU. .nne. 2 362.9 42.6 Streptomyces 40........... 306.2 98.3 TABLE 72 Relative decomposition of galactan in Irish moss by different microorganisms (Waksman and Diehm) Milligrams per flask Organism Found Decomposed Controls ached ee 382.3 — IMUBOMUS. o Becees bet oaaees 259.7 122.6 IAC UCHN OWT: on eatantchouess 263.0 119.3 CHOC CIN re ere 265.7 116.6 LAUSD ER TUL Cotte ey oct tae pica aA 281.3 101.0 SUREDLONIGESs 20" nee ee 263.0 119.3 SUC DLONUUGESHOD eae eee 268.9 113.4 Streptomyces 35........... 287.3 95.0 Streptomyces 40........... 253.8 128.5 241.9 140.4 SUReDLONTI Ges OU mses eee cell material. Most of it is liberated free in the form of ammonia. Although actinomy- cetes also utilize nonnitrogenous organic ma- terials for cell synthesis, such materials do not exert such a depressing effect upon the liberation of ammonia as do bacteria and fungi. Nicolaieva, in a study of protein decom- position by eight cultures of actinomycetes, found that the proteins were completely de- graded. She came to the conclusion that ac- tinomycetes take an active part in soil proc- esses, leading to the mineralization of soil organic matter. As shown previously (Chap- ter 7), Waksman and Starkey demonstrated 246 hemicedluloses decomposed Fer ceat 60 20 THE ACTINOMYCETES, Vol. I SLL LESSEE LE LAE ELDRED ES ISA OANA SNS Mannan y an Galactan Solution Sand / medium medium “n Natural material Solution medium FicurE 93. Rate of decomposition of various hemicelluloses by actinomycetes in different media, in 6 weeks (Reproduced from: Waksman, 8. A. and Diehm, R. A. Soil Sei. 32: 115, 1931). 60 CO, EVOLVED h on T OF mg Sz: A xc S= : = pert=" TABLE 73 yo Influence of rye straw (1 per cent) wpon the / Si, : aie i uf development of fungi and actinomycetes in ae various soils after 10 days (Waksman Ga and Starkey) 4 ee ye | alas Actinomycetes / | Fungi ae) 4 FUNG! Annual soil u thousands treatment Pp = : Start End Start End jae = _) se Manure, min- 5.5) 87,300'750,000| 1,800) 2,800 erals Manure, lime, | 6.7} 19,700) 24,000) 3,360) 2,800 minerals | Untreated 5.1/115, 700/600 ,000} 1,260 200 18 28 42 Lime alone | 6.5} 20,000) 19,000) 2,760) 1,900 DAYS OF INCUBATION NaNO; , min- | 5.8) 73,3800650,000) 1,500) 1,800 | erals FIGURE 94. Decomposition of xylan (Repro- (NH,) »S¢ ee | 6.0 25,700 47,000. 2,700 1,800 duced from: Waksman, 8. A. and Diehm, R. A. Soil minerals, lime Sci., 32: 113, 1931). DECOMPOSITION OF COMPLEX PLANT AND ANIMAL RESIDUES that actinomycetes actively decompose plant proteins, liberating the nitrogen as ammonia. Decomposition of protein fibers by actino- mycetes has been studied by Goldsmith. The enzymatic mechanisms involved in protein decomposition by actinomycetes are dis- cussed in Chapter 11. Lignin Decomposition In connection with the decomposition of plant materials in soils and in composts the effect of actinomycetes on the lignin is of par- ticular interest. It is now well recognized that the lignins and the proteins contribute greatly to the formation of humus in soils and in composts. As the plant materials are decomposed by fungi and bacteria, there is usually an increase in the concentration of the lignin, since most of these organisms do not attack this complex very readily (Waks- man). This accumulation of the lignin is par- alleled by an increase in ash content and often in the protein content in the case of nitrogen-poor materials, and by a decrease in the total dry material. Through their abil- ity to attack the resistant lignins, the actino- mycetes have the capacity to leave an or- ganic residue with a lower lignin content. Decompostion of Other Organic Complexes Actinomycetes are capable of growing on and decomposing a great variety of other organic materials. These include paraffins (Baldacci, 1947), waxes, rubber, and build- ing materials (McLachlan, 1946). The ability to attack paraffins is characteristic of certain nocardias, as shown elsewhere in the descrip- tion of the individual species in Chapter 23 (Volume IT). Formation and Decomposition of Hu- mus On the basis of the foregoing observations, the conclusion may easily be reached that actinomycetes take an active part in the for- mation and decomposition of organic mat- TABLE 74 Influence of dried blood (1 per cent) upon the numbers of fungi and actinomycetes in various soils after 12 days (Waksman and Starkey) Actinomycetes, Fungi B thousands Annual soil H treatment I Start End Start End | Manure, min- 5.5) 87,300)2,079,950 1,800 190,900 erals | Manure, lime, 6.7) 19,700 minerals | | Untreated 5.1115,700 1,438,300 1,260 2,200 73,3003 ,360 > 6,000 Lime alone 6.5 20,000 125,000 2,760. 500 NaNO; , min- /5.8) 73,300/1,871,650/1 , 500/128, 700 erals | | (NH,)2SO., |6.0) 25,700} 311,60012,700| 42,700 minerals, | | lime | ter or humus in the soil. This comprises both nitrogenous and nonnitrogenous organic sub- stances. Because of their ability to attack native lignin, actinomycetes may even be ex- pected to play a unique role in the forma- tion and transformation of humus materials. The role of actinomycetes in the formation of dark colored compounds and the possible bearing of these compounds upon humus for- mation were first pointed out by Beijerinck. He emphasized that the black pigment pro- duced by some of these organisms on protein media may function as an oxidizing agent. On the basis of this, he tried to postulate their significance in natural processes, notably in the humification of soil organic matter. He correlated this with the abundance of actin- omycetes at different soil depths. The results of Beijerinck were confirmed and further extended by Fousek, Miinter, Kkrainsky, and Waksman, and later by von Plotho, Lantsch et al., and Scheffer et al. The ability of various streptomyces to give rise to dark brown substances, comparable to humic acids, was demonstrated recently by Flaig et al. (1952), Kiister (1952), and Beutelspacher (1952). The formation of such 248 substances depends on the nitrogen source and on the nature of the organism. On pro- longed incubation of the cultures and proper chemical manipulations, preparations were obtained that showed great similarity to the humic acids occurring in natural soils. Cer- tain amino acids can also give rise to brown substances as a result of the growth of some actinomycetes. Pure cultures of an organism belonging to the genus Streptomyces and of the fungus T'r7- choderma were found by Waksman to de- compose more peat material than did a com- plex soil microbiological population (Table 75). The amount of decomposition was meas- ured by the amount of CO. and ammonia formed. The ratio of the carbon decomposed to that of the nitrogen liberated was lower for the pure cultures than for the complex population. This indicated that the pure cultures attacked more of the nitrogenous constituents than did the total soil popula- tion. Actinomycetes are thus shown to be ca- pable of decomposing resistant humus ma- terials in the soil and bringing about the lib- eration of the constituent elements essential for plant growth. The nitrogen stored up in the humus is changed to ammonia, which is later oxidized to nitrate. Liming of soil and draining of swampy areas favor the develop- ment of actinomycetes as well as the decom- position of the soil organic matter. This proc- ess is of considerable importance to soil fertility. According to Fousek, an increase TABLE 75 Decomposition of sedge and reed peat by microorganisms (Waksman and Stevens) On the basis of 20 gm of dry peat decomposed for 28 days under favorable moisture conditions COz Nitrogen as Ratio of Organism formed, ammonia and_ liberated mg C nitrate, mg N C:N Streptomyces....... 87.7 13.4 6.5 Trichoderma....... 88.4 14.1 Gril OU MIME ISLON ele ali 9.6 (a? THE ACTINOMYCETES, Vol. I in plant growth is obtained by inoculating actinomycetes into the soil, thereby bringing about increased decomposition of the organic matter. This observation has not been fully confirmed as yet. Conn also emphasized the importance of actinomycetes in the decomposition of or- ganic residues in the soil. Colonies of these organisms, mostly streptomycetes, were found developing on plates seeded with soil infusions. They made up 20 per cent of the total number of organisms in cultivated soils and 37.5 per cent of organisms from sod soils. The longer the time during which grass was grown in the soil, the larger was the propor- tion of the actinomycetes to the total popu- lation developing on the plate. A soil con- taining 2,900,000 actinomycetes per gram, when treated with grass roots, gave an in- crease in numbers to 6,000,000 in 2 weeks. Both dead grass roots mixed with the soil and grass growing in the soil were found to have a marked stimulating effect upon the development of these organisms. Decomposition of High-temperature Composts In the decomposition of plant materials, especially in composts of stable manures and artificial composts, high temperature or thermophilic conditions are attained. Under these conditions, actinomycetes play an eminent part in the decomposition process, as brought out first by Globig, Miehe, and others. It was later studied extensively by Waksman, Umbreit, and Cordon, who re- ported that at temperatures of 50 to 65°C, the actinomycetes were highly active in the decomposition processes. Soils receiving stable manure contained an abundant pop- ulation of actinomycetes, notably thermo- philic forms, as illustrated in Figure 96. Waksman and Hutchings found that actino- mycetes may be more active in the break- down of plant constituents in mixed pop- ulations than in pure culture. A similar MG CO5/ 100G DENSITY OF MYCELIUM, % INCUBATION - DAYS Ficure 95. Influence of temperature upon growth and CO, production by actinomycetes. Continu- ous line: density of mycelium. Broken line: CO» evolved (Reproduced from: Jensen, H. L. Proc. Linnean Soc. N.S. Wales 68: 70, 1943). Figure 96. Typical growth of actinomycetes in high temperature composts, as illustrated by contact slide method. 250 population picture was obtained by Mvaila. Henssen recognized that certain species of streptomyces and nocardias are found abun- dantly in thermophilic composts. He em- phasized, however, the abundance of specific thermophilic groups of actinomycetes, such as Thermoactinomyces and certain newly created thermophilic genera, as shown in Chapter 29 (Volume IT). Actinomycetes as Agents of Deteriora- tion and Spoilage Through their ability to attack resistant compounds and through their universal oc- currence, actinomycetes may frequently be responsible for considerable damage to food- stuffs and textiles. As a rule, actinomycetes are usually not considered important agents of deterioration and spoilage. It can easily be established, however, that, under certain special conditions, actinomycetes may play a far more important role in these processes than is commonly supposed. It is sufficient to present the following evidence: 1. Certain foodstuffs are known to deteri- orate as a result of characteristic earthy and pungent flavors and odors imparted by ac- tinomycetes, as pointed out previously. This is true of milk, cacao, potable waters, and fish. The flesh of fish is tainted through ab- sorption from the water of the odoriferous substance produced by actinomycetes. Cacao can be damaged in a similar manner. The damage to Brazil nuts by actinomycetes has been suggested; an organism, described as A. brasiliensis, a streptomyces, was isolated by Spencer from the shells of such nuts. 2. Certain fabrics, notably woolens, cot- ton goods, and paper, may be stained or THE ACTINOMYCETES, Vol. I actually destroyed by actinomycetes. Al- though the rate and extent of such destruc- tion cannot be compared with those caused by fungi or certain bacteria, especially under humid and high temperature conditions, the actinomycetes produce a variety of stains (yellow, pink, red, black) on cloth and on paper, especially in books, and thus cause considerable damage. 3. Bredemann and Werner isolated from soils a chromogenic actinomycete capable of actively decomposing salts of butyric acid. The culture withstood heating for 5 minutes at 80°C. The illustrations given in this re- port suggest that the organism was a Mvcro- monospora. The culture was warty and brown in color. It produced a soluble rose pigment. 4. As a result of extensive studies carried on in connection with the deterioration pro- gram during the Second World War in the Pacific, were isolated. Their exact part in causing rarious cultures of actinomycetes deterioration of service materials has not been fully established. References to numerous other forms of potential deterioration of essential materials by actinomycetes are found in the literature. Galli-Vallerio and Reiss pointed out the ability of actinomycetes belonging to the streptomyces to attack photographie paper, They found such cultures in the wash water used both developed and undeveloped. in photographie work. The ability of actino- mycetes to attack rubber, paraffin, and other complex materials has already been men- tioned. The nature of the damage that may thus be caused has not been determined. A Par : KR l 4 Causation of Animal Diseases Saprophytism and Parasitism The substrate on which microbes normally live has frequently been used as a basis for classification and differentiation of these or- ganisms. The normal existence of an organ- ism on dead organic and inorganic residues has come to indicate its saprophytic nature. Parasitism has come to indicate the normal existence of an organism on living bodies of higher plants and animals and of microor- ganisms. On the basis of their ability to live exclusively or electively on living substrates, some of the parasites are classed either as obligate or as facultative. A parasite may also be virulent, if it has the capacity to infect a living organism. Vir- ulence varies greatly in nature and intensity, depending not only upon the species of the infecting organism, but also upon the strain and its previous history, as well as upon the nature of the host. The mode of infection, the ability of the parasite to spread through the various tissues of the host, its toxic mani- festations, the degree of communicability, all contribute to the intensity of virulence. An organism may be made to increase or decrease its virulence by serial animal pas- sage or by growth of the culture under sap- rophytic conditions. Phenomena of dissocia- tion in the culture and the development of resistance to a particular treatment also con- tribute to the degree of its virulence. Often such changes are accompanied by a change in the morphology of the organism or in its immunological properties. o The above considerations have a particu- lar application to the analysis of the patho- genic properties of actinomycetes. The most important and most highly significant eom- ment to be made in this connection is that although actinomycetes are abundant and widely distributed in nature, they are able to cause only very few human and animal diseases. On the contrary, many actinomy- cetes are able to produce antibiotic sub- stances which have found extensive applica- tion in the treatment of such diseases, especially those caused by bacteria. Actinomycetes as Causative Agents of Disease Although actinomycetes have been jso- lated from various organs and excretions of diseased human and animal bodies, they have not always been the causes of the dis- eases. At present, there are two major dis- eases with which actinomycetes are usually associated. One is caused by anaerobic or- ganisms and is known as actinomycosis. The other is caused by aerobic organisms and is known as nocardiosis. Various other syno- nyms, such as streptothricosis and madura- mycosis were once used to designate these or similar diseases, but these terms were grad- ually discarded (Foulerton, 1910). Cope insisted upon adopting the generic name Actinomyces for the ‘whole group of 9 organisms” and actinomycosis for the dis- ease caused by them, since the name “is 252 THE ACTINOMYCETES, Vol. I Figure 97: A. bovis. (a) 48 hour, X300; (b) 100, L. Proc. Staff Meet. Mayo Clinic 25: 84, 1950.) graphic in character, descriptive in nature, and sanctioned by long usage.” As reviewed previously (Chapters 1 and 4) the “lumpy jaw” disease of cattle was recognized for many years prior to modern developments of microbiology. A similar, if not the same, disease was also known to oc- cur in man. In 1876, Bollinger observed that a branched organism was constantly associ- ated with the diseased jaw of a cow. He rec- ognized the organism as the cause of the dis- ease, and placed this material in the hands of the botanist Harz. The latter examined the granules, observed the characteristic ra- diation of the organism, and named it A ctzno- myces bovis and the disease ‘‘actinomycosis.”’ As pointed out previously, Harz never iso- lated this organism in culture. A comprehensive review of the human and animal diseases caused by actinomycetes is found in the work of Pinoy (1913), Poncet and Berard (1928), Cope, Dodge, Bullock, Conant (1944), Topley and Wilson (1946), Emmons, Gonzalez-Ochoa, Mariat, and nu- ce merous others. Isolation of Specific Actinomycetes from Human and Animal Diseases Simultaneously with the work of Bollinger and Harz, J. Israel (1878) studying pathological material from pyemia and sup- puration in the neck of man; he observed Was granules which contained mycelium similar to that described by Bollinger in cattle. The (c) 6 to 7 days, X20 (Reproduced from: Thompson, presence of a staphylococcal infection pre- vented him from establishing definitely that an actinomyces was the causative agent of human actinomycosis. Ponfick (1879) is usually credited with having reported the first accurate observation of the human in- fection. Johne (1882) took serious exceptions to Ponfick’s claims, however. He stated that Langenbeck was the first to observe, in 1845, the occurrence of actinomycetes in the hu- man body, but he emphasized that it was Israel (1878), in spite of Ponfick’s claims, who first recognized and described an ac- tinomyces as a causative agent of human diseases. In the case of animals, Hahn (1870) was said by Johne to have observed such an organism on cattle tongue, but it was Bol- linger (1876) who first recognized and de- seribed its infectious nature. Johne gives Ponfick only the credit for recognizing the identity of the human and animal pathogens. In 1885, Israel published the results of a study of actinomycosis based upon 38 cases; he thereby definitely elucidated the clinical aspect of the disease. In 1885, Bostroem claimed to have succeeded in isolating pure cultures of the organism from cattle; later, in 1890, he claimed further to have isolated such cultures also from human lesions. Since these cultures were aerobes, it is now gen- erally assumed that he isolated air contami- nants. Bostroem’s identification proved to be incorrect and highly misleading. In 1889, CAUSATION OF ANIMAL DISEASES 253 Bujwid obtained from human actinomycosis pure cultures of the anaerobic organism, the true causative agent of the growth on glycerol agar resembled that of the tubercle bacillus. In 1891, Wolff and Israel reported on a comprehensive investigation of the morphol- ogy and pathogenicity of the organism caus- disease; its ing actinomycosis. They considered the mi- croaerophilic actinomyces to be the only causative agent of the human and bovine forms of the disease. It may be of interest to quote here the results on the cultivation of the organism as reported by them (Tr. by Wright): “Tt grew best under anaerobic conditions and did not grow at room temperature. On the surface of anaerobic slant cultures on the third, fourth and fifth day numerous minute isolated dew-drop-like colonies appeared, the largest pin head in size. These gradually became larger and formed ball-like, irregularly rounded elevated nodules varying in size up to that of a millet seed, exceptionally attaining the size of a lentil or larger. As a rule the colonies did not become confluent, and an apparently homogeneous layer of growth was seen to be made up of separate nodules if examined with a lens. In some instances the colonies presented a prominent center with a lobulated margin and appeared as rosettes. A characteristic of the colonies was that they sent into the agar root-like projections. In aerobic agar slant cultures no growth or a slow and very feeble growth was obtained. In stab cultures the growth was sometimes limited to the lower portion of the line of inoculation or was more vigorous there. In bouillon, after three to five days, growth appeared as small white flakes, partly floating and partly collected at the bottom of the tube. Growth oc- curred in bouillon under aerobic conditions, but was better under anaerobic conditions. The micro- organism in smear preparations from agar cultures appeared chiefly as short homogeneous, usually straight, but also comma-like or bowed rods, whose length and breadth varied. In many cultures short-clump rods predominated, and in others longer, thicker, or thinner individuals were more numerous. The ends of the rods often showed olive or ball-like swellings. In egg cultures growth oc- curred in the form of white opaque granules, the largest about the size of a pin head. Microscop- ically the growth was characterized by the de- agar Figure 98. Stained dental scum (Reproduced from: Ndeslund, C. Acta Pathol. Microbiol. Seand. 2: 140, 1925). velopment of long filamentous forms forming a network. The longer filaments were arranged more or less radially, were straight 6r wavy or spiral and sometimes branched. Cultures on potato or in milk are not mentioned. Some twenty guinea-pigs and rabbits were inoculated, most of them in the peritoneal cavity with pieces of agar culture. Eighteen animals were killed after four to seven- teen weeks, and four were still alive seven to nine months after the inoculation. Seventeen rabbits and one guinea-pig showed at the autopsy tumor growths mostly in the peritoneal cavity and in one instance in the spleen. In the four animals still living tumors were to be felt in the abdominal wall. The tumors in the peritoneal cavity were millet seed to plum size, and were situated partly on the abdominal wall and partly on the intestines, the omentum, and mesentery, and in the liver or in adhesions. Frequently small millet-seed-sized tumors were situated in the neighborhood of the larger tumors. While the surface of the small tumors was always smooth, the surface of the larger tumors showed small hemispherical promi- nences, giving them the appearance of conglomer- ates of smaller tumors. On section the larger tumors presented a tough capsule from which anastomosing septa extended inward enclosing cheesy masses. Microscopical examination of the tumors showed in all cases but one the presence of typical actinomyces colonies, in most cases with typical clubs. The general histological appearance of the tumors was like that of actinomycotic tissue.”’ In 1896, Kruse named this organism Strep- tothrix israeli. Wright, in 1905, suggested changing the name to Actinomyces bovis, since he considered this organism to be iden- tical with that recorded by Harz. It is of interest to quote from Wright: “Branching filamentous micro-organisms have been isolated in pure culture from thirteen cases of actinomycosis in man and two cases in cattle. The micro-organism seems to be all of one spe- cies—grows well only in agar and bouillon cultures and in the incubator; in the other usual culture media and at room temperature, it grows only very little or not at all. It is essentially an an- aerobe. It does not form spore-like reproductive elements.”’ Wright added: “Tn cultures its colonies are similar in char- acter to colonies of the microorganism in the lesions of actinomycosis. If colonies of the micro- organism are immersed in animal fluids, such as blood serum and serous pleuritic fluid, the fila- ments of the colonies in immediate contact with the fluid may, under certain unknown conditions, become invested with a layer of hyaline eosin- staining material of varying thickness, and the filament may then disappear. Thus structures are produced that seem to be identical with the char- acteristic ‘clubs’ of actinomyces colonies in the lesions.”’ Figure 99. N. asteroides in sputum (Repro- duced from: Kirby, W. M. M. and McNaught, J. B. Arch. Internal Med. 78: 8, 1946). THE ACTINOMYCETES, Vol. I Wright emphasized further: “Between Actinomyces from the human and bovine cases I have found no difference which seems to me to be sufficient to justify their classi- fication as separate species. ‘“‘T do not accept the prevalent belief, based on the work of Bostroem, Gasperini, and others, that the specific infectious agent of actinomycosis is to be found among certain branching microorgan- isms, widely disseminated in the outer world, which differ profoundly from Actinomyces bovis in having spore-like reproductive elements. I think that these should be grouped together as a sepa- rate genus with the name of Nocardia, and that those cases of undoubted infection by them should be called nocardiosis and not actinomycosis. The term actinomycosis should be used only for those inflammatory processes the lesions of which con- tain the characteristic granules or ‘drusen.’ “Because the microorganism here described does not grow well on all the ordinary culture media and practically not at all at room tempera- ture, I did not believe that it has its usual habitat outside of the body. It seems to me very probable that Actinomyces bovis is a normal inhabitant of the secretions of the buccal cavity and of the gastro-intestinal tract, both of man and animals, but I have no proof of this to offer at the present time. In these secretions it should not exist in the characteristic forms seen in the lesions, but it probably will be found in the form of fragmented filaments growing in company with bacteria, and net now differentiated from them. I believe that the part played by foreign bodies so frequently found in actinomycotic lesions is not that of the carrier of the microorganism into the tissues from without, but that the foreign body, by its trau- matic and irritative effect furnishes a nidus in the tissues for Actinomyces which enters therein with the secretions from the buccal cavity and gastro- intestinal tract, develops into characteristic colonies, and produces lesions which we call actinomycosis.”’ Colebrook (1920) considered as actinomy- cosis only those cases that showed suppura- tive lesions, the pus of which contained “oranules” visible to the naked eye and com- posed of a framework of a filamentous organ- ism. He did not follow Wright in considering the presence of “clubs” (“ray-formation’’) at the periphery of the granules as an abso- CAUSATION OF ANIMAL DISEASES 255 lute requirement in the diagnosis of actino- mycosis. In isolating pure cultures, Cole- brook used the method of Gordon, which consisted of implanting a granule into a tube of blood-broth under an oil seal. Growth of the organisms was always slow, the primary culture requiring 3 to 8 days and subcultures 2 to 6 days. Growth never occurred at 22°C. All strains isolated showed preference for anaerobic growth, but several were capable of aerobic growth after some subculturing. Occasionally, even primary aerobic cultures were obtained after 10 to 14 days’ incuba- tion. The organism isolated from 21 cases was definitely of the A. bovis type. It showed coarse agglutination with the serum of heav- ily infected patients, as well as with the se- rum of infected rabbits. Colebrook dismissed the idea that infection with such a fragile organism and of such slight viability could occur from outside ‘‘natural’’? sources, as claimed by Bostroem and others. He con- sidered the organism as a common inhabitant of the human alimentary tract. Lieske tried to Justify the apparently con- flicting observations on the aerobic and an- aerobic organisms by emphasizing the fact that the anaerobic forms tend to become aer- obie after several subcultures. This led him to suggest that one type might be converted into the other. Naeslund (1931) finally established the fact that the great majority of actinomycosis cases are caused by a preferentially anaerobic organism. Certain cases, however, affecting the Jung and skin may be caused by aerobes, which come from the inhalation of dust con- taining the organism (Biggart). Cope suggested recognition, for clinical purposes, of two main groups of actinomy- cetes: 1. A very large group including those forms which grow in the natural state in soil and on organic residues. These are hardy organisms growing easily and quickly at nutrient asteroides on (Reproduced from: Kirby, W. M. M. and Me- Naught, J. B. Arch. Internal Med. 78: 8, 1946). Fiaure 100. N. agar room temperature on all ordinary media in the ordinary atmosphere. They are then aerobic in nature. Very few of them are path- ogenie. 2. Those that are preferentially anaerobic. They comprise a much smaller group. Clin- ically they are more important. They are much more delicate and more difficult to grow than the aerobic type. They grow best in the absence of oxygen or with only a limited supply. They are not found outside the body, and are responsible for nine-tenths of the cases of actinomycosis in man. The first group comprises those organisms that are now recognized as members of the genera Nocardia and Streptomyces. Numer- ous reports are found in the literature con- cerning the isolation, from human or animal blood or pus, of actinomycetes belonging to this group. Many investigators were inclined to consider them largely as dust contam- inants. Some (Thjotta and Gundersen) looked upon them not as etiologic agents of particular diseases but as saprophytes found in the respiratory tract (in the throat and on the tonsils) and gaining entrance into the blood of the patient when the body defenses were low. There were numerous other reports of the occurrence of streptomyces in human or- gans, as in the ear (Odom et al.), in tumors (Leyton and Leyton), and in tear ducts and glands (Gruter). Mackinnon and Artaga- veytia-Allende (1956) examined 38 strains of aerobic actinomycetes producing localized mycetoma in various zones of the world. They identified these organisms as species of Streptomyces and Nocardia. The isolation of Micromonospora cultures from animal and human cases has also been reported. Morquer and Comby found (1943) M. caballi to be parasitic on the horse, but not on rabbits, rats, or mice. Acton and McGuire observed the occur- rence in India of a red actinomycete that produces keratolytic changes in the skin of the hands and feet, causing lesions known as keratolysis plantare sucatum, mango toe, cracked heel, paronychia, onychomycosis, and vesicular eruptions. It is commonly be- lieved that these lesions are caused by walk- ing barefooted on damp soil, particularly soil contaminated by horse manure. The organ- ism was actually recovered from both horse and cow manure; it had a marked lytic action on the horny layer of the epidermis of the soles of the feet and sometimes on the palms of the hand. In culture, the colonies were red or black, with deep mycelium penetrating the media. On microscopic examination the organism showed fine hyphae, about 0.8 y in diameter. The conidia were small and round, and formed along the course of the aerial hyphae, at the ends or growing out laterally. At first they were single, but in old cultures they were grouped and surrounded the aerial hyphae. The name A. keratolytica was pro- posed, but a study of the illustrations shows the organism to be a A/icromonospora. The organism causing lumpiness of matted wool in sheep is definitely a Nocardia and not an Actinomyces, as claimed by Bull. Heymer (1957) found a Streptomyces (S. coelicolor) in relatively great abundance in THE ACTINOMYCETES, Vol. I the microflora of human _ beings, nota- bly in the sputum, tonsilar crypts, and skin. This organism exerted an antagonistic effect upon various human pathogenic fungi and yeasts. It was suggested that such organisms may play an important role in keeping the microbiological equilibrium in the human body. Further information, some of which is highly fragmentary, on the causation of dif- ferent animal diseases by actinomycetes is found elsewhere in this treatise, notably in Chapters 24, 25, and 30, Volume IT. In other cases, where some degree of certainty exists of the disease causation, the organisms have not been sufficiently studied to enable ascer- tainment of their exact relationship to others now well recognized. The introduction of new systems of classification has complicated fur- ther the recognition of some of the disease- producing forms. It was simple enough when the organisms could be classified under ‘‘Ac- tinomyces” or ‘‘Streptothrix.”’ It was still relatively simple to place the anaerobes un- der Actinomyces and the aerobes under No- cardia. With the introduction of the newer genera, notably Streptomyces and Micromon- ospora, it became very difficult to decide when an aerobic organism should be placed in the Streptomyces or in Nocardia. In the excellent work of Erikson, for example, the accurate descriptions permit one to decide where certain cultures might preferably be placed. Other, more recent investigators were so uncertain of the systematic position of the particular cultures as to designate them by two generic names, such as Nocardia (Strep- tomyces). Still others (Gordon) were not averse to lumping many cultures, showing only minor variations from one another, un- der a particular species. The present discussion may be limited, however, to two types of disease caused by actinomycetes and most frequently desig- nated as actinomycosis and nocardiosis. CAUSATION OF ANIMAL DISEASES 257 Fiaure 101: Mycetoma pedis (Reproduced from: Pijper, A. and Pullinger, B. D. J. Trop. Med. Hyg. p. 2, June 15, 1927). Actinomycosis An extensive literature has accumulated, since the early work of Bollinger and Harz, on the etiology of actinomycosis. Among the more recent investigations are the work of Chiarolanza (1910), Harbitz and Grondahl (1911), Klinger (1912, 1921), Galli-Vallerio (1912), Ligniéres and Spitz (1924), Magnus- son (1928), Feit (1928), Naeslund (1929), Lord (1933), Grooten (1934), Lord and Trevatt (1936), Mohler and Shahan (1937), Lentze (1938, 1948), Emmons (1937), Cope (1938), Gins and (1940), Davis (1941), Slack (1942), Thompson (1950), Gonzalez-Ochoa and Sandoval (1955), and Paasch many others dealing especially with the oc- currence of actinomycetes in connection with special infections. Actinomycosis in animals was discussed by Lord (1910), Sforza (1940), and various others. The use of animals as diagnostic aids for the identification of A. bovis has been dis- cussed by Meyer and Verges (1950). In general, from a historical point of view, our concepts of the nature of the organisms that cause actinomycotic infections in men and in animals are closely related to the de- velopment of our concept of actinomycetes in general. The animal pathogen Actinomyces bovis has contributed the name ‘‘actinomy- cetes” to the whole group of these organisms, “Actinomycetales” to the taxonomic order, and “actinomycosis” to the major disease. A very extensive literature has accumulated on the pathogenic nature of actinomycosis; the identity of the specific agent has been the subject of much speculation. 258 Ficure 102. Granules and clubs of N. pre- toriana (Reproduced from: Pijper, A. and Pul- linger, B. C. J. Trop. Med. Hyg. p. 2, June 15, 1927). The specific disease affects both man and cattle, usually involving the jaw. Thus, the expressions “lumpy jaw” and “pig jaw”’ are frequently used to designate this condition. The disease is not contagious, but once ac- quired, it is difficult to eradicate. It is char- acterized by a swollen jaw and a hard board- like induration, accompanied by destruction of the normal tissue and formation of granu- lation tissue. “Sulfur granules” are fre- quently present in the pus. They consist of cellular debris and of radially arranged hyphae; these terminate at the periphery in the form of “‘clubs,”’? which are composed of eosinophilic material forming a sheath around the hyphal tip. Immons emphasized that while various actinomycotic infections give rise to clubs, certain forms of the disease caused by actino- mycetes, notably by N. asteroides, do not form such clubs (see Gibson). Even A. bovis may fail to produce clubs in some tissues and under certain conditions. Emmons, therefore, defined actinomycosis as ‘‘an in- fection caused by invasion of the host by some species of Actenomcyes.”’ Several forms of the disease were recognized: actinomyco- sis of the skin, actinomycotic meningitis, lung infection, actinomycotic types of my- THE ACTINOMYCETES, Vol. I cetomas (Kanthack), although some of these may be more properly classified with ‘‘no- cardiosis.”’ The presence of sulfur granules is frequently considered as a diagnostic symp- tom of actinomycosis. Granules may be pro- duced, however, by various organisms, whereas some actinomycetes do not form any granules. As pointed out previously, various at- tempts to isolate the causative agent yielded aerobic cultures that were found later to be ar contaminants. Wolff and Israel are cred- ited with being the first to isolate from maxil- lary actinomycosis in cattle a culture which they found to be a microaerophilic form. This culture was identical with A. bovis. Minute pinpoint and dewdrop-like colonies appeared on the surface of anaerobic agar slant cul- tures. The colonies gradually became larger and formed ball-like, irregularly rounded, elevated nodules; they did not become con- fluent and homogeneous. Some of the colo- nies presented a prominent center with a lobulated margin, appearing in the form of rosettes. In stab cultures, growth was more pronounced and was limited to the lower por- tion of the line of inoculation. In liquid broth, small white flakes appeared under aerobic conditions, some floating on the sur- face and some falling to the bottom of the tube. In general, anaerobic conditions were favorable to the growth of the organism. Microscopie examination of the culture grown on agar showed long filaments form- ing a network. These were arranged more or less radially; they were straight, wavy, or spiral, and sometimes branched. Smear prep- arations gave short, homogeneous, usually straight, but also comma-like, rods of varied length and width; the ends of the rods often showed club-like swellings. The tumor-like growths of infected ani- mals were situated partly on the abdominal wall and partly on the intestines, in the liver, and in other tissues. Microscopic examina- CAUSATION OF ANIMAL DISEASES 259 tion showed typical actinomyces colonies. The histological appearance of the tumors was similar to that of actinomycotic tissue. In 1905, Wright made a detailed study of actinomyecosis in man and in animals. He suggested that the word ‘‘actinomycosis”’ be restricted to a suppurative process com- bined with granulation tissue formation, the pus of which contains the characteristic granules. These are made up of dense ag- gregates of branched filamentous microor- ganisms and of their transformation or de- generation products; the latter include the characteristic club-shaped bodies radially disposed at the periphery of the granule. Cultures isolated from human and bovine cases were found to show insufficient differ- ence to justify their classification as separate species. Wright further suggested that organ- isms different from A. bovzs and which were associated with other forms of actinomycosis be grouped together under Nocardia, and that those cases of undoubted infection caused by them should be designated as ‘‘no- cardiosis.” The presence of actinomycetes in sputum and in the contents of carious teeth was studied by Lord. Emmons also obtained or- ganisms of the A. bovis type from the oral cavity. He isolated from tonsils two micro- aerophilic types of actinomyces: one, mor- phologically and physiologically similar to A. bovis; and another somewhat different morphologically, but also considered as a strain of A. bovis. Slack differentiated be- tween the exogenous and endogenous types of infection in actinomycosis: in the first, awns of grass and grain frequently observed in actinomycotic lesions suggested their role in the infection; in the second, the anaerobic organisms isolated from normal mouth, from tonsils, from carious teeth, and from pyor- rhea pus suggest their etiology. The oral cavity was looked upon as the source of in- fection, possibly accompanied by sensitiza- Figure 103. Lumpy jaw in a cow (Reproduced from: Mohler, J. R. and Shahan, M. S. U.S.D.A. Circ. No. 438, 1937, p. 4). tion. Numerous investigators (Magnusson, Negroni and Bonfiglioli) reported consid- erable variation in the strains isolated from different forms of clinical actinomycosis. Maxillary actinomycosis is believed to be caused by organisms living in the mouth, since the contents of the mouth and tonsils were found capable of causing actinomycosis in experimental animals. It was suggested that A. bovzs is often present in oral cavities, where it may exist as a saprophyte. Emmons also suggested the possibility that there are atypical strains found in certain lesions, in sputum, in carious teeth, and in tonsils. Hu- man actinomycosis as influenced by mode and source of infection has been studied by Acland (1886), Shiota (1909), Mattson (1922), Shapiro (1931), Thompson (1950), and numerous others. Actinomycotic endocarditis has been studied by Wedding (1947); actinomycosis of the eye by Herrenschwand (1927), of the face and neck by Lamb et al. (1947), by Glahn (1954), and by others. Skin actino- mycosis has been studied by Namyslowski (1909, 1912) and Daines and Austin (1932); actinomycosis of the knee by Moore et al. and many others; liver abscess by Bloom- 260 field and Bayne-Jones (1915); actinomycosis of the esophagus by Langer, of tonsils by Davis (1914), of the oral cavity by Naeslund (1925), and Sullivan and Goldsworthy (1940), cervicofacial by Glahn (1954), of the nervous system by Jacobson and Cloward, and of the heart by Cornell and Shookhoff (1944). Pulmonary actinomycosis has re- ceived much attention (Warthin and Olney, 1904; Sartory and Sartory, 1925; Penta, 1941; Lynch and Holt, 1945; Poppe, 1946; Vawter, 1946; Garrod, 1952). The earlier literature on various forms of actinomycosis is found in the work of Schlegel (1928). Erikson suggested that the anaerobic or- ganisms should be divided into the human and the bovine types. Lentze also concluded that actinomycosis in man and in animals represents two different types of anaerobic gram-positive organisms. Those involved are: (a) one (R-type) capable of growing on the surface of the medium, forming leathery irregular colonies and producing a sediment in liquid media; (b) another (S-type) pro- ducing smooth, easily broken colonies on solid media and causing turbidity in anaero- bie liquid media. The first represents the classical type of Wolff-Israel and the other resembles corynebacteria. According to Wright, however, no significant differences exist between human and bovine strains. Gradually, an extensive amount of litera- ture has accumulated on the etiology of in- fections caused by actinomycetes. Naeslund grouped these infections under the anaerobic and aerobic types. The first, or A form, can be readily isolated from the mouth; it is a typical A. bovis and can bring about the true actinomycotic infection. The second, or B form, is a pathogenic aerobe, considered to be less important than the anaerobe; it is commonly found in nature, usually produc- ing reddish or yellowish colonies, is acid- fast, and usually forms spores; it comprises the forms now included under Nocardia. Rosebury isolated four strains of A. bovis THE ACTINOMYCETES, Vol. I from cervicofacial actinomycosis, and 11 from gingival scrapings taken under oral pathological conditions in the absence of actinomycosis. Optimum conditions for growth of these organisms were provided by anaerobiosis in the presence of 5 per cent carbon dioxide. Considerable variation was observed in oxygen tolerance among the dif- ferent strains at different times. Pure cul- tures were maintained by cultivation under anaerobic conditions. Cope summarized the data of 1330 cases of actinomycosis: of these, 56.8 per cent affected the cervicofacial re- gion, 22.3 the abdomen, 14.9 the thorax, and 5.9 per cent other sites. The clinical features of actinomycosis were described by Conant and Rosebury as fol- lows: ‘““Actinomycosis is a subacute or chronic, gen- erally progressive disease of man, cattle, swine, horses and other animals, characterized by the development of indurated granulating swellings chiefly in connective tissue, by suppuration usually of limited extent, and by the presence in the pus or lesions of Actinomyces bovis, demon- strable microscopically or culturally. In man the lesions are found chiefly in the cervicofacial con- nective tissues and in the thoracic or abdominal viscera, and develop over periods ranging from a few weeks to a year or more. The lesions spread widely by contiguity, sometimes pointing toward the skin and forming fistulae that tend to heal and reform elsewhere; rarely pointing toward mucous or serous membranes. The organism may be dis- seminated through the blood, or, in the lungs, through the bronchi. The lymphatic system is only rarely involved. Bone may be eroded in the path of the lesion, but is seldom affected interstitially except in the jJaws.”’ Different forms of actinomycosis are fur- ther differentiated by Conant and Rosebury: “Cervicofacial actinomycosis accounts for more than half of all cases in man. It apparently originates from the mouth, but affects the soft tissues and skin of face and neck, and the tongue, and secondarily, the maxillary bones. The salivary glands, larynx, thyroid, and lacrymal glands, the orbit and even the brain may more rarely be in- volved. The commonest lesions appear on the CAUSATION OF ANIMAL DISEASES cheek or submaxillary skin, and are characterized by indurated or edematous swellings, bluish or reddish in color, with a tendency to form a series of irregular folds separated by furrows, the healing lesions forming scars as new lesions develop. “Thoracic actinomycosis accounts for about fifteen per cent of human cases. It is found mainly in the lungs, with the formation of abscesses and cavities which are usually small. Extensive lesions may be found in the bronchi, and their rupture may lead to dissemination of the infection by way of the bronchial tree. Actinomycotie pleurisy and empyema have been observed, as has involvement of the heart and pericardium. Thoracic lesions may originate from the mouth or throat by aspira- tion, by extension from the abdomen, or by me- tastasis. “Abdominal actinomycosis comprises about twenty per cent of human cases. The lesions may be found in any organ but are most common in the region of cecum and the appendix. From here they may extend with suppurating foci and the formation of fibrous adhesions to the abdominal wall, where skin lesions may appear similar to those of cervicofacial actinomycosis. Or the lesion may remain circumscribed, forming a fibromalike mass. The liver is commonly attacked, and lesions of the genital tract are relatively frequent. The stomach, small intestine and kidney are seldom affected. Infection is probably derived in most instances directly or indirectly from the intestinal or genital mucous membranes which, however, are not themselves involved. In the skin, actinomycosis, secondary to lesions of underlying tissues or organs, is relatively common, as has been noted; but itis doubtful whether true actino- mycosis is ever primary in the skin.’’ Nocardiosis Nocard was the first to describe, in 1888, a pathogenic actinomyces of the aerobic type. This organism was found to be the cause of “farcin du Boeuf,” a disease of cattle in the Guadeloupe Islands. Trevisan named this organism Nocardia, in honor of its discov- erer, the species being N. farcinica. Soon af- terwards, Eppinger described a filamentous organism found in the pus of a cerebral abscess; he designated it as Cladothrix asteroides. This organism was transferred to the genus Nocardia by Blanchard and No- eard (1896). MacCallum reported in 1902 261 that N. nitis in experimental animals. According to Benbow, Smith, and Grimson, about 90 per asteroides produces a diffuse perito- cent of all clinical cases of actinomycosis are caused by A. bovis; the remaining 10 per cent are caused by N. asteroides, most strains of which are partially acid-fast. Benbow et al. excluded from this classification the myce- tomas which are caused by other species of Nocardia. Following these early studies, much work was done on nocardiosis and the organisms involved. It is sufficient to mention that of Nakayama (1906), Evans (1918), Drake and Henrici (1943), Gonzalez-Ochoa (1945), Gonzalez-Ochoa and Hoyos, Binford and Lane (1945), and Kirby and McNaught (1946). Pijper and Pullinger (1927) emphasized the affinity for iron among the Nocardia or- ganisms. Henrici differentiated between three well- defined types of infection caused by actino- mycetes in man and in animals: 1. The lumpy jaw type, which is the most common infection and is produced by an organism be- longing to A. bovis studied by Israel. 2. The madura foot type, caused by an aerobic form which is usually designated as Nocardia madurae, and more recently recognized as Streptomyces madurae (Mariat, 1958). 3. A rare type of infection caused either by N. asteroides, most frequently in man, or by N. farcinica, which occurs in cattle. Glover et al. (1948) suggested that the term ‘‘actinomy- cosis” be restricted to infection due to the microaerophilic A. bovis and that ‘‘nocardio- sis’ be used for infections caused by the aerobic N. asteroides and other species of Nocardia. As many as 13 species of Nocardia isolated from white, yellow, or red granules found in the pus in cases of mycetoma have been described, but some of these names are now recognized as synonyms. Vincent cultivated the organism now known as S.(V.) madurae. 262 THE ACTINOMYCETES, Vol. I He considered it to be the most common causative agent of the disease. These aerobic organisms cause specific types of mycetomas. Infections of the lungs and of the skin are frequently produced. The organisms are cultivated much more readily than the ana- erobic types and are pathogenic to labora- tory animals. N. farcinica, isolated from cattle, forms a yellowish, wrinkled growth on solid media. N. caprae, isolated from the lung of a goat, gives a more whitish growth and greater fragmentation of the mycelium. N. canis, which produces infection in dogs, is similar to N. caprae. Conant and Rosebury described the clin- ical features of nocardiosis as follows: “Nocardiosis 1s a chronic suppurative, purulo- granulomatous disease of the subcutaneous tissues and bones (mycetoma) characterized by multiple tumefactions and draining sinuses from which granules (yellowish-white, red, or black) are ex- pressed in the pus or found in the tissues; or, a pseudotuberculous infection (systemic) of the lungs and pleura with hematogenous spread throughout the body, especially to the brain and meninges, in which filamentous, bacillary or coccoid, acid-fast forms may be found in the sputum, spinal fluid, or pus from subcutaneous abscesses. “Mycetoma of the extremities results in the clinical picture of Madura foot, although other subcutaneous tissues of the body also may become infected. The characteristic lesion with pain, swelling, and sinus formation, and eventual club- bing and marked deformity of the infected member is developed only after months or years. Infection spreads by extension through adjacent tissues with bone destruction, multiple abscesses with rupture, and with no systemic reaction unless secondary bacterial invasion is’ established. Histologically, sections of the sinus and abscess walls may show only a chronic inflammatory re- action. Further development of the acute purulent abscess results in a surrounding layer of granula- tion tissue infiltrated with round cells and fat- Jaden macrophages enclosed by a fibrous capsule. Diagnosis, however, depends on the presence of granules, surrounded’ by neutrophils, centrally located in the abscesses. “Systemic nocardiosis is caused by NV. asteroides and is chiefly pulmonary in origin. Of thirty-four polymorphonuclear cases, including two new cases, reviewed by Kirby and McNaught (1946) the lungs were infected in twenty-nine and, of these, eleven had metastases to the brain. Occasionally the presenting symp- toms of headache, nausea and vomiting may indi- cate either brain tumor or brain abscess; or, the symptoms may be those of an infectious meningitis (tuberculous) with minimal or no findings in the lungs. Symptoms referable to a pulmonary infec- tion include general malaise, fever, productive cough with sputum, night sweats, anorexia and loss of weight. Roentgenograms of the lungs usually show a progressive infiltrative process which may lead to multiple cavity formation. Hematogenous spread results in metastatic lesions throughout the body. Histologically, such lesions may be of a purulent nature, containing centers composed of polymorphonuclear neutrophils and a few mycelial fragments, or such areas may show a more advanced granulomatous reaction leading to granulation tissue, giant cells and searring.’’ Granules are not formed by N. asteroides. Mariat (1957) made a detailed study of the causative agents of chronic subcutaneous lesions, known as mycetomas. Only five species were recognized: 1. Nocardia asteroides. An infrequent caus- ative agent of mycetoma. Semi acid-fast or- ganism. Enzymatic reaction reduced or ab- sent; pathogenic to experimental animals. 2. Nocardia brasiliensis. Prevalent in Central and South America. Semi acid-fast. High enzymatic potential. No agreement on experimental pathogenicity. 3. Streptomyces madurae. World-wide dis- tribution. Causative agent of mycetoma. Forms large granules, white to reddish white; surrounded by long, club-shaped swellings. Hyphae not acid-fast. Nonpathogenie to ani- mals. 4. Streptomyces pelletiert. Found largely in Africa. Granules small, numerous, and red, often fragmented. Hyphae not acid-fast. High enzymatic potential, but not patho- genic to animals. 5. Streptomyces somaliensis. Frequently found in Africa. Granules yellowish white. Hyphae not fragmented. High enzymatic po- tential. Not pathogenic to animals. The nu- CAUSATION OF ANIMAL DISEASES tritional properties of these organisms have been presented in Chapter 7. Further analyses of nocardiosis, or the aerobic actinomycotic diseases due to aerobic actinomycetes, largely of the Nocardia type, are found in the numerous textbooks on bac- teriology (Plehn). Gruter made a comprehensive study of the occurrence of actinomycetes in eye in- fections, especially in tear glands and duets. He came to the conclusion that a nocardia was involved. He designated his culture A. discofoliatus. According to Gordon and Hagan, certain acid-fast actinomycetes isolated from soils and plant material are similar to those found in lesions of men and animals. The pigments produced by these organisms range from yellow through orange to coral. One of the soil forms, soon after isolation, was found to be pathogenic to rabbits but not to guinea pigs. Allergic Reactions Various attempts have been made to ex- amine the immunological reactions of ac- tinomycetes. Goyal compared 11 cultures obtained from collections and as fresh isola- tions. Most of them appeared to be members of the genus Nocardia. When inoculated into rabbits, they proved to be either entirely nonpathogenic or only slightly virulent, ex- cept for N. eppingert. The cultures were grown in glycerol broth, at 38°C for 30 days; extracts, designated as ‘‘streptothricin, were prepared in a manner comparable to oe 9 tuberculin (Helzer). Animals sensitized to the nocardia extracts (‘“‘nocardin’’) were also sensitive to tuberculin, and vice versa (Ble- tey). Serologic studies confirmed the conclu- sions reached on the basis of allergy tests; a common antigen was demonstrated for the tubercle bacillus, the diphtheria organism, and the nocardias. These results led to the conclusion that there is a definite antigenic 263 relationship between the actinomycetes and the mycobacteria. Further studies of the allergic reactions of actinomycetes have been made by Mathie- son et al. (1935). Therapy of Actinomycotic Diseases The therapy of actinomycosis has been given rather limited consideration (Menning, 1933; Heuber, 1940). According to Cope: “The prognosis of actinomycosis varies greatly according to the part of the body affected. It is most favourable with those cases which affect the head and neck, less favourable with abdominal cases, and most unpromising with thoracic disease “The great majority—probably 97 per cent—of those cases in which the cheek, jaw, tongue, and neck are involved come to a satisfactory issue. The disease may last for several years but nearly always ends satisfactorily. The 3 per cent of cases in which death results are those in which the pathological process either extends deeply towards the base of the skull and causes cerebral complica- tions, or extends downwards to the superior and posterior mediastinum, or more rarely, becomes generalized.” Treatment of actinomycosis consisted first of radiation therapy and use of vaccines. More recently, with the advent of the sulfa drugs (given internally or applied locally) and especially the antibiotics, chemotherapy of actinomycotic infections took a new turn. Previously use was made of specific vaccines (Scott), and iodine was considered to be by far the most important drug which had a definite effect in the treatment of actinomy- cosis (Cope). But today the sulfa drugs and the antibiotics have taken the place of iodine in chemotherapy, of both actinomycosis and nocardiosis. This was demonstrated by Cut- ting and Gebhardt (1940), Dobson et al. (1941), Hollenbeck and Turnoff (1943), Lyons et al. (1943), Hendrickson and Leh- man (1945), Farris and Douglas (1947), Kay (1947), Holm (1948), Benbow et al. (1949), Boand and Novack (1949), Strauss e¢ al. 264 (1951), Fischer and Harvey (1956), Bianco et al. (1957), and numerous others. Among the antibiotics used were penicillin (Dobson and Cutting, 1945; Drake, 1946; Holm, 1948; Nakhimovskaia et al., 1957), streptomycin (Pemberton and Hunter, 1949; Torrens and Wood, 1949), chloramphenicol (Littman et al., 1952), the tetracyclines (Martin et al., 1956; Lane and Kutsaber, 1956), and various others (Banerjee ef al., 1954; Hanf, 1956). In a comparative study of the inhibitory effect of antibiotics upon the growth of the anaerobic A. israeli, Garrod found penicillin to be active in a concentration of 0.1 unit and streptomycin in 23.7 units, with the tetracyclines and chloramphenicol falling between (2.2 to 4.2 units). This bears out the sensitivity of anaerobes to penicillin and their relative resistance to streptomycin. Frequently an antibiotic is active only in vitro or in vivo. The most effective antibiotics against N. asteroides, for example, were THE ACTINOMYCETES, Vol. I found to be, when tested zn vitro, erythro- mycin and novobiocin; however, the only therapeutic action in experimental animals was exerted by cycloserine. Mackinnon et al. made a detailed study of the effect of various chemotherapeutic agents upon and nocardiosis. They found that strains belonging to the same species show sufficiently similar sus- ceptibility as to prove species sensitivity. Three species of Streptomyces (S. somaliensis, S. pelletiert, and S. madurae) were susceptible to at least two of the following antibiotics: penicillin, streptomycin, chlortetracycline, and chloramphenicol. These species could be distinguished by their relative sensitiveness to these antibiotics. Diaminophenylsulphone is active upon N. brasiliensis, N. asteroides, and S. somaliensis, but less so upon S. pel- letiert. The pathogenic actinomycetes were found to show development of resistance to streptomycin and to aromatic diamidines. mycetoma KR i 8 Causation of Plant Diseases Very few forms among the actinomycetes are capable of causing plant diseases. In spite of the great abundance of actinomycetes in nature, especially in the soil, the number of plants attacked by them, as compared to the number of plants attacked by bacteria, fungi, and viruses, is rather limited. Except for two species—the Irish potato and the sugar beet—plants subject to infection by actinomycetes do not occupy a very promi- nent place in human economy. Potato Scab An extensive amount of literature has ac- cumulated dealing with the causation of po- tato scab, the development of the disease, the organisms involved, the effect of environ- ment, and methods of control. Our main concern here is the causative agents of the disease. Organisms The organisms capable of causing com- mon scab of potatoes were at first believed to comprise only a single actinomycete. Grad- ually it came to be recognized that a number of species, or at least a number of races or strains, all belonging to the genus Strepto- myces, are capable of causing the infection. In 1890, Thaxter first described the organ- ism causing potato scab under the name Oospora scabies. This was later changed to Actinomyces chromogenus, then to a @ oO (o} oOo RELATIVE NUMBER OF SCABBED TUBERS, % nN ° to) 5.5 6.0 mn ° +—TUBERS SCABBED 269 40 WwW oO pH FREQUENCY +—pH OF SOIL fe} 6.5 7.0 15 pH Figure 105, Relation of soil reaction to the occurrence of potato seab (Reproduced from: Dippenaar B. J. Union of South Africa Dept. Agr. Sci. Bull. 136: 52, 1933). found difficult if not impossible to do this. It has been observed in western Nebraska that potato scab is always severe following heavy, packing rains when the field cannot be cultivated subsequently. Scab was also commonly found in flooded areas, at the lower ends of irrigated fields, and in poorly drained portions of dry-land fields. This is the reverse of what one might expect from the work of Sanford and Dippenaar on soil moisture and of Sanford on the necessity of abundant aeration for the best development of the scab organism. A study was made to determine whether the effect of soil sterilization on the occur- rence of scab was due to chemical or physical changes in the soil or to lack of competing organisms. Sterilized soil was treated with an extract of unsterilized soil. The effect of the time of inoculation was determined by adding the inoculum either at the time of planting or at the beginning of tuber forma- tion. In sterilized soil there was a greater amount of scab. This was due to a lack of FriaurE 106. Corky seab of potatoes (Repro- duced from: Millard, W. A. Common scab of pota- toes. Univ. of Leeds Pam. No. 118, 1921, p. 2). competing soil microorganisms that would antagonize the scab-producing forms. The numbers of actinomycetes in the soil, as de- termined by the plate method, checked well with the incidence of scab in the various tests. The largest number of actinomycetes, 82 days after sterilization, occurred in the soil which had been inoculated for the long- est time and in which the inoculum had ap- parently become well established before the soil became infested with competing organ- isms. Further tests of the effect of additions of sterilized and nonsterilized organic matter TABLE 76 The effect of the competition of soil microorganisms wpon the occurrence of scab in inoculated soils (Goss) Tubers in various classes, determined by percent- o Num-| age of scabby surface Treatment of soil* uw | ber of 3 |tubers S ES ell oS Sho | O37 |) Daag Ss Z a Sterilized soil, inoculated with S. scabies 1. No treatment 58 | 249 2 6 | 34 | 58 2. Filtrate of un- | 34 | 176 fy | dle |) a || aks} sterilized soil 3. Filtrate of ster-) 40 | 205 ilized manure 4. Sterilized ma- | 20 | 102 PZ) ila | 4G) |] Bry ~J 19) ise) fo ws leo) nure Se hultrate of un= oO le2lon 2002220 34 | 24. sterilized ma- nure 6. Unsterilized 20 78 54 | 17 | 23 6 manure 7. Penicilliwm sp. | 30 | 160) 1) 41] 85 | 60 8. Bacteria 30 | 160 9) 9 | 38 | 44 9. Streptomyces 20 | 103 7| 7 | 48 | 39 sp. (sapro- phytic) 10. Mixture of 19 94 2 2 o2 ses treatments of 1185.9 Sterilized soil, not inoculated 11. No treatment 20 85 | 100 0 0 0 Unsterilized soil, inoculated with S. scabies , 12. No treatment EXON, PAL |p lee ay | ats) | ale) * All treatments were made at the time of inocu- lation. All inoculations were made at the rate of 200 ml of inoculum per pot, equal to 146 Petri dish cultures. Saprophytic organisms were added at the same rate. The filtrates were obtained by soaking 4 parts of soil or manure in 6 parts of water over- night, filtering through cheesecloth, and adding 200 ce per pot. The manure in Sets 4 and 6 was added at the rate of 200 gm per pot. THE ACTINOMYCETES, Vol. I and of the filtrates of both to sterilized inocu- lated soil confirmed the above observations. Three types of organisms were isolated from the unsterilized soil, cultivated, and added to the soil in amounts approximately equal to the S. scabies content of the soil. This was for the purpose of determining whether any one of these particular groups was an effec- tive competitor of the scab organisms in the soil. These tests were made during different years in the greenhouse, but the results were of the same general nature (Table 76). Goss stated that in emphasizing the num- bers of actinomycetes and their relation to soil moisture and other factors, one must consider the fact that these organisms must be growing vegetatively if they are to cause infection. High plate counts often indicate sporulation or fragmentation of the myce- lium, whereas low counts may indicate vege- tative development, and this in turn may be correlated with high infection. Dippenaar suggested that germination, growth, and sporulation may all occur at their maximum under similar conditions. Conditions favor- able for sporulation would tend to increase the scab-producing power of the soil. If this were followed by conditions favorable for vegetative development, more infection might result. The effect of moisture and ster- ilization upon scab development is brought out further in Table 77. Numerous other investigations have been carried out throughout the world on the ef- fect of various environmental factors on growth of the potato scab organisms and on the causation of scab (Shapovalov, Jan- chen, Cocchi, Blodgett and Howe). The effect of the variety of potato on scab develop- ment also has received considerable atten- tion (Umbreit, 1938). Relationship of Host to Parasite Schaal and his collaborators made an extensive study of the effect of phenolic sub- stances upon the degree of infection of pota- CAUSATION OF PLANT DISEASES 271 toes (pustule type). The formation of chloro- genic acid in the cells of the lenticel area was the causative mechanism and Schaal, 1952; Schaal and Johnson, 1955; Schaal et al., 1953). They found that, in cells adjacent to the periderm, chlorogenic (Johnson acid was present in larger quantities in po- tato tubers of scab-resistant varieties than in those of susceptible varieties. A test that would not require destruction of the whole tuber was devised so the tested tuber could be used for seed purposes. The test was based on the fact that chlorogenic acid is localized in the cells directly under the epi- dermis and corky covering and in cells around the lenticels of the highly resistant varieties. Scab resistance was found to be associated with the amounts of chlorogenic acid present. The test consisted in placing several drops of 2 per cent aqueous ferric chloride solution on the surface of a tuber and macerating with a stainless steel knife the tissue covered by the test solution. By pricking this area un- der a drop of ferric chloride solution, the presence of chlorogenic acid in or near the lenticels could be determined. The distribu- tion of chlorogenic acid in the tuber was measured by spreading the ferric chloride solution over the freshly cut surface of a half of a tuber. In resistant varieties a green color reaction was found near the surface. In some highly resistant varieties the chloro- genic acid was present throughout the tuber, its greatest concentration being in the cells directly under the corky covering. The color reaction was found to be greater in the im- mature than in the mature tubers. Different varieties showed differences in color reaction. The effect of six phenolic compounds on the growth of S. scabies was tested at pH 6.0, 7.5, and 8.5. Four orthodihydricphenols (chlorogenic acid, caffeic acid, catechol, and tetrahydroxybenzoin) were effective upon autoxidation in inhibiting the growth of S. scabies in culture medium. The inhibition increased with increases in the pH. These results were said to support the theory that the mechanism of scab resistance in potato tubers involves enzymatic oxidation of chlor- ogenic acid, which produces a quinone toxic to the scab organism. The mechanism was believed to depend on the amount of chlorogenic acid in the periderm and on the presence of tyrosinase in the same tissue. The localization of this acid around the lenticels that serve as the normal entrance points for the scab organism was important. A tendency for the acid to accumulate in cells adjacent to injured areas was observed. Tyrosinase was found in high concentration in the tissue containing chloro- genic acid and was found to become oxidized when the tissue was injured. The oxidation products formed, such as quinones, were be- lieved to be toxic to the invading organism. Johnson and Schaal (1957) brought out further that the concentrations of chloro- genic acid and of total o-dihydricphenols were much higher in the periderm of tubers of certain scab-resistant varieties than in tubers of scab-susceptible varieties. The greatest difference between the varieties was found at the stage where the tubers were growing most rapidly. The tubers were analyzed at three stages of growth and after 5 months of storage at 35 to 38°F. The re- sults indicated that the ferrie chloride spot test for scab resistance could be made on im- mature or on freshly harvested mature tu- bers. Effect of Treatment Addition of stable manure to soil has usu- ally been found to increase the incidence of scab, because of the resulting alkaline reac- tion and the accumulation of humus. Similar results were also reported from excessive use of potassium. Application of certain fresh organic materials, such as green manures, results, however, in the reduction of seab. Millard and Taylor saw in this effect a kind 272 THE ACTINOMYCETES, Vol. I TABLE 77 Effect of soil sterilization and moisture content on scab following inoculation with S. scabies (Goss) | | | fost in various eee devenis | re t * Set No. Soil treatment \No. of tubers| Mees He eee Ea cael | | a0 02 | 225 | 25-75 | 75-100 1 __Sterilized-high moisture 33 lekle) S460 15/|) h4&%)| 1 88ccln Se ee 2 Sterilized-low moisture 900 0} 0 BB i 97 3 Not sterilized-high moisture 36 1795 | é 12° |. 84 1 0 4 \Not sterilized-low moisture 38 | 1210 0 0 21 ey 64 * Data based upon tuber weights. of competition between the soil saprophytes and parasites. Further studies indicated that several factors may be involved. These com- prise an increase in soil acidity, an increase in the buffering and moisture-holding ca- pacities of the soil, anda possible stimulatory effect upon those soil microbes which exert an inhibitory effect upon the scab organisms. Soil sterilization and subsequent inocula- tion lead to increased infection. This is apparently due, according to Goss, to a result- ant lack of competition of soil microorgan- isms rather than to changes in the physical or chemical structure of the soil. Addition to such soils of a filtrate from unsterilized soil tends to counteract this effect; the effect of sterilization is greatly reduced if the inocu- lum is not added until after saprophytic or- ganisms have become established in the soil. Various other factors affect the pathogen- icity of the scab organism, such as passage through the digestive tract of (Morse), spread by potato residues (Lut- man), the influence of the potato variety (Longree), and the presence of antagonistic animals organisms (Daines). According to Fellows, if the scab disease is to occur, the potato tubers must be in- creasing in size. Stomata or young unsub- erized lenticels must be present through which the infection can take place. There must also be dividing cells or cells which can sasily be incited to division by the products of the organism, thus permitting the produc- tion of the typical corky scab lesions. According to Goss and Werner, seed treat- ments are effective in controlling seed-borne scab; however, even when healthy or treated seed potatoes are used, the disease may be very severe because of infection from the soil. Crop rotation reduces the incidence of the disease, but the fact that potato scab may cause serious loss in soils never before planted to potatoes suggests that other fac- tors than the time interval between potato crops affect the occurrence of the disease. Biological Control of Potato Scab No attempt will be made here to review in detail the practical methods of control of potato scab (Berkner and Schréder, Noll). Suffice it to say that crop rotation (Werner et al.) and acidification of soil (Martin, Waksman, 1922, Duff and Welch, Cook and Nugent, Blodgett and Cowan) were found to be most effective. A complete review of the literature, especially of the environ- mental factors bearig on scab formation, has been made by Hollrung. One of the most interesting aspects in the production and control of scab on potatoes is the possible effect of other soil microorgan- isms. Reference has already been made to the work of Millard. Sanford (1926) has shown that green rye plants, plowed into the ground at the rate of 50 tons per acre, showed no effect upon the reduction of scab in a well- CAUSATION OF PLANT DISEASES 213 infested soil at pH 5.0 to 5.4. There was no noticeable increase in soil acidity during a 58-day period. In artificial media, however the growth of certain bacteria made condi- tions unfavorable for the growth of S. sca- bies. The conclusion was reached that when scab is controlled in some soils by green ma- nure crops, this may be due to the ‘‘antibi- otic’ qualities of certain predominant soil microorganisms. Kenknight attempted, unsuccessfully, to control scab through the antagonistic ac- tivities of other microorganisms. Addition of organic matter to the soil tended to aggra- vate the development of scab except in ex- treme cases of scab infection. The findings of Millard and Taylor that green manure was effective in controlling scab in the presence of S. praecox were not confirmed. When this and other species of actinomycetes were in- troduced into scab-infested soil in green ma- nure and in various other media no control was obtained. KenKnight thus agreed with Goss, who also failed to obtain control of scab with S. praecox. MeCormick observed that S. praecox was antagonistic on solid media to S, viridis and S. intermedius, but not to certain other para- sitic actinomycetes. This observation sug- gested specific differences in the action of the antagonist. No control of scab was obtained with B. megaterium, an organism antago- nistic to certain actinomycetes. Pseudomonas fluorescens, which was antagonistic to certain actinomycetes and to Trichoderma lignorum, reported to be antagonistic to many fungi, exerted no effect on the scab organism. Daines reported (1937) that Trichoderma lignorum produces a diffusible substance which is toxic to S. scabdes in an artificial liquid medium. Because of the rapid destruc- tion of this toxie principle by aeration at the pH of potato soils and because of removal of the toxic material from solution by charcoal and by the soil itself, the efficacy of this fungus in combating potato scab was con- sidered as doubtful. Although Trichoderma might be of some assistance in this capacity in poorly aerated soils which possess low ad- sorptive capacities, its function in many soils was questioned. The physical as well as the biological en- vironment in many cultivated soils was con- sidered a strong barrier against the establish- ment of the 7’richoderma. When introduced into a 5-day-old S. scabies culture, T'richo- derma was strongly inhibited by the seab. A soil-inhabiting bacterium was also found to produce a material that was toxic to T’richo- derma and to the actinomycete alike. Daines argued that ‘in such complex physical, chemical and biological environments, as are afforded by soils, these antagonistic relation- ships may be modified or even entirely de- stroyed.” KkenkKknight argued that the absence of proper biological controls in the above ex- periments may have been due to failure to establish the introduced organisms in the soil, or to the failure of these organisms to show antagonism toward any or all of the parasitic actinomycetes under soil condi- tions. He found an analogy between the in- troduction of organisms not well suited to the soil conditions and efforts to obtain a stand of wheat by planting the seed in un- broken prairie sod. He believed that Iies- sling’s mixed cultures of bacteria that had the capacity to control scab, except for one type of scab caused by a species other than S. scabies, suggested that if practical control of potato scab is ever obtained by biological means in soil infested with several parasitic actinomycetes, it will be with mixed cultures, because it appears unlikely that a single organism will be found that is antagonistic to all strains of the scab organism. The relation of soil fungi to the develop- ment of potato scab has been studied further by Pratt (1918) and by others. Detailed studies have been made of the re- lation of the soil population to the develop- 274 ment of the scab organism. The question was raised whether actinomycetes diminish in numbers and whether the parasitic potato- scab-producing strains tend to die in soil in which no potatoes have been grown for a long time. The actinomycetes were found to remain fairly constant in their numbers and in their percentage relation to the total num- ber of organisms. The pathogenic types, how- ever, were gradually reduced in numbers. Tests were made by planting a susceptible potato variety in the soil; if the scab-produc- ing strains were still present, scabbing would result. The formation of russetted tubers sug- gested the possibility that the scab organism, after a long period of deprivation of its host, was weakened in pathogenicity and produced the true, deep scab only in rare instances. S. scabies is also capable of causing necrosis of subterranean stems of potatoes. The stems may become girdled and rotted at the base with vascular discoloration extending up the stem six to eight internodes (Hooker and Kent). Sugar Beet and Mangel Scab Certain actinomycetes are also capable of causing scab on various root crops, nota- bly sugar beets and mangels. Kriiger was the first to establish, in 1904, that the production of scab on sugar beets is due to actinomy- THE ACTINOMYCETES, Vol. I cetes. Under the influence of the generic des- ignation of the actinomycetes used by Thax- ter, he described several actinomycetes as species of Oospora, namely, O. cretacea, O. rosella, O. intermedia, O. tenax, O. nigrificans, and O. violacea. All of these were, however, typical actinomycetes, belonging to the genus Streptomyces. Kriiger studied particu- larly the type of scab known as “‘girdle’”’ seab of sugar beets. The strains of the organisms he isolated were believed not to be identical with the potato scab form of Thaxter. Various other isolations of actinomycete cultures were made from beet scab. Some of these cultures were found to be parasitic but varying in the degree of virulence. Mil- lard and Beeley recognized two distinct types of mangel scab, the raised and the pit- ted forms. The raised scab was subdivided into the mound and knob types, which were found to develop particularly on yellow- skinned varieties of mangels. The pitted scab was similar to the common scab of potatoes, whereas the raised scab was not formed from ¢ the cambium of the vascular rings, but re- sulted from the proliferation of the pericycle. A culture was isolated from mound scab which reproduced the same type of scab in artificial inoculation experiments; it was de- tumulz. From pitted scab a culture was isolated which also reproduced seribed as A. Fiagure 107. Mangel scab (Reproduced from: Millard, W. A. and Beeley, F. Ann. Appl. Biol. 14: 311, 1927). CAUSATION OF PLANT DISEASES its own type in inoculation experiments. This culture also attacked the roots and rootlets of the inoculated mangel plants, on which it produced numerous characteristic dark brown, nodular outgrowths. It was identical with S. scabies. Sweet Potato Pox or Soft Rot An actinomycete, designated by Tauben- haus as A. poolensis, was found to be a con- tributing factor to the causation of soft rot of sweet potatoes. It was considered as a superficial wound parasite, usually follow- ing the pox spots produced by a fungus. Other cultures of actinomycetes were la- ter isolated from sweet potatoes. One of them, designated as A. pox, was believed to be the cause of the pox disease (Adams). Still another organism causing sweet po- tato rot was described as S. zpomoea. It pro- duced aerial mycelium and was thus a mem- ber of the genus Streptomyces. Sweet potato rot does not develop in soils of pH below 5.2; above that reaction, the disease develops readily. According to Person and Martin, sweet potato rot is more serious in dry soils and in wet seasons than under normal mois- ture conditions. The disease has been pro- duced in the greenhouse and in field inocula- tion experiments with pure cultures of S. ztpomoea. The optimum reaction for growth is pH 5.6 or above, and the optimum tem- perature 32°C. Other Plant Diseases Caused by Actin- omycetes A number of other plant diseases have been reported to be caused by actinomy- cetes. Banga described a strawberry disease. Godfrey listed a form causing citrus gum- mosis. Hooker reported that seedling plants rep- resenting eight families developed root ne- crosis when grown in soil-extract agar arti- ficially infested with pure cultures of S. 275 scabies. Ten cultures of actinomycetes were tested on seedlings of wheat, garden pea, soybean, corn, radish, and cucumber, and on potato sprouts. Six of these cultures caused neither appreciable necrosis of potato stems nor injury to seedling roots. Four cultures caused severe necrosis of roots as well as a reduction in root weight of wheat, pea, soy- bean, and radish; necrosis was most. pro- nounced on root tips, and the development of secondary roots was almost inhibited. Corn roots were only slightly necrotic, but their weight was markedly reduced. Three of the four cultures also caused scab of po- tato tubers and necrosis of potato stems. Palm described the occurrence in South Sweden of actinomycotie infections of Beta vulgaris, Brassica sp., Raphanus sativus, and Daucus carota. He concluded that the infec- tion of these plants may be caused by the same pathogen. The disease appears as sunken spots, delimited by the more promi- nent veins of the bulb seales, and are of a greenish mother-of-pearl-like color. The pathogen lives intracellularly, filling the cells completely with its very thin (1-1.2 yw in diameter) mycelium. The mycelium is non- septate, of a strong yellow color, and spore formation in or on the host is not seen. The organism undoubtedly belongs to the chro- mogenic actinomycetes, of the genus Strep- tomyces. Mycorrhiza Formations The associations of certain actinomycetes with the root systems of certain plants are of particular interest. These associations are believed to be comparable to mycorrhiza for- mations by true fungi. Peklo (1910) made : detailed study of the endophytes of the alder bush, Alnus glutinosa, and of sweet gale, Myrica gale. Two species of actinomycetes, A.alni and A. myricae, were isolated. These organisms produced, in culture, swellings comparable to those formed by animal path- ogens. The significance of these associations 276 for plant growth has not been fully eluci- dated. Formation of small tubers on the roots of the oleander has been demonstrated by Ro- berg (1934). The organism is similar to A. alni and was described as A. elaeagni. All three of these actinomycetes definitely belong to the genus Nocardia. Only one of them is included in the classification, namely, N. alni. A further study of these associations has been made by von Plotho (1941). It was sug- gested (Lieske) that the role of actinomy- cetes consists in enabling the plant to fix atmospheric nitrogen. Actinomycetes and Plant Development Lutman suggested that the occurrence of actinomycetes in the outer layers of roots and tubers of the potato plant corroborates his theory that these organisms play an im- portant role in plant growth. The stems above ground are also infected; but the tips of young roots and stems contain only a few strands between the cells. These facts sug- gested that the infection is systemic and he- reditary. Young potato plants grown from disinfected seed and in disinfected soil are found to contain numerous actinomycete fil- aments. According to Lutman, potato scab lesions are associated with strands of actinomycetes extending from the abnormal cells of the cork cambium to the interior of the tuber. Similar strands have been found in clean tubers grown on land never known to pro- duce scabby tubers. The strands found under the scabs seem to be unusually large and numerous, especially those about five to ten cells below the pathological tissue. The cell walls of Jerusalem artichoke tu- bers and the enlarged roots of beets, carrots, parsnips, and turnips contain gram-positive filaments which seem to be of the same sort as those occurring in the potato plant. The suggestion was made that since actinomy- THE ACTINOMYCETES, Vol. I cetes are abundant in the roots of plants, they may take part in the synthesis of alka- loids and proteins. Since large numbers of soil actinomycetes are pectin-dissolving, the different varieties found in the various host plants may be only modifications of one large species. The walls of higher plants were be- lieved to be living, through the presence and action of strands of actinomycetes. The ef- fects of actinomycete filaments surrounding every cell suggested the theory that the ma- terials they withdraw from the cells and the products which they excrete and which must be absorbed by the cells change the charac- teristics of the cells. Richards outlined in detail the method of staining the petato scab organism. The or- ganism can be selectively impregnated with carbol-auramin and when exposed to ultra- violet radiation, it fluoresces bright yellow. The hyphae are stained bright yellow. This permits ready localization and study of the micropathology of the tissue with a simple fluorescence microscope. The staining tech- nic is done at room temperature. No counter- stain is used. The results obtained tended to confirm Lutman’s conclusion that the fila- ments are intercellular and grow within the middle lamellae. After complete removal of the paraffin, the sections are strained 4 min- utes in carbon-auramin (distilled water 97 ml, liquefied phenol 3 ml, certified auramin 0.1 gm), washed, destained in a 0.5 per cent solution of NaCl in 70 per cent aleohol with 0.5 ml of HCl (cone.) per 100 ml, washed, and mounted in glycerol. Vords et al. reported that streptomycin exerts a protective effect upon the potato plant, rendering it resistant to Phythophtora, via the polyphenol-polyphenolase system of the host plant. The fact that polyphenolases are copper enzymes, their activity depend- ing upon the copper supply of the plant, and the fact that copper and streptomycin were found to exert a synergistic effect may help to explain the above effect of streptomycin. EPILOGUE TO VOL. I These then are the actinomycetes, dismissed only about two decades ago as a “little-known group of microorganisms,’’ and considered by some as fungi and by others as bacteria. I have presented in these pages my personal experiences with the actinomycetes, especially their occurrence in nature, their structure and functions, and their role in natural processes. I have attempted to summarize some of the reactions whereby they carry out their characteristic biochemical activities, their varied biochemical potentiali- ties, which are at present being taken advantage of for the benefit of the human race. The subsequent volumes will deal with the problems of how to recognize them and how to utilize them for the production of valuable drugs that are saving millions of human lives. 277 Reterences Acker, R. F. ann LecHeva.iger, H. Some nutri- tional requirements of Streptomyces griseus 3570 for growth and candicidin production. Appl. Microbiol. 2: 152-157, 1954. AcuaNpb, T. Actinomycosis hominis. Trans. Pathol. Soc. London 37: 546, 1886. Acosta, E. Nueva propriedad del Cladothrix in- vulnerabilis. Crénica Méd. quirurg. Habana No. 18, 1894 (Centr. Bakteriol. 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Calif. 5: 239-292, 1944. 18123, INDEX TO SPECIES OF ACTINOMYCETES Actinobacillus (Bacillus) oligocarbophilus, 130 Actinomyces albido-flavus, 5 albus, 11, 42, 48, 49, 50, 62, 70, 156 alni, 275, 276 arborescens, 5 asteroides, 5, 52, 70 bovis, 5, 7, 8, 13, 29, 44, 47, 51, 52, 57, 61, 62, 70, 80, 87, 88, 102, 132, 166, 252, 254, 255, 257-261 bovis albus, 5 bovis farcinicus, 5 bovis luteo-roseus, 5 bovis sulphureus, 5 brasiliensis, 250 canis, 5 caprae, 52 carneus, 5 catt, 5 chromogenes, 42, 70 chromogenus, 5, 49, 210, 265 citreus, 5 cloacae, 41 cuniculi, 5 cyaneus, 199 discofoliatus, 263 elaeagni, 276 elastica, 155 ferrugineus, 5 flavus, 50 Foerstert, 5 fuscus, 155 graminis, 4 hanseni, 40 Hoffmanni, 5 invulnerabilis, 136 israeli, 7, 44, 52, 70, 87, 129, 166, 264 keratolytica, 256 madurae, 52 monosporus, 135 mutabilis, 104 myricae, 275 naeslundii, 129 nocardi, 52 319 odorifer, 42, 49 oligocarbophilus, 42, 130, 132 pelogenes, 38 pleuriticus canis familiaris, 5 pluricolor, 5 poolensis, 275 pox, 275 roseolus, 156 scabies, 212, 265 spinae, 157 sulphureus, 4 thermophilus, 135 tumuli, 274 verrucosus, 156 vesicae, 52 violaceus, 5, 50 Bacillus farcinicus, 5 oligocarbophilus, 114, 130 Chainia antibiotica, 66 Cladothrix asteroides, 5, 49, 261 Leptothrix buccalis, 3 Microbispora rosea, 64 Micrococcus cytophagus, 188 Micromonospora caballi, 256 chalcea, 93, 188 fusca, 93 proptonici, 155 vulgaris, 938, 135 Micromyces Hoffmanni, 5 Mycobacterium butyricum, 68 chalcea, 188 eos, 217 fortuitum, 191 leprae, 68 phler, 68, 191, 229 rhodochrous, 191, 192 smegmatis, 68, 191, 229 stercoris, 68 tuberculosis, 209, 214, 215, 219, 22 Mycococcus cytophagus, 188 , 230, 234, 235 320 Nocardia actinomyces, 62 alba, 184 alni, 276 aquosa, 152 astleroides, 67-69, 81, 84, 90, 120, 124, 127, 159-162, 170, 178, 181, 184, 191, 192, 254, 255, 258, 261, 262, 264 atlantica, 125 blackwellii, 67-69 brasiliensis, 67, 68, 124, 127, 181, 191, 262, 264 butyricum, 67 calcarea, 157 caprae, 68, 69, 262 caviae, 68 cellulans, 33, 157 citrea, 169, 188 coeliaca, 152 convoluta, 68, 69 convolutus, 160 corallina, 67-69, 91, 102, 120, 156, 158, 160, 189, 192, 200 cuniculi, 38, 67-69 eppingert, 263 erythropolis, 67-69, 152 farcinica, 55, 61, 62, 160, 175, 182, 261, 262 flava, 125, 184 foersterz, 62 gardneri, 68 globerula, 67-69, 152 intracellularis, 67, 68 leishmanii, 67, 68 leprae, 67 madurae, 67, 68, 124, 127, 181, 191, 261 minima, 67-69 opaca, 67-69, 152, 153 paraffinae, 67-69, 160 paraguayensis, 180, 181 pelletiert, 67, 68, 124, 127, 192 petroleophila, 130 polychromogenes, 67-69, 90, 101, 154, 160-162, 170, 172, 199 pretoriana, 258 rangoonensis, 67, 68 restricta, 152 rhodnii, 45, 195 ruber, 184 rubra, 68, 69, 160-162, 170, 172 rugosa, 192, 194, 195 sebivorans, 135 somaliensis, 124, 127 sylvodorifera, 68, 69 viridis, 184 Oospora cretacea, 274 INDEX TO SPECIES OF ACTINOMYCETES Guignardi, 5 intermedia, 274 Metschnikowi, 5 nigrificans, 274 rosella, 274 scabies, 7, 8, 48, 265 tenax, 274 violacea, 274 Proactinomyces cytophagus, 189 Streptomyces albido-flava, 42 albidoflavus, 146 alboflavus, 155, 160 albogriseolus, 236 albus, 38, 42, 44, 112, 121, 125, 135, 152, 154, 155, 160, 167, 173, 174, 188, 193, 195, 222 albus var. ochraceus, 209 antibioticus, 20, 125, 160, 189, 222, 226, 228, 231 aurantiaca, 42 aurantiacus, 156 aureofaciens, 105, 115, 116, 123-125, 145, 152, 160, 164, 177, 189, 193, 194, 223, 224, 237 aureus, 68, 154, 160, 185, 186 autotrophicus, 130 bikiniensis, 141, 178 bobiliae, 38, 121, 160-162, 170, 172, 185 cacaoi, 43, 179 californicus, 120, 122, 160, 236 candidus, 179 carnea, 42 cellulosae, 185 chromogena, 42 chrysomallus, 116, 160, 231 citreus, 154 clavifer, 266 coelicolor, 51, 100, 101, 104, 105, 110-112, 115, 121, 122, 131, 145, 148, 156, 158, 160, 170, 188-190, 197, 199, 200, 220, 222, 256 craterifer, 160 cyaneus, 181 diastaticus, 125, 179, 187 diastatochromogenus, 185 erythreus, 127, 160, 165, 190, 238 erythrochromogenus, 121, 185 exfoliatus, 186 flaveolus, 105, 108, 121 flavovirens, 20 flavus, 160, 220, 266 fragilis, 123 floridae, 120, 122, 236 fradiae, 54, 110-112, 116, 126-129, 141-145, 150, 152, 154, 158-162, 170, 171, 185, 186, 190, 193, 194, 223, 224, 226, 230, 236, 237 fulvissimus, 160, 185 gardnert, 20 INDEX TO SPECIES gelaticus, 121, 152 globisporus, 108, 157, 192, 197, 222 globisporus vulgaris, 157 108, 157, 160 griseoflavus, 185 121, 186 griseoviridis, 123 20, 66, 68, globosus, griseolus, 74, 77, 78, griseus, 87, 96, 98, 104-107, 110-112, 115-117, 119-123, 125, 127, 129, 131, 138-143, 145, 147-155, 157-162, 164, 167, 170- 172, 175, 176-179, 181, 185-187, 189, 190, 193, 194, 196, 197, 202, 203, 222-226, 230, 233, 235, 240-242 griseus var. purpureus, 236 halstedii, 185 humidus, 235 intermedius, 273 tpomoea, 160, 275 kanamyceticus, 236 lavendulae, 20, 50, 105, 106, 1238, 125, 128, 129, 145, 150, 152, 153, 157, 158, 160-162, 167, 178, 185, 222, 225, 232 lipmanii, 160 longisporus, 222 longisporus ruber, nigrificans, 209 madurae, 261, 262, 264 marinus, 125 nigra, 42 niveus, 238 oligocarbophilus, 146 olivaceus, 121, 148, 160, 179, 181, 185, 193, 194, 230 olivochromogenus, 121 omiyaensis, 236 parvullus, 231 parvus, 160 pelletiert, 262, 264 phaeochromogenes, 127 pheochromogenus, 121, 185 poolensis, 121, 230 praecox, 160, 210, 211, 273 puniceus, 122, 236 purpurescens, 160 reticuli, 83, 99, 160 reticuloruber, 83 rimosus, 59, 111, 145, 154, 160, 165, 206, 223, 237 roseochromogenes, 127, 154, 160-162 roseochromogenus, 121 roseodiastaticus, 202 roseus, 222 ruber, 68, 121, 202, 222 rubescens, 38, 152 156 226, OF ACTINOMYCETES rubrireticuli, 58, 155 scabies, 104, 106, 144, 149, 155, IL, 197, 210) 211, 218, 265, O7K “aio 160, ee 267, 268, 27( 175, somaliensis, 262, 264 spectabilis, 238 spheroides, 238 105 199, 209 (ate, UN allo sterilis, tricolor, venezuelae, 181, 236 verne, 200 verlictllatus, 21 vinaceus, 122, 226 violacea, 42 130, 131, 145, violaceoruber, 199, 200 violaceus, violaceus niger, 201 violaceus-ruber, 185 viridis, 200, 273 viridochromogenes, 160, 189, 19 Streptothrix actinomyces, 7, 51 alba, 30, 42, 62, 70 albido-flava, 5 Albus, 5 aurantiaca, carnea, 5 chromogena, 9 chromogenus, 5 cuniculr, 5 eppingert, dl Eppingerii, 5 erystpeloides, 51 erythrea, 41 flava, 70 Foersteri, 2, 5, 47, 62 israeli, 7, 254 leucea, 41 2, 193, 200 51 leucea saprophytica, 41 madurae, 51 mihi, 51 niger, 5 odorifera, 118 rubra, 51 violacea, 5, 70 Thermoactinomyces monosporus, 63 thalpophilus, 63 vulgaris, 40, 63 Thermopolyspora bispora, 64 64 Waksmania rosea, 152, 321 ser PAG 160, 105, 136, 155, 160, 173, 185, 200, 222 GENERAL INDEX Acetate, utilization by cells griseus, 150 of Streplomyces Acid production, 157, 158 reaction of Streptomyces griseus, as affected by metals, 141 utilization, 158 Actinomyces, historical significance of name, 52 Actinomycetales, classification and description, 56, 65 Actinomycetin, action on bacteria, 173, 174 Actinomycins, production and properties, 203, 231 Actinomycosis, 1, 7, 47, 251, 257 Actinophage effect on Antinomycetales, 181 plaque formation, 176 streptomycin production, 176, 177 isolation from soils, 179, 180 mechanisms, 175-181 production, 175 Actinorhodin, 205 Actinorubin, 142 Adaptation to antimicrobial agents, 109 Aerobie actinomycetes, metabolism, 129, 130 Aldolase, 189 Alginase, 188 Allergies caused by acinomycetes, 263 Amino acid content of cell walls, 162, 163 synthesis, 163 Amylase hydrolysis of mannosidostreptomycin, 187 production, 187 Anaerobic actinomycetes, metabolism, 131, 149 Antagonisms in mixed populations foreed, 209 types, 207 Antagonistic organisms isolation from natural substrates, 211, 213, 215 soil as source, 209, 211-215 Antagonistic properties antiphage, 219 bactericidal, 208, 218 distribution among actinomycetes, 210, 213 effects on pathogenic organisms, 209, 211, 216- 218, 220 of fungi and bacteria, effects on actinomycetes, 221 fungicidal, 208, 209, 215-218, 220 relation to pigmentation, 217 screening methods to determine, 212 Anthocyanins, 200, 201 Antibacterial substances as chemotherapeutic agents, 208 production by organisms, 207, 208 Antibiotic-producing organisms activity of soil actinomycetes, 211-215 isolation and testing 226, 227 mutations caused by irradiation, 107 variants, 105 Antibioties activity against pathogens, 241 antiphage activity, 24, 219 antiprotozoan, 240 antitumor, 240 antiviral, 219, 240 autoantagonisms, 222, 223 bactericidal, 216 biosynthesis, 163, 241, 242 chemical structure, relation to activity, 242 classification, 204, 22¢ concentration, effect on resistance, 238, 239 effect of metals on production, 142, 145 of phages on production, 177, 178, 181 historical studies on production, 14 identification by bacterial stains, 240 isolation and testing methods, 211 media for production, 229 principles of production, 225 production in natural substrates, 45, 221 properties, 207 screening programs, 211, 217, 241 spectra, 228-230, 241 test organisms, 218, 219 Antifungal action, 197, 232 Antigenic properties, 166-167 Antimicrobial activity of streptomycetes, as af- fected by metals, 142 Antimicropia! spectra of Streptomyces fradiae strains, 237 Antiphage factors, 24, 219 GENERAL INDEX Antitumor activity of actinomycetes, 219, 240 Aryl sulfatase production, 191, 192 Aureothricin, 205 Autoinhibition, 222, 224, 227 Autolysis mechanisms, 168 relation to antibiotic production, 171 Autotrophy, 130 By production, see Vitamin By production Bacteria, relation to actinomycetes, 1, 18, 53, 71 Bacteriolysis, mechanisms, 171 Biochemistry, historical studies, 14 Biology, historical studies, 11 Biotin production by Streptomyces olivaceus, 194 Bromtetracycline, 145 Cancer, actinomycins in treatment, 203 Candicin, production as affected by metals, 142 Carbohydrate composition of cell walls for taxo- nomic use, 160 Carbomycin, 238 Carbon-dioxide fixation by anaerobic actinomy- cetes, 149 Carbon sources, utilization agar, 125 by antibiotic producers, 122, 123 as basis of classification for antibiotic-producing streptomyces, 121 cellulose, 124 chitin, 125 efficiency and metabolic changes, 125 glucose, 1382, 133 of proteins, 124 relation to nitrogen utilization, 128 to streptomycin production, 119, 133 in species characterization, 119, 120 unusual compounds, 125 by various actinomycetes, 118, 120, 122, 123 Carotinoids, 193, 202 Catalase activity, relation to streptomycin pro- duction, 190 Cell walls, chemical composition, 158-163 Cellulase production, 188 Cellulose decomposition, 155, 244 Chitin decomposition, 155 Chitinase production, 186 Chloramphenicol, production and properties, 145, 236 Chlortetracycline, production and properties, 145, 164, 237 Cholie acid utilization, 152 Chromopars, 199 Chromophores, 199 323 Classification systems carbon utilization by streptomyces, 20, 201 cell wall composition, 69 Buchanan’s, 56 generic, 47 group characteristics, 67-70 group identification by sporulation, 67 historical background, 55 pigmented antibioties, 204 specific, 49 Club formation, 7, 81 Coelicolorin, 205 Coenzyme A production, 192, 195 Colony formation, 8, 76 variants of Streptomyces griseus, 96 Constancy of characters, 95 Coremia formation, 80, 84 Cortexone conversion, 152 Crop yields in relation to actinomycete population of soil, 33 Cross-resistance, 222, 223 Cultivation of actinomycetes, techniques, 17 Cultural variations, 103 Decomposition of plant materials cellulose, 155, 244 complex, 247 hemicelluloses, 155, humus, 247 lignin, 244, 247 polysaccharides, 244-246 proteins, 245 thermophilic composts, 248 Deguanidases, 186 Diastase production, 185 Diketopiperazine, effect on rice germination, 196 Diseases of animals actinomycetes isolated from, 252-257, 262 actinomycosis, 7, 251-253, 257 allergic reactions, 263 mycetoma, 261, 262 nocardiosis, 261, 263, 264 therapy, 263 causation, 6, 13 of plants -aused by chromogenic organisms, 275 citrus gummosis, 275 mangel scab, 274 necrosis of seedlings, 275 potato scab, 7, 265-274 strawberries, 275 sugar beet scab, 274 sweet potato pox, 275 244, 246 324 GENERAL INDEX Distribution of actinomycetes in air, 41 animal tissues, 6, 44 foodstuffs, 42 fresh waters, 38, 40 geologic formations, 45 manures and composts, 10, 40 plants, 43 sea water and marine sediments, 37 soil, 9, 12, 830-32, 34-36 Drying, influence on actinomycetes, 135 Enolase production by Streptomyces coelicolor, 189 Enzyme systems of Nocardia corallina, 189, 192 Enzymes, extracellular and endocellular, 183 Erythromycin, 2388 Esterase, 189 Ethanol dehydrogenase production by Strepto- myces coelicolor, 189 Fermentation of glucose, 149, 150, 157 by Streptomyces griseus sarbon balance, 151 nitrogen balance, 154 F-forms, 101 Flavoprotein production, 193 Foodstuffs, actinomycetes in, 42 Fungi, relation to actinomycetes, 1, 13, 53, 71 Generic interaction in Streptomyces, 110 Grisein production, 142, 145, 195 Growth as affected by salt concentrations, 146, 147 -promoting effects on animals, 195, 196 bacteria, 196 in soil, as affected by rye straw and dried blood, 246, 247 of various genera Actinoplanes, 62 Micromonospora, 61 Nocardia, 48 Streptomyces, 50, 51, 54, 55, 58, 59, 138-145 Streptosporangium, 63 Glucose utilization, effect on acidity, 132 streptomycin production, 133 Hemicellulose decomposition, 155, 244, 246 Hemolysin production, 181] Heterokaryosis in Streplomyces, 110-112 Humus, formation and decomposition, 247, 248 Hydroactinochromes, 201 Identification of species morphological properties in, 18 physiological and cultural properties in, 19 techniques, 17, 22 Invertase production, 185, 188 Irradiation, effect on mutations, 107 vitamin By production, 194 Isolation techniques, 17 Keratin decomposition, 153 Keratinase production, 185 Lab production, 185 Lacease, 189 Laminarinase, 188 Leucocyte-stimulating factor produced by Strepto- myces griseus, 193 Life cycle Actinomyces bovis, 87 Micromonospora, 94 Nocardia, 89, 90, 91 Streptomyces, 75 Light, influence, 136 Limestone precipitation, 156 Lipase, 189 Lipoactinochromes, 202 Luminescence, relation to antibiotic activity, 206 Lysis of Streptomyces fradiae, as affected by raleium, 145 Lysozyme action on cell walls, 159, 160, 170, 172, 173 enzymatic properties, 183 Macrolides, 238 Magnamyecin, 238 Mangel scab, 275 Mannosidostreptomyein, hydrolysis by amylase, 187 Mannosidostreptomycinase, 187 Media for growth of actinomycetes, 18 Melanin, 203 Mesophilic organisms, 134 Metabolic processes of Streptomyces, phases, 116 Metabolic reactions, role of metallic elements, 146, 147 Metabolism of aerobic actinomycetes, 129, 130 of anaerobic actinomycetes, 131, 149 carbohydrate, by Streptomyces, 148, 149 fatty acids, by Nocardia opaca, 152, 153 historical studies, 114 role of antibiotie biosynthesis in, 241, 242 Methymycin, 288 Mineral requirements of Streptomyces griseus, 138 GENERAL INDEX 320 Morphological characteristics Actinomyces, 86 constancy in species and genera, 86 in identification, 18 Micromonospora, 79, 93 Nocardia, 88 relation to those of bacteria and fungi, 13 Streptomyces, 92 variations in, 101 Motility, 81 Mutations as affected by irradiation, 107 as affected by physical and chemical agents, 109 development of phage-resistance, 110 genetic studies on Streptomyces, 110 saltations, 106 Mycelium, aerial coremia, S4 formation, as affected by medium, 67 pigmentation, 83 secondary, S4 structure and growth, 75, 82 verticil formation, 21, 83 zonation, in Streptomyces colony, 25 substrate structure and growth, 73, 74, 75, 80 suspensions, as affected by shaking, 25 Mycetoma, 261, 262, 264 Mycolysate, clinical use, 209 Mycorrhizal associations, 275 Neomyecin production and properties, 230, 236, 237 relation to autolysis, 171 by Streptomyces fradiae, as affected by metals, 142 Neomycin B, effect on growth of broad beans, 196 Niacin production by Streptomyces olivaceus, 194 Nitrate reduction, 156, 157 Nitrification, 156 Nitrogen fixation, 157 sources, utilization amino acids, 126 plant proteins, 127 relation to antibiotic production, 124, 127 relation to carbon utilization, 128 by various actinomycetes, 122, 126 Nocardiosis, 261, 263, 264 Nomenclature Actinomyces, historical significance, 52 generic, systems, 47 specific, 49 Streptothriz, historical significance, 51 Odor production, 43, 156, 250 O-forms, 96 Oleandomycin, 238 Oxidation of steroids and lipids, 152 Oxygen consumption, 129 Oxytetracycline, production and properties, 165, 237 Pantothenic acid, olivaceus, 194 Paraffin decomposition, 156, 247 Parasitic actinomycetes, 251 Pathogenicity, variability of Streptomyces scabies, 104; see also Diseases of animals, Diseases of plants Penicillinase production, 191 Phage, effect on lytic properties, 110 Phenol oxidases, 189 production by Streptomyces Phosphatase production by anaerobie actino- mycetes, 192 Phosphofructokinase production by Streptomyces coelicolor, 189 Phosphoglyceryl kinase production by Strepto- myces coelicolor, 189 Physiological variations, 103 Picromycin, 238 Pigment production as affected by environment, 203 relation to antibiotic production, 198, 202-204 relation to antimicrobial activity, 217 by Streptomyces, as affected by metals, 139, 144 by tetracyclines, 206 use in classifying actinomycetes, 198 Pigments anthocyanins, 200 brown-black, 202 carotinoids, 202 chemical and physical properties, 198, 200 chromopars, 199 chromophores, 199 green, nature of, 200 hydroactinochromes, 201 hpoactinochromes, 202 prodigiosin, 202 rhodomyein, 202 solubility, 199 use in classifying antibiotics, 204 variations, 103, 105 Plant development, as affected by actinomycetes, 276 residues, decomposition, 243 Poisons, antimicrobial effects, 137 Polyene antibiotics, 238-240 Polysaccharidase production, 188 326 Population studies, methods microscopic, 20 plate, 24 selective culture, 25 Porphyrin biosynthesis, 194, 195 Potato scab biological control, 272 causative organisms, 265 control by Streptomyces praecox, 210, 211 environmental effect, 267-270, 272 relation of host to parasite, 270 treatment, effect of, 271 Preservation of cultures, methods, 26, 128 Prodigiosin pigments, 202 Progesterone metabolism, 152 Protease production, 140, 183, 185 Proteolytic activities, 153 Psychrophilic organisms, 154 Pyocyanase, 207 Pyridoxine, production by Streptomyces olivaceus, 194 Relation of actinomycetes to bacteria and fungi, iL, WS}, Gah, 7 Rennet production, 185 Reproduction, methods, 75, 78, 84, 86 Resistance to metabolic products and antimi- crobial substances, 109 Respiration of Nocardias, carbohydrates in, 151 R-forms, 96 Rhodomyein, 202 Riboflavin production, 193, 194 Rubber decomposition, 155, 247 Saprophytic actinomycetes, 251, 255 Sarkomycin, effect on growth of broad beans, 196 Scab, see Potato scab, Sugar beet scab, Mangel scab Sclerotia formation in a streptomyces, 81 Screening methods antifungal surveys, 215 relation between antimicrobial pigmentation, 217 for specific antibiotic producers, 217 in study of antagonistic properties, 211 test organisms, 216, 219 Sectoring, 98, 107 Serological relations among actinomycetes, 166, 167 Sexuality, 74 S-forms, 95 Soil actinomycetes antagonistic, 209, 211-215, 222 activity and GENERAL INDEX factors controlling, 37 seasonal variation, 30, 34 variation with depth, 31 enrichment with pathogens, 222, 223 processes, role of actinomycetes in, 9 Spiromycin, 238 Spoilage, agents of, 250 Spores chains, 85 distribution of types among groups of Strepto myces, 86 formation of different types, 71, 77, 79, 82, 84 germination, 19, 73 role in characterization of species, 85 Sporophores of Streptomyces, 54, 55, 58-60, 82, 83 Sporulation, as affected by cobalt, 143 Staining, 77 Starch decomposition, 155 Steroid oxidation, 191 Stimulants, microbial, 137 Streptomycin antibacterial activity, 2380 biosynthesis, 163-166 destruction by deguanidases, 186 effect on Mycobacterium tuberculosis, 234 production as affected by actinophage, 176, 177 as affected by catalase activity, 190 by Streptomyces bikiniensis, 141 by Streptomyces griseus, 115, 119, 133, 138-141, 145, 151 relation to autolysis, 171 properties, 230, 233 protective effect on potato plant, 276 Streptothricin, production and properties, 222 Streptothrix, historical significance of name, 51 Streptovaricin, production and properties, 238 Sugar beet scab, 274 Sweet potato pox, 275 Symbiosis with insects, 195 Temperature, influence, 133 Tetracyclines pigment production in presence of metals, 206 production and properties, 237 Thermophilic organisms, 134 Thiamin production, 193, 194 Thioaurin, 205 Thiolutin, 205 Triose phosphate dehydrogenase, production by Streptomyces coelicolor, 189 Triose phosphate isomerase, production by Strep- tomyces coelicolor, 189 Tyrosinase, 191 GENERAL INDEX 327 Ultraviolet radiations factors affecting, 142, 143, 145, 193, 194 antibacterial action, 136 relation to antibiotie production, 193 mutagenic and lethal effeets, 102, 103, 105, 107 Vitamin synthesis, relation to growth-stimulating Urea production from guanidine, 186 effect, 193, 195 Urease production, 186 Wool digestion, as affected by magnesium, 186 Variations, 97-99, 101, 103, 110 Viomycin, production and properties, 122, 236 Xanthomycins A and B, 205 Vitamin By production Xylanase, 188 wrey veverlers Lee Pelee he Sitteele bole deperereiageveqoneions . wees Pieleiese shee eeetbeye etna ie lei wrereceue Srepeiel wae . ‘ ine fae fate Pirveyeretes peeveieyeuee eircebey syeregel eee Ph ererey Pee rereeee sepa eal leleteie eheceleiee ee eyelelaye aeyeee ~eree srepeieist Veheleieolenyel eye rerereeeerte,y ae Vetelepepaley Seem apm alee lelecalelerece APIS Ble arash erate jeieieres wlelere aleveyelele pert ebeieiereraue peierneadeiere worielete Pareyetelereeeeier eins Vee eree cereale qiese ete ’ 4 . rT sales a . peer cwers eieiee yeheler sieeaepayeley Bie eyareteiene hevegete alee beveebelecetove ony tite be Bem eta iecaye eee recaet ‘ eee eee eyetey ee oe ecerssetase oie ere seegecabeyapetal Pee et ee de Steet SE et 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