S^^E S^QS^^^S Marine Biological Laboratory Library Woods Hole, Mass. Presented by John Wiley and Sons,Inc, J1LL7 22, 1961 ES^^^E l^^^SE MICROBIAL CELL WALLS C I B A LECTURES IN MICROBIAL BIOCHEMISTRY 1956 H. A. Barker, Bacterial Fermentations 1957 E. P. Abraham, Biochemistry of Some Peptide and Steroid Antibiotics 1959 E. F. Gale, Synthesis and Organisation in the Bacterial Cell 1960 M. R. J. Salton, Microbial Cell Walls C I B A LECTURES IN MICROBIAL BIOCHEMISTRY LIBRARY MASS. r^li^V MICROBIAL CELL WALLS By M. R. J. SALTON 1960 JOHN WILEY & SONS, INC. New York • London The CIBA Lectures in Microbial Biochemistry were established In 1955 at the Institute of Microbiology, Rutgers, The State Univer- sity of New Jersey, through the support of CIBA Pharmaceutical Products inc.. Summit, N. J. The lectures are given in the spring of each year at the Institute of Microbiology, New Brunswick, N. J. Copyright © 1961 by John Wiley & Sons, Inc. All rights reserved This book or any port thereof must not be reproduced In any form without the written permission of the publisher. Library of Congress Catalog Card Number: 61-11497 Printed in the United States of America PREFACE Science, like art, music, and literature, is susceptible to fashions, and it has been my good fortune to be actively engaged in a field of research that has attracted many in- vestigators in the last ten years. This has added much personal stimulus to the fascination of scientific research, and it is always a great pleasure to talk "shop" with an ever-growing circle of microbiologists and biochemists. An invitation to present three lectures at the distinguished Institute of Microbiology at Rutgers was an extremely happy event for me, as it enabled me to meet and talk with both old and new friends and to lecture on a topic I espe- cially enjoy. For this two-fold pleasure I should like to express my warmest appreciation to CIBA Pharmaceutical Products Inc., whose generous support made these Lectures in Microbial Biochemistry possible. The structure of the microbial cell has intrigued most microbiologists, and what has been particularly fascinating has been the discovery that their biochemical apparatus and vi PREFACE Structural and functional elements are so neatly packaged into cells of such small dimensions. Because many of the anatomical parts of microbial cells were beyond the limits of resolution of the light microscope, little detailed knowl- edge of microbial structure could emerge until the introduc- tion of electron microscopy. It was this coincidence of the development of electron microscopy with the accumulated wealth of biochemical information that paved the way for the investigators of the major structural components of microbial cells. This book, based on the lectures delivered at Rutgers, illustrates the successful application of the tech- niques of biophysics, chemistry, and biochemistry to one facet of microbial anatomy. The development of the studies on microbial walls has been a rapid one and has occurred in a number of laboratories. Thus we have al- ready reached the stage where we can but survey the general field in three lectures. The material in this book, therefore, does not represent a complete record of investigations on microbial walls. It has been selected with the hope that it will give an orientation to the newcomer or interested reader and a more detailed record on several aspects of wall chemistry for the initiated investigator requiring a sum- mary. In a field advancing with some rapidity it is inevi- table that important papers will have appeared in the interim between the lectures and this published account, and it is the constant nightmare of all authors and reviewers that their works will be out of date by the time they are printed. This does not, I hope, negate the usefulness of a summary of events leading to the latest exciting addition to the study of microbial cell walls. For my own small part in the development of this field of endeavor I owe much to the broad introduction to micro- biology I received in Australia and the many years of in- terest, stimulation, and encouragement I enjoyed as a visitor PREFACE vii and later as a member of Professor E. F. Gale's unit in the Department of Biochemistry at Cambridge. For the prepa- ration of electron micrographs used in the lectures and this book I am most grateful to Dr. J. A. Chapman of the Rheu- matism Research Department, University of Manchester, Professor A. L. Houwink of the Technical Physics Depart- ment, Delft, Professor E. Kellenberger of the Laboratoire de Biophysique, Geneva, Dr. V. Mohr of the Department of Biochemistry, The Technical University of Norway, Profes- sor R. G. E. Murray, Department of Bacteriology, London, Ontario, Dr. D. H. Northcote, Department of Biochemistry, Cambridge, and Professor R. C. Williams, University of California, Berkeley. I should also like to thank Dr. M. Ikawa and Professor E. E. Snell for their kind permission to quote their results prior to publication. It is a great pleasure to thank the members of the Institute of Micro- biology at Rutgers for their hospitality during the presenta- tion of these lectures. M. R. J. Salton Department of Bacteriology, University of Manchester, England. March 1961 CONTENTS CHAPTER 1 Isolation and General Properties of Microbial Cell Walls 1 CHAPTER 2 Chemistry of Cell Walls 25 CHAPTER 3 Enzymic Degradation and Biosynthesis of Mi- crobial Walls 57 INDEX 89 78556 CHAPTER 1 ISOLATION AND GENERAL PROPERTIES OF MICROBIAL CELL WALLS Microbial anatomy, that specialized branch of the study of the structure of microorganisms, has emerged in the last ten to twenty years and has gieatly attracted the attention of the biochemist and the biophysicist. There are very good reasons for distinguishing between the cytologists of former periods and the modern microbial anatomists, for the latter now have to attempt to explain their observations in terms of the biochemical functions of the cell and the molecular structures of cellular subunits. Our interest in microbial cell structure has, of course, a long history and really stems from Antonie van Leeuwen- hoek's observations on the shapes and forms of various microorganisms. Just as Leeuwenhoek's microscope re- vealed a new and exciting world of small "animalcules," so in our day the electron microscope with all its associated techniques has taken us inside the cell itself and revealed many fascinating details of the macromolecular complexity of living organisms. Thus in the last two decades a great 1 MICROBIAL CELL WALLS deal has been learned about the structure, functions, and chemistry of the principal morphological entities (flagella, capsules, walls, membranes, and nuclei) and various sub- cellular particles and organelles of microorganisms.^- ^ That most bacteria, yeasts, fungi, and algae are sur- rounded by a rigid wall was apparent to the early cytologists. Indeed, it would seem that Leeuwenhoek was sufficiently perspicacious to realize that his "animalcules" were bounded by some sort of structure. From his letter to the Royal Society (Dobell ^) it is evident that he looked and expected to resolve what it was that "held them together." Little time was lost between the introduction of methods for grow- ing microorganisms in pure culture and the first attempt to discover the chemical composition of a microbial cell wall. Vincenzi,* as long ago as 1887, was the first to in- vestigate what he believed to be the wall of Bacillus sub- tilis. Most of the early studies of cell-wall composition were based on analysis of material that resisted various solvents and extraction procedures designed to remove cellular com- ponents. We now know, of course, that the carbohydrate chemist's addiction to extracting tissues with alkali to ob- tain wall polysaccharides removed other constituents and really left only part of the "native" cell wall. Methods used for the isolation of chitin from higher organisms have been applied to microorganisms, and X-ray data, together with chemical analysis, have substantiated the presence of a chitin-like polymer in the walls of some fungi.^-^ Thus Blank ^ found that the "chitin" fraction of a number of dermatophytes gave X-ray results and nitrogen values simi- lar, if not identical, to those expected for pure chitin. It is now generally conceded that the polymers isolated as "wall" or mycelial residues by extraction procedures used in the earlier studies do not represent the entire chemi- ISOLATION AND PROPERTIES OF WALLS cal Structure of the wall as it occurs in the intact cell. Con- sequently, more refined and less drastic methods for isolat- ing walls have been evolved, and mechanical disintegration of cells and tissues has become the universal starting point. Isolation of Cell Walls The isolation of microbial structures as homogeneous morphological entities has resulted from the application of the methods of biochemistry, biophysics, and electron microscopy. Weibull ^ was one of the first to use a combi- nation of such methods for the isolation of a bacterial struc- ture when he separated and characterized flagella from Proteus vulgaris. Although mechanical methods have been available for the disintegration of microorganisms for some time, they were not applied to the problem of isolating wall structures until Mudd, Polevitsky, Anderson, and Cham- bers ^ showed by electron microscopy that sonic disintegra- tion of bacteria left a resistant wall. Dawson ^ later demonstrated the complete separation of cytoplasm from the wall of Staphylococcus aureus by disintegrating the cells with glass beads. It thus became apparent to several of us (Mitchell and Moyle,io Salton and Horne,^^ Salton ^2) that such procedures could be used in conjunction with differential centrifugation to obtain homogeneous prepara- tions which could be submitted to the techniques of analyti- cal chemistry for the elucidation of their nature. The methods for isolating microbial cell walls follow well-known recipes, and as we are all familiar with what good and bad cooks can do with recipes we need not dis- cuss the isolation procedures in any detail. Cells may be disintegrated and deprived of their cytoplasm by one of the following three methods: 1. Mechanical disintegration (disruption by violent agita- tion with beads,^'^^'^^ sonic and ultrasonic disintegra- MICROBIAL CELL WALLS tion/3,14 decompression rupture/^ pressure cell disinte- grator 16) 2. Osmotic lysis 3. Autolysis ^^ Of the three methods, mechanical disintegration is prefer- able, and all of the major methods listed under (1) have been used successfully in wall isolation. The method of choice will depend on the particular organism, but it may be worth emphasizing that of the mechanical procedures tried disintegration by sound and supersound can lead to a greater breakdown of the wall structure than that en- countered with the other methods. ^^ Even the robust walls from Staphylococcus aureus can be rendered nonsediment- able by exposure in the 10-kc Raytheon for 30 to 60 min- utes.i^ Marr and Cota-Robles ^o have also pointed out that concomitant with the disruption and release of ribo- somes and intracellular particles from Azotobacter vine- landii there is a disintegration of the "envelope" structure. These effects of sonic disintegration of wall, or envelope, may account for the rather low yields of walls encountered by some investigators. Disintegiation is generally performed under conditions that minimize enzymic modification of the walls, and the methods devised by Shockman, Kolb, and Toennies ^i and Ribi, Perrine, List, Brown, and Goode ^^ have great ad- vantages in that the temperature can be controlled accu- rately during disruption. Many organisms contain en- zymes capable of completely digesting their own cell walls. Strange and Dark -^ had difficulty in obtaining wall prepara- tions of Bacillus spp. free of cell-wall degrading enzymes. Because of the risk of degrading the wall enzymically, lytic and autolytic methods of cell disintegration are not recom- mended. On the other hand, various enzymes have been ISOLATION AND PROPERTIES OF WALLS used with considerable advantage in the removal of cyto- plasmic materials from crude cell-wall fractions. Thus ribonuclease, trypsin, and lipase can be used without de- stroying the rigidity of or apparently degrading the wall structure. Owing to the small dimensions of microbial structures, the only satisfactory method of establishing their morpho- logical homogeneity has been by electron microscopic ex- amination. Cell walls can thus be differentiated from other structures such as flagella, fimbriae, ribosomes, and intra- cellular particles. Electron Microscopy of Isolated Cell Walls Microbial walls isolated by the foregoing procedures generally retain the shape and outline of the organism from which they had been derived. This fact, together with the morphological changes accompanying enzymic removal of walls with protoplast formation (Weibull ~^), makes it certain that it is the wall that confers the shape on a par- ticular organism. Walls of rod-shaped organisms are typi- cally cylindrical in shape on examination in the electron microscope and those of Streptococcus faecalis are ellip- soidal.^^ Some of the first microbial walls isolated by mechanical methods showed no evidence of fine structure. The wall of baker's yeast isolated by Northcote and Home ^^ appeared as a thick amorphous structure on examination in the electron microscope. However, by treatment with alkali and acid successively, Houwink and Kreger ^^ removed some of the matrix from the walls of Candida tropicalis and showed a microfibrillar structure in the walls of this yeast (Fig. 1). By using more selective methods of extracting wall compounds, Nickerson and his colleagues ^6. 27 were MICROBIAL CELL WALLS Fig. 1. Microfibrillar structure in the wall of Candida tropicalis (X 18,500). By courtesy of Drs. Houwink and Kreger (Ref. 25). able to show that the glucan component of baker's yeast wall possessed the fibrillar structure. The microfibrils in the yeast wall (Fig. 1) are arranged at roughly 90° to one another. However, around the bud scars the fibers are oriented differently, and Falcone and Nickerson ^s have proposed an explanation for the fiber orientation, based on a local explosion or "blow-out" of the wall during cellular division. Northcote, Goulding, and Home ^9 have also shown that by degradation of the isolated wall of Chlorella ISOLATION AND PROPERTIES OF WALLS pyrenoidosa with dilute solutions o£ sodium hydroxide a microfibrillar layer is revealed, and again the fibers lie at approximately 90° to one another (Fig. 2). The presence of microfibrils in fungal cell walls has been reported by several investigators (Frey-Wyssling and Miihle- thaler,^^ Roelofsen,^! Shatkin and Tatum ^^). Roelofsen ^^ found that the fibrils on the outer and inner layers of the li: («) (P) Fig. 2. Electron micrographs showing microfibrillar structure in the wall of Chlorella pyrenoidosa. (a) Walls treated with 0.5% NaOH for 30 minutes at room temperature (x 21,000). (fo) Walls treated with 3% NaOH for 30 minutes at room temperature (x 39,000). By courtesy of Drs. Northcote, Goulding, and Home (Ref. 29). MICROBIAL CELL WALLS developing wall of Phycomyces blakesleeanus sporangio- phores were 150 to 250 A thick. The average fibril direction was too uncertain to suggest a spiral structure, but the inner layer showed a roughly transverse orientation. Thin sections of Neurospora crassa prepared by Shatkin and Tatum ^~ showed a wall containing fine fibrils in a homogeneous matrix. The wall structure is much more readily shown in isolated mycelial fragments prepared by disintegration of Neurospora crassa by the methods used for bacterial cell- wall isolation. Figure 2>a illustrates the appearance of an isolated mycelial wall with a rough outer texture and the more detailed microfibrillar structure of the wall in Fig. 36 (Chapman and Salton ^^). No such fibrillar layer has been detected in the walls of bacteria, although the walls of Bacillus megaterium give a vague impression of being fibrous (Fig. 4). The walls of many Gram-positive bacteria, such as those of Staphylococ- cus aureus and Streptococcus faecalis, have a homogeneous appearance, and only thickened bands at what is presumed to be the site of new wall formation can be seen. A type of fine structure differing from that observed in yeast walls and various algae 2^- ^^ was first reported by Houwink ^^ on examination of the wall of a large Spirillum species. The cell wall of this organism was a multilayered structure, with one layer composed of spherical macromole- cules*^ packed hexagonally. Such a macromolecular layer was also observed in the wall of Spirillum serpens, and Salton and Williams ^^ found a similar type of fine structure in the wall of Rhodospirillum rubrum. This spherical macromolecular type of structure is apparently not uncom- mon, for Houwink ^^ detected it also in the wall of Halobac- terium halobium. Figure 5 illustrates the hexagonally packed macromolecular fine structure found in the wall of Halobacterium halobium. Fig. 3. Electron micrographs of (a) cell walls of Neurospora crassa (X 3,800); (b) microfibrillar structure in isolated cell wall of Neurospora crassa (X 9,500). By courtesy of Drs. Chapman and Salton (Ref. 33). Fig. 4. Isolated cell wall of Bacillus megaterium (x 21, 000). By cour- tesy of Drs. Salton and Williams (Ref. 36). Fig. 5. Electron micrograph of Halobacterium halobium showing hexagonally packed macromolecules in the cell wall (x 42,500). By courtesy of Drs. Houwink, Mohr, and Spit. 10 ISOLATION AND PROPERTIES OF WALLS A different kind of microstructure in a bacterial wall was observed by Labaw and Mosley.^^ A rectangular array of niacromolecules was found in the wall of an unidentified organism. More recently, yet another type of fine structure has been discovered in the wall of Lampropedia hyalina from observations made by Dr. J. A. Chapman (Rheumatism Research Department of the University of Manchester) and the author and independently by Dr. R. G. E. Murray. The outer layer of the wall of this organism possesses macro- molecular subunits arranged to give the appearance of either a honeycomb network or an array of "knobs" spaced on a basal sheet— rather like a rubber mat. This type of struc- ture gives rise to a "perforated edge" and lattice appearance as seen in isolated cell-wall fragments (Fig. 6). In general, the niacromolecules or their spacings in the fine-structured walls are of the order of 100 A. The diam- eters of the large spherical niacromolecules of the Spirillum sp. wall were 120 A.^^ Although bacteria such as Escherichia coli have shown no fine structure in the isolated walls when examined in the electron microscope by the usual methods, the thin sec- tions prepared by Kellenberger and Ryter ^^ have clearly established the multilayered nature of the wall. Thus, as shown in Fig. 7, it has been possible to differentiate a multi- layered wall from the underlying membrane (presumably the protoplast membrane). As prepared for electron mi- croscopy, the wall consisted of three layers, two of which were electron dense and one electron transparent, each of about 20 to 30 A in thickness. Thin sections of yeast ^^ and Chlorella pyrenoidosa walls 2^ have also confirmed the presence of several layers; they are probably double-layered structures. Thus, with microfibrillar layers in their walls, the yeasts, Chlorella, and some fungi closely resemble the wall structures found in Fig. 6. (a) Isolated wall fraction from disintegrated Lampropedia hyalina (x 37,000); (b) wall fragment showing typical lattice appearance (X 102,000). By courtesy of Drs. Chapman and Sal ton, to be published. 12 Is Fig. 7. Thin sections of Escherichia coli infected with bacteriophage. (a) Coccoid and lysed cells showing multilayered wall and underlying membrane (x 32,500). By courtesy of Drs. Kellenberger and Ryter (Ref. 39). {b) Cell showing complete differentiation of wall, qtoplas- mic membrane, and residual cytoplasm upon phage infection (X 34,000). By courtesy of Drs. Kellenberger and Boy de la Tour, unpublished electron micrograph. 13 14 MICROBIAL CELL WALLS higher red, brown, and green algae and plants. ^^ The types of structures detected in microbial walls are summarized in Table 1. Multilayered walls are encountered more fre- TABLE 1 Physical Properties of Microbial Cell Walls Revealed by Electron Microscopy Type of Fine Structure Algae Chlorella pyrenoidosa Fungi Phycomyces Neurospora crassa Yeast Saccharomyces cerevisiae Bacteria Escherichia coli Halohacterium Spirillum sp. Spirillum serpens Rhodospirillum rubrum Lampropedia hyalina Bacillus megaterium Staphylococcus aureus Streptococcus faecalis Double-layered, microfibrillar polysaccharide (fibers at 90° to one another) + amorphous matrix Microfibrillar components Multilayered wall, microfibrillar layer (fibers 90° to one another); fibers oriented around bud scars Multilayered (2 electron dense: 1 electron transparent layers); macromolecules not visible in intact wall Multilayered structures with spheri- cal macromolecules (80-120 A di- ameter) visible; hexagonal packing Structure giving crystalline lattice appearance Fibrous? Amorphous structure— thickened bands at zone of wall formation References 24 to 40. ISOLATION AND PROPERTIES OF WALLS 15 quently in the Gram-negative group of bacteria, a difference in the level of complexity that could have been predicted from the early studies of chemical constitution. General Physical Properties The majority of microbial cell walls are fairly robust structures, and in many instances they must obviously be strong enough to withstand high pressures exerted upon them by those organisms capable of achieving a high con- centration gradient across the wall-membrane (envelope). Mitchell and Moyle ^^ found that the solute concentration in Micrococcus lysodeikticus and Sarcina lutea corresponded to an osmotic pressure of 20 atmospheres. The wall must therefore possess sufficient tensile strength to protect the cell against osmotic explosion. However, the walls of certain halophilic organisms are apparently not strong enough to prevent osmotic lysis when these bacteria encounter envi- ronments of low solute concentrations. *- The thickness of microbial walls has been reported by a number of investigators, either from thin sectioning of the cells or isolated walls or from direct measurement of the height of the shadows cast in specimens examined by elec- tron microscopy. Some typical examples for various micro- bial walls are given in Table 2, together with data on the contribution of the wall to cell mass. There would seem to be some anomalies in the data for wall thickness, cell size, and weight contribution for the yeasts and Chlorella in particular, and the final assessment of the accuracy of these measurements will have to await further determina- tions. It is evident that the wall accounts for a considerable proportion of the cell weight, the actual contribution de- pending on the phase of growth in the case of a bacterium such as Streptococcus faecalis.^^ Toennies and Shockman ^^ 16 MICROBIAL CELL WALLS TABLE 2 Cell-Wall Thickness and Contribution to Cell Dry Weight Wall Cell Walls as % Dry Organisms Thickness A Weight Bacteria Escherichia coli 100 15 Staphylococcus aureus 150-200 20 Streptococcus jaecalis 200 27 (exponential phase) 38 (stationary phase) Myxococcus xanthus 250 7-8 Chlorella pyrenoidosa 210 13.6 Yeast 1600 15* * Based on 90% recovery of various fractions from disintegrated yeast. References 1, 24, 29, 39, 40, 43, 45. have clearly shown that the nutritional status of the organ- ism is of some importance in governing the amount of wall formed. When Streptococcus jaecalis was grown under con- ditions of threonine depletion, the wall accounted for as much as 44% of the weight of the cell. No detailed investi- gations comparable to those of Shockman and Toennies appear to be available for microorganisms other than bac- teria. General Chemistry of Microbial Cell Walls Before discussing in any detail the nature of the chemical constituents of microbial cell walls, let us first consider the major classes of substances encountered in these structures. It is generally agreed that nucleic acids are not major con- stituents of walls, although, as Barkulis and Jones *^ have ISOLATION AND PROPERTIES OF WALLS 17 pointed out, small amounts of nucleic acid can be extracted Irom streptococcal (group A) wall preparations. Whether the nucleic acid or nucleic acid derivatives extractable from the wall are associated with it for "biochemical purposes" is not known. Contrary to the earlier views of Stacey/^ it is now generally conceded that the nucleic acids are thus of minor importance in a consideration of the types of structural polymers in cell walls. In addition to the nucleic acids, the cellular pigments also appear to be of intracellular origin, and there is no evi- dence of their being associated covalently with structural compounds encountered in nature. Although many pig- mented organisms give wall fractions devoid of pigments, there is a number of instances in which these compounds persist in the wall fraction during isolation. Cell-wall frac- tions of several photosynthetic bacteria contain both carote- noids and photosynthetic pigments, although the latter are obviously much more abundant in the chromatophore frac- tions.^-^^ The presence of pigments in the wall fractions can, with some justification, be regarded with suspicion, and their presence may be an artifact of the isolation procedures. However, it may well be that in some organisms certain pigments are located in the wall of the intact cell. Isolated walls of the two blue-green algae, Anacystis nidulans and Microcoleus vaginatus, contained carotenoids, but the chlo- rophylls separated in a small particle fraction quite cleanly from the wall fractions (Salton, unpublished data). In selecting results to illustrate the general features of the chemistry of cell walls, I have confined my choice largely to studies in which the wall structures have been isolated by mechanical disintegration and differential centrifuga- tion. It became apparent during the search for this data that, apart from bacteria, little information is available for 18 MICROBIAL CELL WALLS Other groups of microorganisms. Although there have been many studies of what has been assumed to be wall material after extraction of whole microorganisms with alkali, these studies have not been included in the present survey. The only comparative study of the old methods of isolating walls by alkaline digestion and the new methods by mechanical disintegration is that of Aronson and Machlis ^^ for the walls of the fungus Allomyces macrogynus. Their results are presented in Table 3 and show a loss of wall constituents when isolation is performed by extraction with alkali. Several typical analyses of the isolated walls of a yeast, a green alga, and a Gram-positive and a Gram-negative bacterium are summarized in Table 4. One conspicuous feature illustrated in Table 4 is the high amino sugar con- tent of the wall of the Gram-positive organism in compari- son to the other microorganisms. The major classes of sub- TABLE 3 The Composition of Walls of Allomyces macrogynus isolated by Alkaline Digestion and by Sonic Oscillation %of Dry Walls Alkaline Sonic Component Digestion Oscillation Nitrogen Acetyl Moles N/moles acetyl 4.7 15.5 0.9 5.5 Protein — 10 Chitin 68 58 Glucan 8 16 Ash 10 8 Reference 49. ISOLATION AND PROPERTIES OF WALLS 19 TABLE 4 Comparative Cell-Wall Composition for Organisms from Several Microbial Groups % Dry Weight Cell Wall Yeast Alga Bacteria Chemical (Baker's {Chlorella [Escherichia (Streptococcus Constituent yeast) pyrenoidosa) coli) faecalis) Nitrogen 2.1 4.6 10.1 5.6 Phosphorus 0.31 0.67 1.52 1.88 Lipid 8.5 9.2 22.6 2.3* Protein 13.0 27.0 60.0 1 Glucan 28.8 Mannan 31.0 a-cellulose 15.4 Hemicellulose 31.0 Reducing value 16.0 61.0 Hexosamine 1-2 3.3 3.0 22.2 * Ether extractable material after HCl hydrolysis. f An approximate figure. References 12, 24, 29, 50. stances encountered in walls from the main microbial groups are listed in Tables 5, 6, and 7. Nature seems to have utilized the polysaccharides as the principle type of structural polymer. Some of these micro- bial wall polysaccharides have been identified as chitin [^(1 -> 4) N-acetylglucosaminide] and cellulose. That both cellulose and chitin can occur together has been clearly established by Fuller and Barshad.^ Both types of polysac- charides were found in the cell wall of the aquatic Phyco- mycete, Rhizidiomyces sp. It is now evident from studies of the chemistry of cell walls that although the walls of a 20 MICROBIAL CELL WALLS TABLE 5 Chemical Constituents of Microbial Cell Walls Green Algae Chlorella pyrenoidosa Platymonas subcordiformis Gonyaulax polyedra Dunalliella Diatoms e.g., Phaeodactylum Red and Brown Algae Polysaccharide, protein, and lipid Polysaccharide * (galactose, uronic acid) Polysaccharide (glucose) Lipoprotein (membrane ?) Silica, polysaccharide Polysaccharides (glucose, xylose, arabinose, uronic acids) * Traces of amino acids. References 29, 51-56. TABLE 6 Chemical Constituents of Microbial Walls Fungi Penicillium spp. Aspergilus spp. Rhizopus stolonifer Tricophyton mentagrophytes Neurospora crassa Yeasts Saccharomyces cerevisiae Candida albicans Candida pulcherrima Polysaccharide Polysaccharide Polysaccharide Polysaccharide Polysaccharide (glucosamine, glucose, galactose, mannose) * (glucosamine) (glucosamine) (glucose, glu- cosamine) * Polysaccharide, protein, lipid Polysaccharide, protein Polysaccharide, protein * Amino acids detectable. References 5, 24-28, 57, 58. ISOLATION AND PROPERTIES OF WALLS 21 TABLE 7 Chemical Constituents of Microbial Walls Bacteria Eubacteria Gram-positive Mucocomplex (mucopeptides, mucopoly- saccharides) and teichoic acids Gram-negative Protein, polysaccharide, lipid, mucocomplex constituents Myxobacteria Myxococcus xanthus Protein, lipid, polysaccharides, mucopeptides, carotenoids Blue-Green Algae Anacystis nidulans \ Microcoleusvaginatus\^^'''^''P^P''^^ constituents, carotenoids Nostoc sp. Protein References 45, 48, 50, 61-63 number of microorganisms are predominantly polysaccha- ride they contain in addition significant protein and lipid constituents. Furthermore, the investigations of Nickerson and his colleagues ^^-^^ have shown that in the yeast wall glucans and mannans occur as protein complexes and that they are not present as simple polysaccharides. Comparative studies of cell-wall chemistry have also estab- lished the presence of a new type of structural heteropoly- mer, the mucocomplexes,^^ in walls of all bacteria so far examined, and in many of the Gram-positive bacteria they constitute the entire wall. The essential similarity of this class of cell-wall substance to other mucopolysaccharides was first pointed out as a result of the investigations of the wall of Streptococcus faecalis (Salton '^^), and their distinc- tion from known mucoproteins was also emphasized. This 22 MICROBIAL CELL WALLS became even more evident when it was discovered that the -w^all of Micrococcus lysodeikticus was composed solely of hexosamine, glucose, and the four amino acids: alanine, glu- tamic acid, glycine, and lysine (Salton ^*'). The mucocom- plexes can be separated into further groups, depending on whether they are predominantly peptide, as in mucopep- tides, or predominantly polysaccharide, as in mucopolysac- charides. (See Table 7.) In addition to the mucopeptides and mucopolysaccharides, Baddiley, Buchanan, and Carss «- discovered that some bacterial cell walls also contain major components of ribitol- and glycerolphosphate polymers. These polymers have been called the "teichoic acids" (from Greek x^lyoz, = wall) by Armstrong, Baddiley, Buchanan, Carss, and Greenberg.^^ REFERENCES 1. Bacterial Anatomy, Symposium Soc. Gen. Microbiol., 6 (1956). 2. Gale, E. F., Synthesis and Organisation in the Bacterial Cell, Wiley, New York, 1959. 3. Dobell, C, Antony van Leeuwenhoek and His "Little Animals," Russell and Russell, New York, 1958, p. 118. 4. Vincenzi, L., Hoppe-Seyler's Z., 11, 181 (1887). 5. Blank, F., Biochim. et Biophys. Acta, 10, 110 (1953). 6. Fuller, M. S., and I. Barshad, Am. J. Botany, 47, 105 (1960). 7. Weibull, C, Biochim. et Biophys. Acta, 2, 351 (1948). 8. Mudd, S., K. Polevitsky, T. F. Anderson, and L. A. Chambers, /. BacterioL, 42, 251 (1941). 9. Dawson, I. M., Symposium Soc. Gen. Microbiol., 1, 119 (1949). 10. Mitchell, P., and J. Moyle, /. Gen. Microbiol., 5, 981 (1951). 11. Salton, M. R. J., and R. W. Home, Biochim. et Biophys. Acta, 7, 177 (1951). 12. Salton, M. R. J., Biochim. et Biophys. Acta, 8, 510 (1952). 13. Salton, M. R. J., /. Gen. Microbiol., 9, 512 (1953). 14. Bosco, G., /. Injections Diseases, 99, 270 (1956). 15. Fraser, D., Nature (London), 167, 33 (1951). ISOLATION AND PROPERTIES OF WALLS 23 16. Ribi, E., T. Peirine, R. List, W. Brown, and G. Goode, Proc. Soc. Exptl. Biol. Med., 100, 647 (1959). 17. Weidel, W., Z. Nalurforsch., 6b, 251 (1951). 18. Slade, H. D., and J. K. Vatter, /. Bacteriol., 71, 236 (1956). 19. Salton, M. R. J., unpublished results. 20. Marr, A. G., and E. H. Cota-Robles, /. Bacteriol., 74, 79 (1957). 21. Shockman, G. D., J. J. Kolb, and G. Toennies, Biochim. et Biophys. Acta, 24, 203 (1957). 22. Strange, R. E., and F. A. Dark, /. Geyi. Microbiol., 16, 236 (1957). 23. Weibull, C, /. Bacteriol., 66, 696 (1953). 24. Northcote, D. H., and R. W. Home, Biochem. ]., 51, 232 (1952). 25. Houwink, A. L., and D. R. Kreger, Antonie van Leeuwenhoek, 19, 1 (1953). 26. Nickerson, W. J., 4th Intern. Congr. Biochem., Vol. XIV, 1959, p. 191. 27. Nickerson, W. J., and G. Falcone in Sulfur in Proteins, Academic Press, New York, 1959, p. 409. 28. Falcone, G., and W. J. Nickerson, 4th Intern. Congr. Biochem., Vol. VI, 1959, p. 65. 29. Northcote, D. H., K. J. Goulding, and R. W. Home, Biochem. J., 70, 391 (1958). 30. Frey-Wyssling, A., and K. Miihlethaler, Vierteljahresschr. Nalur- forsch. Ges. Ziirich, 95, 45 (1950). 31. Roelofsen, P. A., Biochim. et Biophys. Acta, 6, 357 (1951). 32. Shatkin, A. J., and E. L. Tatum, /. Biophys. Biochem. Cytol., 6, 423 (1959). 33. Chapman, J. A., and M. R. J. Salton, in preparation. 34. Preston, R. D., Science Progress, 46, 593 (1958). 35. Houwink, A. L., Biochim. et Biophys. Acta, 10, 360 (1953). 36. Salton, M. R. J., and R. C. Williams, Biochim. et Biophys. Acta, 14, 455 (1954). 37. Houwink, A. L., /. Gen. Microbiol., 15, 146 (1956). 38. Labaw, W., and V. M. Mosley, Biochijn. et Biophys. Acta, 15, 325 (1954). 39. Kellenberger, E., and A. Ryter, /. Biophys. Biochem. Cytol., 4, 323 (1958). 40. Bartholomew, J. W., and R. Levin, /. Gen. Microbiol, 12, 473 (1955). 41. Mitchell, P., and J. Moyle, /. Gen. Microbiol, 15, 512 (1956). 42. Christian, J. H. B., and M. Ingram, /. Gen. Microbiol, 20, 32 (1959). 24 MICROBIAL CELL WALLS 43. Shockman, G. D., J. J. Kolb, and G. Toennies, /. Biol. Chem., 230, 961 (1958). 44. Toennies, G., and G. D. Shockman 4th Intern. Congr. Biochem., Vol. XIII, 1959, 365. 45. Mason, D. J., and D. Powelson, Biochim. et Biophys. Acta, 29, 1 (1958). 46. Barkulis, S. S., and M. F. Jones, /. Bacteriol., 74, 207 (1957). 47. Stacey, M., Symposium Soc. Gen. Microbiol., 1 (1949), p. 29. 48. Salton, M. R. J., unpublished observations. 49. Aronson, J. M., and L. Machlis, Am. J. Botany, 46, 292 (1959). 50. Salton, M. R. J., Biochim. et Biophys. Acta, 10, 512 (1953). 51. Lewin, R. A., /. Gen. Microbiol, 19, 87 (1958). 52. Hastings, J. Woodland, unpublished observations. 53. Brown, A. D., unpublished observations. 54. Fogg, G. E., The Metabolism of Algae, Methuen, London, 1953, p. 104. 55. Lewin, J. C., R. A. Lewin, and D. E. Philpott, /. Gen. Microbiol., 18, 418 (1958). 56. Cronshaw, J., A. Myers, and R. D. Preston, Biochim. et Biophys. Acta, 27, 89 (1958). 57. Cummins, C. S., and H. Harris, /. Gen. Microbiol, 18, 173 (1958). 58. Salton, M. R. J., and M. P. Hatton, in preparation. 59. Falcone, G., and W. J. Nickerson, Science, 124, 272 (1956). 60. Kessler, G., and W. J. Nickerson, /. Biol. Chem., 234, 2281 (1959). 61. Salton, M. R. J., in The Bacteria, Vol. 1, Academic Press, New York, 1960, p. 97. 62. Baddiley, J., J. G. Buchanan, and B. Carss, Biochim. et Biophys. Acta, 27, 220 (1958). 63. Armstrong, J. J., J. Baddiley, J. G. Buchanan, B. Carss and G. R. Greenberg, /. Chem. Soc, 4344 (1958). CHAPTER LIBRARY CHEMISTRY OF CELL WALL! '* MASS. Now we shall turn to the more detailed studies of the chemical constituents of microbial cell walls. For this discussion our selection of material is confined almost ex- clusively to yeast and bacterial cell walls. Some ten years ago very little was known about the chemistry of the walls of bacteria. This situation has been rapidly changed so that more is known about the chemical constitution of walls of bacteria than those of any other microorganism, and only a condensed account of the chemistry of bacterial walls can now be given in a single lecture. Chemistry of Yeast Cell Walls Long before the yeast wall had been isolated as a single morphological entity yeast polysaccharides had been puri- fied and their structures investigated. Glucan from Sac- charomyces cerevisiae and from Candida albicans both con- tain /?(1 -^ 3) and ^(1 -^ 6) glycosidic linkages, but the 25 26 MICROBIAL CELL WALLS polysaccharide from the latter appears to be more highly branched.^' -'^'^ Some differences in the linkages and degree of branching have been suggested for mannans derived from various yeasts.^' °'^ Isolation of walls by mechanical disintegration led to the discovery of protein and lipid components in addition to the polysaccharides. ''^'^'^° Not all yeast species contain appreciable quantities of lipid in the wall, for Kessler and Nickerson^ found as little as 1% total lipid in the walls of strains of Candida albicans and as much as 10% in the wall of Saccharomyces cerevisiae. A clearer understanding of the molecular architecture of the yeast cell wall has begun to emerge from the important discovery by Falcone and Nickerson ^ that the wall polysaccharides occur as protein complexes. Further investigations by Kessler and Nickerson ^ have established the presence of a glucan-pro- tein complex and two types of glucomannan-protein com- plexes in a variety of yeast walls. The percentage of the wall accounted for by the various polysaccharide-protein complexes for several yeasts is illustrated in Table 8. The presence of a mannan-protein complex in baker's yeast wall has been confirmed by Korn and Northcote/^ and, from alterations in the surface charge of yeast walls degraded with various enzymes, Eddy ^^ has suggested that the man- nan-protein complex forms part of the outside layer of the wall. However, this suggestion, based on microelectro- phoresis data, must await more definitive biochemical and chemical investigations. The nature of the bonding be- tween the polysaccaride-protein complexes is not known, but Kessler and Nickerson ^ suggest the possibility of esterifi- cation of carboxyl groups of the protein with hydroxyl groups of the polysaccharides. CHEMISTRY OF CELL WALLS 27 TABLE 8 The Percentages of Various Polysaccharide-Protein Complexes in the Walls of Several Yeasts * Recoveries of Cell Wall Complexes Glucan Glucomannan- Glucomannan- Protein Protein I Protein II Organism % % % Baker's yeast 41.6 13.6 34.7 Saccharomyces cerevisiae 18.29 28.3 55.8 11.9 Candida albicans RM806 46.7 7.5 41.5 Candida albicans 582 47.4 3.0 27.2 * From the data of Kessler and Nickerson.^ Chemistry of Bacterial Cell Walls In discussing the chemistry of bacterial cell walls, it be- comes necessary to distinguish between the two major groups of organisms differentiated by the Gram stain re- action. Early comparative studies of wall composition indi- cated the greater complexity of the walls isolated from Gram-negative bacteria. ^^-^^ This chemical heterogeneity was later found to be paralleled by a more complicated physical structure of multilayered walls with macromolecu- lar subunits, as mentioned in Chapter 1. Whether the greater chemical complexity of the "wall" of Gram-negative bacteria is due to the presence of a single structure possess- ing the functional units of a true wall and a membrane has not been satisfactorily resolved. However, the thin sections 28 MICROBIAL CELL WALLS of Gram-negative bacteria give strong support to the belief that there are indeed two separate structures, a complex wall and a membrane.^^'^^-^^ (See Fig. 7.) It appears likely, then, that the differences in wall composition between the Gram-positive and Gram-negative bacteria amply demon- strated in many investigations ^^-^^'^^ are real and are not an artifact of a major structural difference between the two groups of organisms. What is worth emphasizing here is that both groups of organisms possess mucopeptide con- stituents in common, a finding that has led to the idea of a "basal" structure being present in all bacterial cell walls (Work 20). The nature of the basal structure has become more apparent, and it is likely that one of a variety of mucopeptides can perform this function. ^i' 22 What is uncertain at the moment is the variety of monomeric con- stituents in the mucopeptides from both Gram-positive and Gram-negative bacterial walls. At the present time there is insufficient evidence to suggest that the term "basal struc- ture" means any more than a class of mucopeptides con- taining some common building units of amino sugars and amino acids. Constituents of Walls of Gram-Positive Bacteria Analysis of the walls of Gram-positive bacteria revealed the presence of both nitrogen and phosphorus, and in Bacil- lus suhtilis walls the content of P was very high.^s Qn hydrolysis the walls contained reducing substances and amino sugars, and some of the typical results are shown, together with N and P determinations, in Table 9. The first fascinating detail to emerge from the early studies of cell-wall chemistry was the small variety of amino acids in the walls of some bacteria. ^^ Thus the wall of Micrococcus lysodeikticus isolated by mechanical disintegra- tion and receiving no treatments other than washing with CHEMISTRY OF CELL WALLS 29 TABLE 9 Composition of the Walls of Several Gram-Positive Bacteria % Reducing % Amino % N % P substances sugar Bacillus 5.3 0.42 48 18 jiiegaterium Bacillus 5.1 5.35 34 8.5 sub ti lis Micrococcus 8.7 0.09 45 16 lysodeikticus Sarcina lutea 7.6 0.22 46 16 Streptococcus 5.6 1.88 61 22 faecalis Reference 13. M NaCl and water contained the four amino acids (alanine, glutamic acid, glycine, and lysine) together with hexosamine and glucose. 13 since then a great deal of qualitative and quantitative work on walls has been performed by Cummins and Harris,23.24 Work,^^ Snell, Radin, and Ikawa,-^ Ikawa and Snell,26 Strange,^' Baddiley, Buchanan and Carss,-^ Armstrong, Baddiley, Buchanan, Carss, and Greenberg,-^ and Abrams,^^ and the following oustanding features have firmly established some of the characteristic chemical prop- erties of the walls: (a) Variety of principal amino acids limited to 3, 4, or 5. (b) Discovery of diaminopimelic acid in certain micro- organisms and its localization in the wall. (c) The detection of D-isomers of amino acids. (d) The isolation and characterization of the amino sugar, muramic acid, from spore peptides and walls. MICROBIAL CELL WALLS (e) The detection o£ ribitolphosphate polymers in walls and the discovery of the teichoic acids. (/) The detection of O-acetyl groups. (g) The presence of ester-linked alanine. The identification of the principal constituents of the walls of Gram-positive bacteria has led to the conclusion that the walls belong to the general class of chemical com- pounds known as mucocomplex substances.^^'^^'^^--^-^^'^^ These mucocomplex polymers can be further subdivided, depending on whether peptide components predominate or whether they are predominantly polysaccharide in nature as below: mucopeptides— composed of amino acids and amino sugars mucopolysaccharides— sugars and amino sugars It is probable that in some cell walls both are covalently joined so that soluble wall compounds derived either chem- ically or enzymically may be essentially either mucopeptide or mucopolysaccharide but containing minor residues of one or the other. In addition to these two classes of sub- stances, we must now add the teichoic acids 2^' ^^ as major wall compounds. The walls of Gram-positive bacteria may therefore be wholly mucopeptide ^3- ^^ or predominantly mucopolysaccharide, with smaller amounts of mucopeptide as in some streptococcal walls,^* or they may contain muco- peptides, mucopolysaccharides, and teichoic acids. Amino-Acid Composition. The distribution of major amino acids has been studied in some detail by Cummins and Harris.23.24,31 Amino acid constituents, and in some cases the monosaccharide components of walls, have been of great taxonomic value. 3^-^- The principal combinations of the major amino acids found in walls are presented in Table 10. It will be seen that in none of the walls so far CHEMISTRY OF CELL WALLS 31 TABLE 10 Principal Combinations of Major Amino Acid Constituents Found in Walls of Gram-Positive Bacteria Groups Amino Acids Staphylococci Micrococci Streptococci Lactobacilli Aerococci Bacilli Coynebacteria Mycobacteria Nocardia Micrococci Clostridia Proprionibacteria Streptomyces Micromonospora . Alanine, glutamic acid, lysine, glycine, and serine in some Alanine, glutamic acid, lysine, and aspartic acid in some Alanine, glutamic acid, DAP Alanine, glutamic acid, DAP, glycine References 23, 24, 31, 37. 41, 51 studied do diaminopimelic acid (DAP) and lysine occur to- gether as major amino acid constituents. More and more information on the quantitative amino acid composition of bacterial walls has become available (Perkins and Rogers, ^^ Rogers and Perkins,^* Strominger, Park, and Thompson,^^ Hancock ^6), and on the whole there is good agreement for various organisms, although it is now apparent that there will be significant differences between various strains.'^ The molar ratios of the principal amino acids of walls from various species investigated by Salton 32 MICROBIAL CELL WALLS and Pavlik ^~ are presented in Tables 11 and 12. It is evi- dent from these results that the peptide composition may vary widely from one group to another, although in some TABLE 11 Relative Molecular Proportions of the Principal Amino Acids in Cell Walls Glutamic Walls from Lysine Acid Glycine Serine Alanine Bacillus sp* 1 1.7 0.5 0.3 2.3 Corynebacteriumsp. 1 1.0 Of 0.7 3.9 Micrococcus citreus 1 3.0 0.8 0 2.1 Micrococcus lysodeikticus 1 1.0 1.0 0 2.6 Micrococcus roseus 1 1.1 0 0 5.1 Micrococcus tetragenus 1 1.2 1.2 0 2.3 Micrococcus urea 1 1.3 1.0 0 2.3 Sarcina flava 1 1.4 . 1.0 0 2.2 Sarcina lutea 1 1.6 1.0 0 2.0 Sporosarcina ureae 1 1.7 1.0 0 2.0 Staphylococcus albus 1 1.1 4.8 0.4 2.9 Staphylococcus aureus 1 1.1 4.8 0.45 3.0 Staphylococcus citreus 1 1.0 4.0 0.5 3.1 Staphylococcus saprophyticus 1 1.0 4.6 0.6 3.3 Streptococcus faecalis I 0.9 0 0 4.0 * A high proportion of threonine (0.7) was also present f "0" used to designate absence or only faint traces of amino acids. Reference 37, N. B. Serine values for the four staphylococci in- correctly shown above. Salton and Pavlik 3" have been corrected CHEMISTRY OF CELL WALLS 33 TABLE 12 Relative Molecular Proportions of the Principal Amino Acids in Cell Walls Glutamic Walls from DAP * Acid Glycine Alanine Bacillus cereus I 1.3 0 2.6 Bacillus megaterium ] 1.8 0 2.8 Bacillus pumilis ] 1.6 0 4.6 Bacillus stearothermophilus 2.0 0 3.8 Bacillus subtilis 2.4 0 4.3 Bacillus thuringiensis 1.4 0 2.8 Micrococcus varians 4.3 1.8 2.6 Lactobacillus arabinosus ] 1.1 0 2.9 * a,e-diaminopimelic acid. Reference 37. instances very similar ratios of amino acids were observed. No one would claim that these results represent anything more than the gross amino acid composition of the walls, as they give no indication of the distribution in the various wall components such as the mucopeptides and teichoic acids or other special structures that may yet remain to be discovered. Apart from the peptide residue of the nucleotide isolated from penicillin-inhibited Staphylococcus aureus (Park,^^ Park and Strominger ^^), there are no published accounts of the sequence of amino acids in peptides derived directly from cell walls. In addition to the known amino acids, bacterial walls have yielded upon hydrolysis several unknown ninhydrin- reacting constituents.^3,23,24,37 Cummins and Harrises 34 MICROBIAL CELL WALLS found an unknown compound in the walls of lactobacilli. This substance w^as found to be a peptide of lysine and as- partic acid (a-aminosuccinoyllysine), which was more re- sistant to acid hydrolysis. The compound in which the aspartic acid is joined to the eNHs group of lysine was also encountered in hydrolysates of the antibiotic bacitracin. *« Appreciable amounts of ammonia have been found on hydrolysis of cell walls by Ikawa and Snell/^ and if this is not due simply to destruction of wall compounds such as the amino sugars it indicates the possibility that some amino acids may be present as amides. Typical results for the amino acid composition of several lactic acid bacteria from the studies of Ikawa and Snell ^^ are presented in Table 13. TABLE 13 Amino Acid Composition of Walls from Lactic Acid Bacteria * (mg per 100 mg cell wall) Streptococcus Lactobacillus Lactobacillus faecalis plantarum citrovorum Glutamic acid l 0.6 0 0.9 D 4.6 7.6 10.4 Alanine (total) 4.4 11.6 9.8 D 1.7 3.7 4.6 Aspartic acid (total) 2.4 0.6 8.1 L 0.7 1.8 Lysine (total) 2.5 0.4 5.6 L 2.4 0.5 6.2 DAP 0 5.2 0 a-aminosuccinoyllysine 0 0 4.4 Ammonia 1.1 2.4 3.3 * Data from Ikawa and Snell. ^^ CHEMISTRY OF CELL WALLS 35 Occurrence of D-isomers of Amino Acids. Snell and his colleagues -^' -^ were the first to discover that the D-alanine found in bacterial cells was localized in the wall. A high proportion of the cell-wall alanine was present as the o-iso- mer. Glutamic acid was subsequently found in the wall as the D-isomer.2^ Salton *- also showed that o-alanine occurred quite widely in the walls of various bacterial species. The list of D-amino acids in bacterial walls was extended to aspartic acid when Toennies, Bakay, and Shockman ^^ found that this amino acid occurred partly as the D-isomer in the wall of Streptococcus faecalis. Ikawa and Snell *^ have made an extensive investigation of the proportions of d- and L-isomers of alanine, glutamic, and aspartic acids in the walls of many lactic acid bacteria, and some of the re- sults are summarized in Table 14. Thus about half of the cell-wall alanine occurs as the D-isomer and virtually all of the glutamic acid is in the D-form, whereas D-aspartic acid residues constitute roughly three quarters of the total as- partic acid contents. Park ** has observed that many walls have 1 : 1 ratios of D-glutamic acid to muramic acid. Evidence so far available suggests that only L-lysine is present in walls.*^ However, DAP can occur in bacteria as the LL-, meso(DL)-, or DD-isomers, and occasionally the ll- and meso-isomers together (Hoare and Work ^^). The meso- isomer is most widely distributed in bacteria and the iso- lated walls, being found in members of the Bacillus, Corynebacterium, Mycobacterium, Nocardia, and, less fre- quently, in certain species of Lactobacillus and Micrococcus groups.^^'20.24,37,45 LL-DAP has been detected in members of the Propionibacterium, Streptomyces, and some Clos- tridium species.2*'45 Hoare and Work ^^ found some DD-iso- mer of DAP in hydrolysates of Micromonospora, the pres- ence of this isomer in isolated walls being confirmed later.-* 36 MICROBIAL CELL WALLS TABLE 14 Percentage of Glutamic Acid, Aspartic Acid, and Alanine in the D-configuration in Cell Walls (% of total in D-form) Glutamic Aspartic Acid Acid Alanine Streptococcus faecalis 85 71 39 Lactobacillus casei 100 50 61 Lactobacillus plantarum 100 32 Lactobacillus mesenteroides 73 67 54 Lactobacillus pentosus 94 66 Lactobacillus citrovorum 89 78 47 Lactobacillus bulgaricus 87 72 40 Lactobacillus lactis 94 78 61 Lactobacillus acidophilus 91 67 48 * Data from Ikawa and Snell.^i Identification of N-Terminal and C-Terminal Amino Acids. Attempts to apply some of the classical techniques for determining the chemical structure of proteins to bac- terial cell walls have been complicated by some of the un- usual features of cell-wall composition [see foregoing (rt)-(g)]. The cell-wall constituents possessing free amino groups can be readily identified by reacting the walls with l-fluoro-2,4-dinitrobenzene (FDNB)/^ the method intro- duced by Sanger ^" for the determination of the N-terminal residues in proteins. However, the interpretation of results with bacterial walls is complicated by the presence of ester- linked alanine in the teichoic acids. -^ O-alanyl residues would thus behave as N-terminal amino acids. With walls from Micrococcus lysodeikticus and Sarcina lutea that are CHEMISTRY OF CELL WALLS 37 devoid of the teichoic acids, the DNP-alanine detectable on reaction with FDNB probably represents the N-terminal residue of the wall peptides, and from this data a subunit size can be tentatively suggested.**' The contribution of the teichoic acids to the N-terminal alanine residues can be surmised from a comparison of the amounts of DNP- alanine obtained from the walls of Micrococcus lysodeikti- ciis (23 /xM/g) with those of Staphylococcus aureus (170 fxM/g) and Lactobacillus arahinosus (120 ^M/g), both rich in teichoic acids.*^ The relatively small number of N-termi- nal gi'oups in walls other than those containing large amounts of teichoic acids is perhaps not surprising, as the "free" amino groups of peptides would be required for amide bonding to muramic acid. The comparatively low yields of N-terminal residues could, of course, be equally well explained by cyclic peptide structures or N-acetyla- tion of amino acid residues. The application of carboxypeptidase for the identification of C-terminal residues of walls of Gram-positive bacteria has not been successful (Perkins and Rogers,^^ Salton ^^). This is not at all surprising, since the cell-wall peptides contain D-isomers of several amino acids. Hydrazinolysis,^*^ on the other hand, has been much more successful, and with some walls this method has given very clean results, al- though their interpretation poses several interesting prob- lems of the molecular structure of walls. The yields of C-terminal amino acids (uncorrected for any losses during hydrazinolysis) from several cell walls and lysozyme-digest products are given in Table 15. Whether we are really dealing with C-terminal residues in the protein sense (i.e., at the end of a peptide chain) is not known. It is conceivable that special types of linkages of amino acids in the wall peptides could give false "C-termi- nal" values in just the same way as O-alanyl groups behave 38 MICROBIAL CELL WALLS TABLE 15 ^^ C-terminal Amino Acids of Bacterial Cell Walls Determined by Hydrazinoylsis (aiM/10 mg cell wall *) Glutamic Glycine Acid Alanine DAP Sarcina lutea 1.84 0.47 <0.1 -t Micrococcus lysodeikticus Prep. 1 2.47 0 0.21 — Prep. 2 2.52 0 0.37 — Lysozyme-NDF % 4.1 0 0.21 — Bacillus megaterium — <0.1 0.35 1.29 Micrococcus varians <0.1 <0.1 <0.1 0.14 * Values uncorrected for possible losses during reaction. f Amino acid not present in these walls. :|: Nondialyzable fraction from lysozyme-digested walls. Data from Salton.^s as false "N-terminal" residues. If amino acids occurred as single substituents on muramic acid, or in the form of a side chain linked to the y-carboxyl gioup of glutamic acid, they would also behave as C-terminal substances. The only evidence so far available supporting this suggestion is the report by Perkins and Rogers ^^ that a diffusible compound in partial acid hydrolysates of Micrococcus lysodeikticus walls possessed muramic acid, glucosamine, and glycine in equimolar proportions. The occurrence of substituent groups of glycine on some muramic acid residues would be compatible with the large number of C-terminal groups found in the wall of this organism (see Table 15) and could also explain the origin of free glycine in walls digested with the Streptomyces enzyme complex (Salton and Ghuysen ^i) CHEMISTRY OF CELL WALLS 39 now known to contain an amidase capable of acting on small molecular weight mucopeptides (Ghuysen ^^). Thus in the walls of Micrococcus lysodeikticus the number of C-terminal glycine groups could be due to special groupings on the w^all mucopeptide or could represent the true ends of the peptide chains. If the latter, a subunit size of ap- proximately 4000 molecular weight suggests that this wall possesses relatively short peptide chains on the amino sugar backbone.^^ Amino Sugar Constituents. The key to understanding the structure of the bacterial cell-wall mucopeptides and mucopolysaccharides was provided by the detection and iso- lation of a new acidic amino sugar by Strange -^ and his colleagues. This amino sugar, now known as muramic acid, was first found in the spore peptides isolated by Strange 0 CH C.H:;-CH 0 CH ^ CgH^-CH O CH, L, OR OMe NH-Ac HO>p^ ^ NH3+ R = — CH \ CO^Et .C(0Et)3 or CH CHc CH^ .COo R' = — CH CH< Fig. 8. Synthesis of muramic acid. 40 MICROBIAL CELL WALLS and Powell ^^ and was subsequently found in bacterial cell walls.-^'^*'^^'^*^ The unknown amino sugar in the nucle- otides accumulating in penicillin-treated Staphylococcus aureus discovered by Park and Johnson ^^ in 1949 was later found to be identical to the cell-wall amino sugar.^^ Muramic acid (3-O-carboxyethyl-D-glucosamine) was iso- lated as a crystalline substance by Strange and Dark/^ and the structure was established by the synthetic route worked out by Strange and Kent,^^ starting with the N-acetyl-4 : 6- O-benzylidene-a-methyl-D-glucosaminide, as shown in Fig. 8. Some of the properties of natural and the synthetic stereo- isomers of muramic acid are summarized in Table 16. The TABLE 16 Optical Rotation and Chromatographic Behavior of Natural and Synthetic Muramic Acid and the Synthetic Isomer Average Values Derived from Sev- eral Experiments glucosamine value T Optical on Zeo-Karb Rotation 225 Column Eluted Wd ^f* with 0.33 N-HCl Natural muramic acid +109 0.53 1.10 Synthetic muramic acid +109 0.53 1.10 Stereoisomer of muramic acid + 52 0.44 0.87 * Values obtained with Whatman No. 1 paper and phenol-water as solvent. f Values in this column have been reported by Crumpton (1958). The ^glucosamine value relates the elution characteristics of the sub- stance to those of glucosamine run at the same time. Reference, Strange, and Kent.^^ CHEMISTRY OF CELL WALLS 41 comparison of the optical rotations of the synthetic and naturally occurring compound suggests that the lactic acid residue of the spore-peptide muramic acid possessed the D-configuration. Zilliken ^^ has confirmed the synthesis of muramic acid from D-glucosamine, using several modifica- tions to the procedure developed by Strange and Kent.^^ The structures proposed for muramic acid and that of the muramic acid-nucleotide from Staphylococcus aureus ^^'^^ suggest that the general function of muramic acid in the cell wall is to link peptides (through an amide bond at the carboxyl group of muramic acid) to other amino sugar or sugar residues as shown below: ^.^-'— CHo O H NHCOCH3 CHoCHCONH Peptide Although Park ^* has shown that there is a 1:1 ratio of muramic acid to D-glutamic acid in the walls of a number of bacteria, it should not be assumed from the general type of structure previously suggested that all or nearly all of the muramic acid residues have peptide substituents. As is shown in Chapter 3, in Micrococcus lysodeikticus walls much of the muramic acid is unsubstituted. However, it is not difficult to visualize that in some bacterial walls (pos- sibly those resistant to lysozyme) peptides may form a cross link between parallel chains of amino sugar oligosaccharides, being linked through muramic acid at each end of the pep- tide. This could be especially the case with those walls containing DAP (or lysine), as there is evidence that both 42 MICROBIAL CELL WALLS amino groups of DAP may be unavailable for reaction with FDNB in a high proportion of the residues in some cell walls.^6 Muramic acid has been detected (usually by paper chro- matography) in all of the bacterial cell walls so far exam- ined.^^'21'22,24,42 whether the structures are identical in all cases and whether all muramic acids are the 3-O-D-lactyl ethers of glucosamine remains to be established. It is of interest to note that Agien and Verdier ^° have isolated 6-phosphoryl muramic acid from a protein-bound com- pound in Lactobacillus casei. It will be of great interest to learn whether this compound occurs in the wall as the phosphoryl derivative. In addition to muramic acid, glucosamine is also univer- sally present in bacterial cell walls.13,14,19 Galactosamine has been found, together with muramic acid and glucosa- mine, in some bacterial walls, but it seems to be much less widely distributed.i9'23.24,3i ij- {^ probable that all three amino sugars occur in the walls as N-acetyl or as N-acyl com- pounds. The reaction of walls with FDNB has so far shown that none of the amino groups of the amino sugars is free.^^ Monosaccfiar/des. Some bacterial walls are composed entirely of amino acids and amino sugars being devoid of other sugar components. ^^-s" However, many bacterial walls yield monosaccharides on hydrolysis, and the investi- gations of Cummins and Harris 23,24,31 j^^ve shown that the sugar components are characteristic of certain taxonomic groups. Glucose occurs commonly in many bacterial walls and, as will be seen later, it may also be a constituent of the teichoic acid moiety of the wall. Rhamnose, first found as a wall monosaccharide in Streptococcus faecalis,^^ is the typical sugar of the streptococcal group. Arabinose, de- tected in the wall and isolated cell-wall oligosaccharide of CHEMISTRY OF CELL WALLS 43 Corynebacterium diphtheriae by Holdsworth,^^ ^^s subse- quently found to be confined to a number of related groups. Some of the monosaccharides characteristic of various bac- terial groups are presented in Table 17. The occurrence of mucopolysaccharides in the walls of some bacteria is supported by the isolation of the oligo- saccharide from Corynebacterium diphtheriae by Holds- worth.^2 Fairly drastic conditions were required for the lib- eration of the oligosaccharide from the cell-wall muco- TABLE 17 Principal Combinations of Monosaccharide Constituents Found in Walls of Gram-Positive Bacteria Groups Sugars Staphylococci Sporosarcina Strep tomyces Staphylococci Micrococci Aerococci Bacilli Streptomyces Streptococci Lactobacilli Propionibacteria Clostridia None Glucose, galactose, mannose (singly or in com- bination) Rhamnose, glucose, galactose, mannose (Rhamnose alone or in combination with Rhamnose) Corynebacteria 1 Arabinose, glucose, galactose, mannose Mycobacteria \ (in combination with Nocardia J arabinose) References 22, 23, 24, 31, 37, 62, 63, 64. 44 MICROBIAL CELL WALLS complex,^2 suggesting a firm chemical combination between the polysaccharide and the rest of the wall (as for Strepto- coccus faecalis ^^). Further evidence establishing the pres- ence of mucopolysaccharides in the walls has come from the investigation by McCarty ^^ of the products of enzymic di- gestion of Group A streptococcal walls. The "C" carbo- hydrate fractions from the streptococcal wall still contained small residues of peptide but were composed predominantly of amino sugar and rhamnose.*^^ Further fractionation failed to remove the peptide constituents, and there seems little doubt that these mucopolysaccharides were joined to the mucopeptides in the original wall. Teichoic Acids. Mitchell and Moyle ^^ reported the pres- ence of a poly glycerophosphate compound in the envelope of Staphylococcus aureus, and the status of this material as a wall component remained uncertain until the problem was taken up again following the discovery by Baddiley and his colleagues ^^ of the two nucleotides, cytidine diphos- phoglycerol and cytidine diphosphoribitol. It will be re- called that the wall of Bacillus suhtilis had a very high phos- phorus content (see Table 9), and it was not surprising that an examination of the wall of this organism and that of Lactobacillus arabiiiosus [syn. Lactobacillus plantarum] (the organism from which the two nucleotides were isolated) revealed the presence of ribitolphosphate polymers. ^^ No glycerophosphate polymer was detectable in the walls of either of these organisms. The name teichoic acids was given originally only to the ribitolphosphate polymer,-^ but since the confirmation of the presence of a glycerophosphate polymer in walls of other bacteria, and the detection of both types in yet others, the term teichoic acids has been extended to include both types of polyols.*^^ The distribu- tion of the two types of teichoic acids in various cell walls CHEMISTRY OF CELL WALLS 45 has been studied by Armstrong et al.^^ and is presented in Table 18. Glycerophosphate polymers have been detected in a num- ber of Gram-positive bacteria by McCarty,*^^ but he was un- able to find these localized in the walls. These polymers thus probably differ from the glycerol type of teichoic acid, which in common with the ribitol teichoic acids contain O-alanyl groups.®^ The teichoic acids can be extracted from the isolated walls with trichloroacetic acid (TCA), and Armstrong et al.^^ sug- gest that they may be bound to the other wall constituents by salt linkages. However, conditions for extraction with TABLE 18 Distribution of Teichoic Acids in Bacterial Cell Walls Type of Polymer Glycerol Ribitol Lactobacillus arabinosus 17-5 — + Lactobacillus casei (AT.C. 7469) Lactobacillus delbriickii (N.C.I.B. 8608) Lactobacillus bulgaricus (N.C.I.B. 76) Staphylococcus aureus H Staphylococcus aureus (Duncan) Staphylococcus aureus (Oxford) Staphylococcus citreus Staphylococcus albus (N.C.T.C. 7944) Bacillus subtilis (vegetative form) Escherichia coli Type B Corynebacterium xerosis Streptococcus faecalis (A.T.C. 9790) + + - trace + trace + + + + — + - - + trace — + — + + Reference 68. 46 MICROBIAL CELL WALLS TCA are hydrolytic,^^'*^ and the mode of attachment of the teichoic acids remains uncertain at present. The products of acid hydrolysis of the teichoic acids removed from walls by extraction with TCA have been examined by Armstrong et al.,29 and Table 19 illustrates the variety of compounds detectable in the ribitol type; 1:4 anhydroribitol is one of the main products detectable, but, as pointed out by Salton and Pavlik,^' in 6A^ hydrochloric acid hydrolysates of walls a faster-moving component (possibly dianhydroribitol), not previously reported, is detectable on paper chromatograms. One of the interesting features of the structure of the teichoic acids was the discovery of ester-linked alanine, the first reported occurrence of this type of linkage of an amino acid in a natural product. The detailed structure of the ribitol teichoic acid from Bacillus suhtilis has been proposed TABLE 19 Products of Acid Hydrolysis of Teichoic Acid from Different Bacteria Lactobacillus Bacillus Staphylococcus arabinosus subtilis aureus Alanine + + + Glucose + + — Glucosamine — — + Inorganic phosphate + + + Anhydroribitol -f + + Anhydroribitol phosphate + + + Ribitol + + + Ribitol glucosaminide - - + Reference 29. CHEMISTRY OF CELL WALLS 47 ...O-HgC- OH CHz'O-P-O-HzC OH 0 OH OH HO-HgC HO-HgC OH H H-CHo-O-P... II I =^ II O OH OH o J L CHo-CH-CO. ' I NHo NHc Fig. 9. The structure of teichoic acid from the cell walls of Bacillus sub t His. by Armstrong, Baddiley, and Buchanan/*^ as shown in Fig. 9, and the three general types of teichoic acid are represented in formulas 1, 2, and 3.^^ (1) (2) (3) alanyl-glucosyl-ribitol I 0=P— OH I alanyl-N-acetylglucosaminyl-ribitol I 0=P— OH I I alanyl-glycerol 0=P— OH 48 MICROBIAL CELL WALLS O-ester Groups. The presence of O-substituents in bac- terial walls was first reported by Abrams,3o who discovered O-acetyl groups in the walls of Streptococcus jae calls and those of several other bacteria. Brumfitt, Wardlaw, and Park ^^ subsequently found that a lysozyme-resistant mutant of Micrococcus lysodeikticus contained a much greater amount of O-acetyl in the walls than the parent strain. Removal of the O-acetyl groups restored the sensitivity to lysozyme. The O-alanyl groups of the teichoic acids are the only other O-ester gioups so far reported in bacterial walls. Whether the teichoic acids in Lactobacillus arahinosus are ester linked to other wall components is not known, but it is of interest to note that the lysozyme sensitivity of the isolated walls of this organism is greatly increased after ex- traction with reagents removing O-esters and/or teichoic acid.3^ Armstrong et al.^^ reported that alanine was the only O-ester in the wall of this organism, so it appears that the change in lysozyme sensitivity does not involve removal of O-acetyl groups as in Micrococcus lysodeikticus walls. '^^ Composition of Walls of Gram-Negative Bacteria The status of our knowledge of the chemistry of the walls of Gram-negative bacteria is less satisfactory, although a clearer picture is beginning to emerge from the detailed studies of Escherichia coli walls by Weidel and his col- leagues. The greater complexity of the walls of Gram-nega- tive bacteria has already been emphasized. ^^-^^ In addition to a complete range of amino acids, they also contain sub- stantial amounts of lipid and frequently a variety of mono- saccharide constituents. The amino sugar contents are generally lower than those found for the majority of walls from Gram-positive bacteria. Some typical analyses for amino sugar contents and amounts of lipid in the walls of CHEMISTRY OF CELL WALLS 49 a number of Gram-negative bacteria are given in Table 20. One of the most important recent developments in the study of the chemistry of the walls of Gram-negative bac- teria has been the recognition of mucopeptide constituents of a similar nature to those forming the whole cell-wall structure of Gram-positive bacteria. This discovery has fol- lowed from a nvuiiber of investigations on the occurrence of DAP 20 and the detection of both DAP and muramic acid in the walls of Escherichia coli -i-'^- and those of a variety of Gram-negative bacteria. ^2- ^- Furthermore, Park ** reported the presence of D-glutamic acid in Escherichia coli walls, and a small amount of D-alanine was detected in the wall of Rho do spirillum rubriim^- Additional evidence for the ex- istence of the mucopeptide in Escherichia coli wall came from the work of Weidel and Primosigh ^^.'s when they dis- covered that the phenol-insoluble fraction of the wall con- TABLE 20 Lipid and Amino Sugar Contents of the Walls of Gram-Negative Bacteria % Dry Weight Cell Wall Total Lipid Amino Sugars Escherichia coli 22 3.0 Salmonella pullorum 19 4.8 Salmonella gallinarum 22 3.9 Vibrio metchnikovi 11 1.9 Pseudomonas aeruginosa 11 2.1-2.7 Rhodospirillum rubrum 22 2.0 Chlorobium thiosulphatc yphilum 20 4.2 Organism LCI — 13.0 References 13, 14, 22. 50 MICROBIAL CELL WALLS tained alanine, glutamic acid, DAP, glucosamine, and muramic acid as principal constituents. Material of this general composition was released from the wall on treat- ment with T2 bacteriophage enzyme. Salton ^2 showed that all but traces of the cell-wall DAP and muramic acid were released into the soluble fraction when lysozyme acted on the isolated walls of several Gram-negative bacteria, includ- ing Escherichia coli. The composition of the soluble non- dialyzable constituents released by lysozyme from the walls of the Gram-negative bacteria showed that again alanine, glutamic acid, DAP, and glucosamine were predominant constituents with smaller amounts of muramic acid.22 There seems little doubt now that the mucopeptide is the component that is responsible for the structural rigidity of the walls of Gram-negative bacteria, although it may ac- count for as little as 10 to 20% of the weight of the wall.i^'21.22 That the loss of the mucopeptide brings about a collapse of the rigid cell-wall structure has been directly demonstrated with isolated walls of Rhodospirillum rubrum by the author. Figure \0a shows the appearance of R. ru- brum walls before treatment with lysozyme, and Fig. 106 shows how the structures become spherical on incubation with 100 jxg lysozyme per milliliter under conditions giv- ing a release of mucopeptide constituents. ^2 The actual amounts of mucopeptide in the walls of Gram-negative bac- teria probably vary from one species to another, and the data on amino sugar contents (Table 20) suggest that a whole spectrum of mucopeptide contents exists.^* The bulk of the wall of at least a number of Gram-nega- tive bacteria is made up of protein, lipid, and polysac- charide complexes, undoubtedly forming the surface anti- genic components. The cell walls isolated from Gram; negative bacteria contain the monosaccharide constituents that are characteristic of the purified lipo-polysaccharide Fig. 10. (a) Isolated walls of Rhodospirillum rubrum (X 20,500). (b) Walls of R. rubrum treated with lysozyme, showing conversion from the normal spiral fragments as in (a) to spherical structures (X 11,500). M. R. J. Salton, unpublished. 51 52 MICROBIAL CELL WALLS antigens.^*' ^^ Thus, some of the dideoxyhexoses charac- terized by Westphal and his collaborators "*• " ^ and hep- toses ' ^' ' " are present in the bacterial walls. The charac- teristic spectra of the products of the Dische '^ reaction of heptoses have been used to show that these monosaccharides are located in the lysozyme-insoluble fraction of the wall, clearly indicating that they are not part of the mucopeptide structure."^ A typical result for the walls of Spirillum ser- pens is shown in Fig. II. Much remains to be done in the investigation of the walls of Gram-negative bacteria, and at the moment we have no precise information about the number of different molecu- lar or macromolecular subunits in the walls of this group. At least we are now certain that mucopeptides are common Spirillum serpens walls Untreated A — A Lysozyme-soluble fraction 400 450 500 Wavelength (m^) 550 600 Fig. n. serpens. Spectra of Dische reaction products of walls of Spirillum CHEMISTRY OF CELL WALLS 53 to walls from both Gram-positive and Gram-negative groups o£ bacteria, and these can be identified by characteristic components such as DAP, muramic acid, and o-isomers of alanine and glutamic acid. However, the arrangement of the mucopeptide constituents in the walls of Gram-negative bacteria may differ in that they form a reinforcing net- \vork rather than a continuous sheet of the polymer. The fact that the isolated walls of Gram-negative bacteria can be completely disaggregated by sodium dodecyl sulfate strongly suggests this idea.'^ REFERENCES 1. Hassid, W. Z., M. A. Joslyn, and R. M. McCready, /. Am. Chem. Soc, 63, 295 (1941). 2. Bell, D. J., and D. H. Northcote, /. Chem. Soc, 1944 (1950). 3. Peat, S., J. R. Turvey, and J. M. Evans, /. Chem. Soc., 3862 (1958). 4. Bishop, C. T., F. Blank, and P. E. Gardner, Can. J. Chem., 38, 869 (1960). 5. Haworth, W. N., E. L. Hirst, and F. A. Isherwood, /. Chem. Soc, 784 (1937). 6. Cifonelli, J. A., and F. Smith, /. Am. Chem. Soc, 77, 5682 (1955). 7. Northcote, D. H., and R. W. Home, Biochem. J., 51, 232 (1952). 8. Falcone, G., and W. J. Nickerson, Sciejice, 124, 272 (1956). 9. Kessler, G., and W. J. Nickerson, /. Biol. Chem., 234, 2281 (1959). 10. Masschelein, Ch. A., Revue des fermentations et des industries ali- mentaires, Bruxelles, T. XIV, p. 59 (1957). 11. Korn, E. D., and D. H. Northcote, Biochem. J., 75, 12 (1960). 12. Eddy, A. A., Proc Roy. Soc. (London), B, 149, 425 (1958). 13. Salton, M. R. J., Biochim. et Biophys. Acta, 10, 512 (1953). 14. Salton, M. R. J. in The Bacteria, Vol. 1, Academic Press, New York, 1960, p. 97. 15. Murray, R. G. E., Can. J. Microbiol., 3, 531 (1957). 16. Chapman, G. B., and A. J. Kroll, /. Bacterial., 73, 63 (1957). 17. Kellenberger, E., and A. Ryter, /. Biophys. Biochem. CytoL, 4, 323 (1958). 54 MICROBIAL CELL WALLS 18. Smithies, W. R., N. E. Gibbons, and S. T. Bayley, Can. J. Microbiol., 1, 605 (1955). 19. Cummins, C. S., Intern. Rev. Cytol., Vol. V, Academic Press, New York, 1956, p. 25. 20. Work, E., Nature (London), 179, 841 (1957). 21. Weidel, W., and J. Primosigh, /. Gen. Microbiol, 18, 513 (1958). 22. Salton, M. R. J., /. Gen. Microbiol., 18, 481 (1958). 23. Cummins, C. S., and H. Harris, /. Gen. Microbiol, 14, 583 (1956). 24. Cummins, C. S., and H. Harris, /. Gen. Microbiol, 18, 173 (1958). 25. Snell, E. E., N. S. Radin, and M. Ikawa, /. Biol. Chem., 217, 803 (1955). 26. Ikawa, M., and E. E. Snell, Biochim. et Biophys. Acta, 19, 576 (1956). 27. Strange, R. E., Bacteriol Revs., 23, 1 (1959). 28. Baddiley, J., J. G. Buchanan, and B. Carss, Biochim. et Biophys. Acta, 27, 220 (1958). 29. Armstrong, J. J., J. Baddiley, J. G. Buchanan, B. Carss, and G. R. Greenberg, /. Chem. Soc, 4344 (1958). 30. Abrams, A., /. Biol. Chem., 230, 949 (1958). 31. Cummins, C. S., and H. Harris, Intern. Bull. Bact. Nomen. Tax, 6, 111 (1956). 32. Cummins, C. S., O. M. Glendenning, and H. Harris, Nature (Lon- don), 180, 337 (1957). 33. Perkins, H. R., and H. J. Rogers, Biochem. J., 72, 647 (1959). 34. Rogers, H. J., and H. R. Perkins, Nature (London), 184, 520 (1959). 35. Strominger, J. L., J. T. Park, and R. E. Thompson, /. Biol Chem., 234, 3263 (1959). 36. Hancock, R., Biochim. et Biophys. Acta, 37, 42 (1960). 37. Salton, M. R. J., and J. G. Pavlik, Biochim. et Biophys. Acta, 39, 398 (1960). 38. Park, J. T., /. Biol Chem., 194, 877, 885, 897 (1952). 39. Park, J. T., and J. L. Strominger, Science, 125, 99 (1957). 40. Abraham, E. P., BiochetJiistry of Some Peptide and Steroid Anti- biotics, Wiley, New York, 1957, p. 69. 41. Ikawa, M., and E. E. Snell, /. Biol. Chem., 235, 1376 (1960). 42. Salton, M. R. J., Nature (London), 180, 338 (1957). 43. Toennies, G., B. Bakay, and G. D. Shockman, /. Biol Chem., 234, 3269 (1959). 44. Park, J. T., Symposium Soc. Gen. Microbiol, 8, 49 (1958). 45. Hoare, D. S., and E. Work, Biochem. J., 65, 441 (1957). CHEMISTRY OF CELL WALLS 55 46. Ingram, V. M., and M. R. J. Salton, Biochim. et Biophys. Acta, 24, 9 (1957). 47. Sanger, F., Biochem. J., 39, 507 (1945). 48. Salton, M. R. J., Manuscript in preparation (to be submitted to Biochim. et Biophys. Acta). 49. Salton, M. R. J., VII th Intern. Congr. Microbiol., Abstracts, 1958, 114. 50. Akabori, S., K. Ohno, and K. Narita, Bull. Chem. Soc. Japan, 25, 214 (1952). 51. Salton, M. R. J., and J. M. Ghuysen, Biochim. et Biophys. Acta, 24, 160 (1957). 52. Ghuysen, J. M., Biochim. et Biophys. Acta, 40, 473 (1960). 53. Strange, R. E., and J. F. Powell, Biochem. J., 58, 80 (1954). 54. Cummins, C. S., and H. Harris, /. Gen. Microbiol., 13, iii (1955). 55. Strange, R. E., and F. A. Dark, Nature (London), 177, 186 (1956). 56. Salton, M. R. J., Biochim. et Biophys. Acta, 22, 495 (1956). 57. Park, J. T., and M. J. Johnson, /. Biol. Chem., 179, 585 (1949). 58. Strange, R. E., and L. H. Kent, Biochem. ]., 71, 333 (1959). 59. Zilliken, F., Polysaccharides in Biology, Josiah Macy, Jr. Founda- tion, 5th Conference, 1959. In the press. 60. Agren, C, and C-H. de Verdier, Acta Chem. Scand., 12, 1927 (1958). 61. SaUon, M. R. J., Biochim. et Biophys. Acta, 8, 510 (1952). 62. Holdsworth, E. S., Biochim. et Biophys. Acta, 9, 19 (1952). 63. Romano, A. H., and A. Sohler, /. BacterioL, 72, 865 (1956). 64. Sohler, A., A. H. Romano, and W. J. Nickerson, /• BacterioL, 75, 283 (1958). 65. McCarty, M., /. Exptl. Med., 96, 569 (1952). 66. Mitchell, P., and J. Moyle, /. Gen. Microbiol, 5, 981 (1951). 67. Baddiley, J., J. G. Buchanan, B. Carss, A. P. Mathias, and A. R. Sanderson, Biochem. J., 64, 599 (1956). 68. Armstrong, J. J., J. Baddiley, J. G. Buchanan, A. L. Davison, M. V. Kelemen, and F. C. Neuhaus, Nature (London), 184, 247 (1959). 69. McCarty, M., /. Exptl. Med., 109, 361 (1959). 70. Armstrong, J. J., J. Baddiley, and J. G. Buchanan, Nature (London), 184, 247 (1959). 71. Brumfitt, W., C. Wardlaw, and J. T. Park, Nature (London), 181, 1783 (1958). 72. Weidel, W., and J. Primosigh, Z. Naturforsch., 12b, 421 (1957). 73. Salton, M. R. J., Biochim. et Biophys. Acta, 45, 364. 56 MICROBIAL CELL WALLS 74. Westphal, O., O. Liideritz, I. Fromme, and N. Joseph, Angew. Chemie, 65, 555 (1953). 75. Davies, D. A. L., A. M. Staub, I. Fromme, O. Liideritz, and O. West- phal, Nature (London), 181, 822 (1958). 76. Weidel, W., Hoppe-Seyler's Z., 299, 253 (1955). 77. Maclennan, A. P., and D. A. L. Davies, Biochem. ]., 66, 562 (1957). 78. Dische, Z., /. Biol. Chem., 204, 983 (1953). 79. Shafa, F., and M. R. J. Salton, /. Gen. Microbiol., 23, 137 (1960). CHAPTER ENZYMIC DEGRADATION AND BIOSYNTHESIS OF MICROBIAL WALLS Enzymic Degradation From some of the unusual features of the chemical prop- erties of microbial cell walls outlined in Chapter 2 it is now easy to understand why they resist many of the proteolytic enzymes so active in degrading intracellular proteins and the various enzymes capable of breaking down lipids, poly- saccharides, and other cellular constituents. The resistance of bacterial cell walls to proteolytic enzymes is especially conspicuous, and even if it was purely fortuitous that the D-isomers of amino acids were formed into wall peptides it seems eminently sensible that they should be there. Al- though many microbial walls are unattacked by enzymes degrading the intracellular structures and constituents, they can, as already pointed out, be attacked by enzymes pro- duced by the cells themselves and by a variety of enzymes from other microorganisms and from other cells and tissues. >\/gae. Fungi, Yeasts. The gut of the snail provides a collection of enzymes that have been used in degrading the 57 58 MICROBIAL CELL WALLS wall structures of the alga Chlorella pyrenoidosa,^ Neuro- spora crassa/ and yeast.^ Cellulases and chitinases in the snail-gut enzymes are generally believed to be active in cell- wall digestion, but most investigators have used unfrac- tionated preparations undoubtedly rich in a variety of enzymes. Indeed, Myers and Northcote ^ reported that the snail enzyme preparations contained active lipases and car- bohydrases, including cellulase, xylanase, and mannanase. Only weak proteolytic activity was found in their extracts. Several microorganisms isolated on selective media contain- ing yeast cell walls have been found to produce enzymes digesting the yeast wall structures. 5' <^ The enzymes used for degrading the walls of Chlorella, Neurospora, and yeasts have not resulted in complete digestion. The most effec- tive enzyme so far reported is that prepared from the bac- terium isolated by Masschelein.^ He observed a decrease of 84% in the turbidity of isolated yeast walls incubated with the enzyme preparations. ^ Nor has the nature of the products released by enzymic degradation of these microbial walls been determined in any detail. Northcote, Goulding, and Home ^ reported the release of 70% of the total a-cel- lulose and 43% of the lipid of the wall of Chlorella pyre- noidosa. A mannan-protein complex was released from yeast walls treated with papain. ^ Thus at present there is no indication of the nature of the linkages attacked by the various enzymes used in degrading the walls of these micro- organisms. Baciena. Lysis of bacterial cells and breakdown of the wall has received a great deal of attention, and a variety of enzyme systems is available for various bacterial species. ^-^° Of all the wall-degrading enzymes so far investigated, more is now known about the mode of action of egg-white lyso- zyme than any other system. ^^ The only other enzymes obtained in a purified form and well characterized are the ENZYMIC DEGRADATION AND BIOSYNTHESIS 59 Streptomyces enzymes studied by Ghuysen.^ One of these enzymes (Streptomyces F^) is essentially an N-acetyl-hexo- saminidase ^^ and is therefore similar to egg-white lysozyme; another enzyme (Streptomyces Fsb), an amidase/^ liberates the peptide moiety from low molecular weight mucopep- tides obtained from walls by lysozyme action. ^^ Lysozyme. Although it has been known that lysozyme action on the isolated soluble substrates (usually obtained by chemical fractionation of whole cells) involved the rup- ture of glycosidic bonds with a liberation of N-acetylamino sugar compounds/^' ^*^'^' direct evidence establishing the nature of the linkages broken has become available only in recent years. i-' ^^ The investigation of the nature of the action of lysozyme became simplified when isolated cell walls could be used as "substrate." ^^ Using the isolated cell walls of several sensitive organisms, Salton ^o investi- gated the nature of the products formed on digestion with lysozyme. A complex mixture of fragments resulted, and these fragments were separated into the larger, nondialyz- able compounds of about 10,000 to 20,000 molecular weight. These compounds possessed terminal groups of N-acetyl- amino sugars and contained all of the constituents present in the original wall (but probably in different propor- tions -°). About half the original wall of Micrococcus lyso- deikticus became diffusible upon dissolution with lysozyme. The nature of the diffusible products was investigated, and the most conspicuous "small fragment" detectable was a compound containing glucosamine and muramic acid, prob- ably in the form of a disaccharide.^o This substance was detected in digests of all three cell walls studied, those of Bacillus megaterium, Micrococcus lysodeikticus, and Sar- cina lutea. Evidence suggested that both amino groups of the amino sugars were acetylated and that the disaccharide possessed a free carboxyl group— that of muramic acid. It 60 MICROBIAL CELL WALLS was suggested that the "disaccharide" formed an important structural unit of the cell-wall mucocomplex.-° Additional products reacting more weakly with various spray reagents were detectable in the dialyzable fractions, but their nature remained unknown until their recent isolation and charac- terization.^^-^ In our investigation in 1959 we were able to confirm the nature of the disaccharide and suggest the structure of this compound. The isolated disaccharides from Micrococcus lysodeikticus walls digested with egg-white lysozyme and Streptomyces Fj enzyme were investigated by reaction of the compounds with NaBH4 and by degradation with /?-gluco- sidase. The products of reaction of the compounds with NaBH4 clearly established the identity of the reducing group liberated by lysozyme action as that of muramic acid, thus providing direct experimental evidence for the hypo- thetical structure of the lysozyme substrate proposed by Brumfitt, Wardlaw, and Park. 22 The breakdown of the disaccharides into the free N-acetylamino sugars, N-acetyl- glucosamine and N-acetylmuramic acid, provided evidence of the ^-glycosidic bond. The structure of the disaccharide and the nature of the products formed from reaction with NaBH4 and yg-glucosidase are presented in Fig. 12. The presence of a 1-^6 linkage was suggested from experi- ments performed on [^^C] disaccharide oxidized with NaI04 and determining the recovery of [^*C] formaldehyde.^^, 23 In addition to the disaccharide, an oligosaccharide yield- ing glucosamine and muramic acid on hydrolysis was de- tected, and its structure investigated, by the techniques used in studying the disaccharide. That the compound was a tetrasaccharide was supported by measuring the ratios of glucosamine, muramic acid, and "muramicitol" (the amino sugar hexitol of muramic acid ^2,23^ separated after hydrol- ysis of the substance reduced with NaBH4. Both lysozyme ENZYMIC DEGRADATION AND BIOSYNTHESIS 61 >~^ CO q; c« z CCL-C O O CO 3 bO es CtL X! o> c: iZ rt 62 MICROBIAL CELL WALLS and Streptomyces F^ enzyme yielded disaccharide from the tetrasaccharide, although the activity of the latter enzyme was much weaker than that of lysozyme.^^ The two en- zymes, moreover, are capable of degrading mono- and di- chitibiose (i.e. the di- and tetrasaccharides of N-acetyl- glucosamine), thus clearly showing that they possess /?(!—> 4) N-acetylglucosaminidase activity.^--" This also confirmed the earlier conclusion by Berger and Weiser ^^ that the limited action of lysozyme on purified chitin indi- cated its /5-glucosaminidase properties. The experimental evidence is therefore in accordance with the structure of the tetrasaccharide being a f3{l — > 4) dimer of the disaccharide, as shown in Fig. 13. Independent confirmation that the disaccharide is the simplest product of lysozyme action on its substrate in the cell-wall mucopeptide of Micrococcus lysodeikticus has come from the investigations of Perkins,-*' ^^ and the struc- ture suggested is identical to that proposed by Salton and Ghuysen.^- A disaccharide of N-acetylglucosamine and N-acetylmuramic acid has also been detected in partial acid hydrolysates of walls of Micrococcus lysodeikticus.-'^'-^ The nature of the fragments obtained on digestion of walls with lysozyme has provided us with an idea of the structure of the cell-wall mucopeptide. Thus the wall CH^CHCOOH CH2OH H HO/^ r H H >— — 0' H NHCOCH3 O' H NHCOCH3 CH2OH CH3CHCOOH CKr Fig. 13. Proposed structure of the tetrasaccharide enzymically released from Micrococcus lysodeikticus walls.12, 23 ENZYMIC DEGRADATION AND BIOSYNTHESIS 63 Backbone structure (1-^6) (1-^4) (1-^6) (1^4) (1^6) (1-^4) (1^6) AG AMA AG AMA AG AMA AG AMA- Peptide Lysozyme Peptide Sensitive bonds Fig. 14. Backbone structure proposed for Micrococcus lysodeikticus wall.23 probably possesses a backbone structure of alternating groups of N-acetylmuramic acid and N-acetylglucosamine with alternating ^(1^4) and /?(1 ^ 6) linkages. Some of the muramic acid residues would have peptide substitu- ents, and the possible structure of the wall and the distribu- tion of lysozyme sensitive bonds is shown in Fig. 14. The isolation of a small molecular weight mucopeptide in the dialyzable fraction of lysozyme-digested walls of Mi- crococcus lysodeikticus ^^ has clearly shown that lysozyme can degrade the backbone down to a disaccharide residue possessing a peptide linked through muramic acid, as in the structure in Fig. 15, which shows in addition the linkage sensitive to the Streptomyces amidase.^^ Although it is now possible to understand the manner in which lysozyme degrades the bacterial walls, yielding a variety of products, some containing all of the parent amino acids and amino sugars in the same molar proportions as in the intact cell wall (e.g. the diffusible mucopeptide) as well as the di- and tetrasaccharides, many of the general prob- lems of understanding lysozyme sensitivity remain to be solved. O-acetyl substituents have been shown to be im- portant in governing this sensitivity in mutant strains of 64 MICROBIAL CELL WALLS HO CIIoOH CH. H NHCOCH. O H NHCOCH3 / CH3CHCONH (ala — glu — lys — gly) V 2 111 Streptomyces amidase Fig. 15. Structure of mucopeptide in lysozyme digest and bond sensi- tive to streptomyces amidase. is Micrococcus lysodeikticiis/- but in other walls O-esters can- not account for the greater resistance of the walls to diges- tion with lysozyme. The possibility of different linkages between amino sugars of the backbone has been suggested. ^^ Resistance to lysozyme could also be explained by differences in the ratios of amino sugars, relatively few disaccharide units, branching points, single amino acid substituents at- tached to muramic acid, and a high frequency of cross- linked peptides between muramic acid residues (two types of structures discussed in Chapter 2). There are many in- triguing possibilities, and it will be of great interest to find out the factors responsible for the resistance of the walls of an organism such as Bacillus cereus, which contains such a large amount of amino sugar in the wall (30%). ^^ Biosynthesis of Microbial Walls The biosynthesis of microbial walls is now beginning to attract much attention, and within the brief space of the last couple of years a great deal has been learned. The discovery of the accumulation of uridine nucleotides in ENZYMIC DEGRADATION AND BIOSYNTHESIS 65 penicillin-treated Staphylococcus aureus by Park and John- son -^ and Park -^ and the subsequent recognition of the biochemical significance of these compounds by Park and Strominger ^o stimulated a great deal of interest in the mode of action of penicillin and the mechanism of biosynthesis of bacterial cell walls. Much of the work on wall biosyn- thesis has thus been confined to recognizing wall inter- mediates accumulating in the presence of various antibiotic inhibitors and has been performed mainly with bacterial cells. Yeasts and Fungi. So far as I am aware, there have been no direct studies of the biosynthesis of walls of yeasts or fungi. However, it is well known that possible intermedi- ates in the form of nucleotide anhydrides occur in yeasts. Uridine diphospho-(UDP)-glucose,3i UDP-acetylglucosa- mine,^- and guanosine diphospho-(GDP)-mannose ^^ could all be regarded as potential wall intermediates, since the monosaccharide moieties of all three nucleotides occur in the walls of yeasts. Although there have been no direct observations involving a transfer of the sugar moieties of these nucleotides into wall compounds, it is conceivable that they may well follow the known transglycosylation re- actions ^^-^^ established for uridine nucleotides and follow- ing the general type of reaction given below: UDP-X + ROH — UDP -f RO-X Glaser and Brown ^e have investigated the biosynthesis of chitin by extracts of Neurospora crassa, which is known to contain poly-N-acetylglucosamine in the mycelia.^^ En- zyme preparations catalyzed the synthesis of chitin by the following reaction: UDP-acetylglucosamine + (chitodextrin)^ ^ UDP + (chitodextrin)„+i 66 MICROBIAL CELL WALLS Chitin in an insoluble form was thus synthesized from UDP- acetylglucosamine, soluble chitodextrin, and an activator.^s Higher molecular weight chitodextrin was the most effec- tive primer. The enzyme system was in a particulate form. Bacterial Cell Walls. Three main lines of investigation have been pursued in studies of the biosynthesis of bacte- rial walls. They include (1) biosynthesis of individual wall compounds, (2) synthesis of mucopeptide and incorporation of radioactive compounds into walls, and (3) isolation and characterization of possible intermediates accumulating dur- ing inhibition of wall synthesis. Biosynthesis of Muramic Acid. Attempts to elucidate the origin of the O-lactyl side chain of muramic acid were made by Strominger.^* He discovered that Staphylococcus aureus contained an enzyme catalyzing the transfer of pyru- vate from 2-phosphoenolpyruvate (PEP) to UDP-acetylglu- cosamine by the following reaction: UDP-AG -f PEP -^ UDP-AG-pyruvate + Pi Although the rate of the reaction in this enzyme system was about one fifth of the rate of UDP-acetylglucosamine formation, Strominger ^s has suggested that in 10 minutes at 37° the enzyme could synthesize sufficient substituted N-acetylglucosamine required for the wall of Staphylococ- cus aureus, thus achieving this feat well within the mean generation time of the organism. Strominger and Scott ^^ have also detected a small en- zymic conversion of UDP-acetylglucosamine-[i-^C]-pyruvate to UDP-acetylglucosamine-[i^C]-lactic acid by extracts of Staphylococcus aureus. The net reaction, however, was small, and the mechanism of synthesis of muramic acid and its uridine nucleotides still remains to be established. ENZYMIC DEGRADATION AND BIOSYNTHESIS 67 That the 3-O-carboxyethyl residue of muramic acid is derived from pyruvate was further substantiated in recent experiments performed by Richmond and Perkins ^^ with intact cells of Staphylococcus aureus, incubated under con- ditions favoring only cell-wall synthesis.*^ A cell wall syn- thesized with [^^C] glucose in the absence of alanine showed similar specific activities per microgram of carbon for both glucosamine and muramic acid. However, when the wall was synthesized in the presence of alanine, the muramic acid formed had the specific activity of the side chain re- duced by 75%. Generally labeled [i^C] alanine and [i^C] aspartic acid could act as precursors of the muramic acid side chain. The two noncarboxyl atoms of the side chain of muramic acid yield acetaldehyde when heated at 100° with 86% sulfuric acid for 17 minutes (Strange and Kent ^~), whereas the carboxyl group yields carbon monoxide. These facts enabled Richmond and Perkins ^^ to conclude from experiments with [^^C] alanine that all three carbon atoms of the alanine can act as precursors of the muramic acid side chain without inversion and that these results are con- sistent with the idea that phosphoenolpyruvate is the im- mediate precursor. Synthesis of Wall Mucopeptides and Incorporation of Radioactive Compounds into Walls. The synthesis of cell- wall mucopeptides by washed suspensions of two different strains of Staphylococcus aureus in defined incubation mix- tures was independently reported by Mandelstam and Rog- ej-s4i,43 and by Hancock and Park.*^ The increase in wall mucopeptide content of Staphylococcus aureus incubated in buffers containing glucose and various combinations of the amino acids known to occur in the wall varied from 20 to 150% in one hour at 37°. The results of the experi- ments performed by Mandelstam and Rogers ^^ are pre- 68 MICROBIAL CELL WALLS sented in Table 21. Little mucopeptide synthesis occurred in the presence of glucose alone, but a net increase of about 60% took place when glycine or ammonium chloride was added to the glucose. Thus Mandelstam and Rogers *^ showed that it was possible to study the synthesis of wall mucopeptides dissociated to a large measure from protein synthesis. Hancock and Park ** studied the incorporation of p^q amino acids into cell-wall and protein fractions of Staphylo- coccus aureus in the presence and absence of chlorampheni- col. They showed that the incorporation of typical cell- wall amino acids, such as lysine, glycine, alanine, and glu- tamic acid, into the wall was inhibited only to the extent of 4 to 8% by chloramphenicol when the cells were transferred TABLE 21 Conditions for the Synthesis of Mucopeptide Washed staphylococci incubated 1 hour in buffer containing 1% glucose and one or more amino acids at a final concentration of 400 Mg/ml. Bacteria disintegrated and mucopeptide isolated. Increase in Additions Mucopeptide % None 0-10 DL-Lysine 20 DL-Glutamic acid 25 DL-Alanine 20 Glycine 55-80 Glycine -f- DL-glutamic acid 60 Glycine + DL-lysine 80 Glycine -f DL-lysine -f DL-glutamic acid -|- dl alanine 100-150 Reference 41. ENZYMIC DEGRADATION AND BIOSYNTHESIS 69 to a synthetic growth medium containing the radioactive amino acids. On the other hand, the inhibition of the in- corporation into protein of the "wall" amino acids and leucine, proline, and phenylalanine was as much as 85 to 98%. In agreement with the investigations reported by Mandelstam and Rogers,*^ Hancock and Park ** were also able to demonstrate a doubling of the amount of wall (meas- ured by incorporation of [^^C] lysine and glycine) in a simple incubation mixture containing lysine, glycine, alanine, glu- tamic acid, glucose, and uracil but lacking in some of the amino acids essential for protein synthesis. Under these conditions chloramphenicol had no effect on wall synthesis. An examination of the wall formed in the presence of chlo- ramphenicol suggested that it was normal in that the ratios of increase in glutamic acid, glycine, alanine, lysine, and hexosamine (1:5.8:4.2:2.0:1.8) were similar to those pres- ent in the initial wall (1:6.8:2.8:1.9:1.8). The synthesis of the cell-wall mucopeptide was markedly inhibited by penicillin and bacitracin, neither of which in- hibited protein synthesis.*^ However, a small amount of mucopeptide is synthesized in the presence of penicillin, and Mandelstam and Rogers *^ found some evidence sug- gesting that it possessed an abnormal composition. Nathen- son and Strominger *^ also studied the inhibitory effect of penicillin on the incorporation of [^^C] lysine and P^P] into wall and cellular protein and nucleic acid of Staphylo- coccus aureus and the incorporation of [^H] diaminopimelic acid and [^^C] glucose into the wall of Escherichia coli. The results presented in Table 22 are again in accord with those of other investigators, showing a marked inhibition of amino acid incorporation into the cell wall but allowing both protein and nucleic acid synthesis to proceed in the presence of penicillin. The inhibition by penicillin of p*C] glucose incorporation into the whole cell wall of Escherichia 70 MICROBIAL CELL WALLS TABLE 22 Effects of Penicillin on Incorporation of Isotopes into Cell Wall or into Cell Protein and Nucleic Acid in Staphylococcus aureus and in Escherichia coli Staphyloccus aureus Escherichia coli Cl4-Lysine P32.inorganic phosphate H3-DAP C14- Glucose Isotope Cell- Pro- Wall tein Cell- Nucleic Wall Acid Cell- Wall Cell- Wall Control + Penicillin % Inhibition 34,800 5,100 3,290 4,960 91% 2% 155,000 11,600 48,900 11,600 68% 0 1,040,000 297,000 790/ 389,000 334,000 14% Data are expressed as specific activities (cpm/mg). Reference 45. coli is not marked, and indeed Trucco and Pardee ^^ in earlier experiments had concluded that penicillin did not interfere with the synthesis of the wall of this organism. As Escherichia coli wall possesses major protein, lipid, and polysaccharide components, it seems likely that inhibition of incorporation of the compounds into the mucopeptide fraction of the wall of this Gram-negative organism may have been more spectacular. Inhibition of mucopeptide synthesis by penicillin has been clearly established by at least three groups of investigators for Staphylococcus aureus. The results of interference with wall synthesis in Escherichia coli are conflicting, and Meadow ^^ has recently found that a DAP-requiring mutant of Escherichia coli is unchanged in its ability to incorporate [^^C] glucose [^^C] lysine and [i*C] diaminopimelic acid into wall during the first 30 ENZYMIC DEGRADATION AND BIOSYNTHESIS 71 minutes of exposure to penicillin. To what extent this lack of agreement is a reflection of strain differences has not been determined. It is of interest to note that the partially disrupted cells of Staphylococcus aureus investigated by Gale and Folkes *^ will incorporate a high proportion of the total [^^C] amino acid uptake of an amino acid such as glycine into the tri- chloroacetic acid insoluble, cell-wall fraction (Gale, Shep- herd, and Folkes *^). Whether the mucopeptide was in the form of finished wall or simply TCA-precipitable material was not established. It would be of interest to know whether the disrupted cells retain an intimate contact be- tween the acceptor cell wall and the sites of new mucopep- tide synthesis. In attempting to localize the sites of syn- thesis of mucopeptides, Brookes, Crathorn, and Hunter ^° have investigated the time course of the uptake of [^^C] amino acids (L-alanine, diaminopimelic acid, and L-aspartic acid) into the wall, membrane, and protoplasmic fractions of Bacillus megaterium. They concluded from their results that the mucopeptide components are synthesized at sites on or closely associated with the cytoplasmic membrane. Incorporation of [1-^*C] a, ^-methyl-N-acetyl-D-glucos- aminide into Cell Walls. The organism Lactobacillus bi- fidus var. pennsylvanicus has a specific growth requirement for N-acetyl-D-glucosamine, preferably in the form of ^-gly- cosides.^^ That this requirement was associated with wall- synthesizing systems has been shown by Zilliken's experi- ments ^--^^ in which the proportion of morphologically bi- zarre and bifid forms decreases with increasing amounts of the glucosaminide growth factors; [l-^^C] /3-methyl-N-acetyl- D-glucosaminide is incorporated into the cell walls of Lacto- bacillus bifidus, the specific activity in the muramic acid being 19,000 cpm/mole compared to 21,000 cpm /mole for the starting material. Zilliken ^2, 53 concluded that N-acetyl- 72 MICROBIAL CELL WALLS D-glucosamine is a direct precursor of muramic acid and that the latter compound is indeed a D-ghicosamine deriva- tive. Incorporation of [^^C] Lysine, [^^C] Diaminopimelic Acid, and [^^C] Glucose info Cell-Wall Lysine and DAP. Meadow and Work ^* investigated the incorporation of radioactive compounds into wall fractions of Escherichia coli mutants requiring either lysine or DAP or both amino acids for growth. All of the mutants tested took up [i^C] lysine, and of the radioactivity incorporated 50 to 60% was accounted for in the cell walls. DAP was not labeled. Ten per cent of the cell-wall lysine of the DAP-requiring mutants was derived from that supplied exogenously; [^^C] diaminopi- melic acid was incorporated into both DAP and lysine of the DAP-requiring mutants and the parent strain. The DAP-dependent, lysine-stimulated mutant 173-25 derived 80% of the cell-wall lysine from the exogenous DAP, whereas the corresponding value for the DAP-dependent mutant was 50 to 60%. An alternative route to lysine from glucose was apparent from mutants 173-25 and DAP-de- pendent grown on [^^C] glucose. Labeling of lysine oc- curred, but DAP was unlabeled. Some 10 to 20% of the cell-wall lysine was derived from glucose in these mutants. Accumulation and Identification of Cell-Wall Intermedi- ates. The identification of cell-wall intermediates really commenced with the discovery of the accumulation in Staphylococcus aureus of uridine nucleotides in the presence of penicillin. 28. 29 The significance of these nucleotides as possible cell-wall precursors became apparent when the amino sugar was found to be identical to muramic acid and the complete structure for one of the nucleotides was established, as in Fig. 16.^^ This finding was just pre- ceded by Lederberg's ^^' ^^ suggestion that penicillin acted ENZYMIC DEGRADATION AND BIOSYNTHESIS 73 HC- OH I ■C — I I 1 H 0=C^ CH OH I -C — C— CH9O- I I H H COCHo I ' ^r^/ CH NH I C— C— H H L-ala OH I -C— C H H -CH, -CHoOH OH D-glu I L-lys I D-ala I D-ala COO" Fig. 16. Structure of the uridine nucleotide from penicillin-inhibited Staphylococcus aureus.^o on bacteria (including the Gram-negative Escherichia coli) by blocking wall formation. Thus the biochemical and structural evidence for the inhibition of wall formation by penicillin emerged and has been largely confirmed in many subsequent studies. The anatomical lesion caused by peni- cillin inhibition of wall formation is beautifully illustrated in the thin sections of Staphylococcus aureus shown in Fig. 17a and b, taken from the studies of Murray, Francombe, and Mayall.^^ The consequences of inhibition of the for- mation of the mucopeptide part of the wall of Gram-nega- tive bacteria has become apparent from a number of inves- tigations.^^^^^^ However, the Gram-negative bacteria have major wall constituents unaffected by penicillin action, and the familiar "poached-egg" appearance of "protoplasts" of Vibrio metchnikovi formed in the presence of penicillin is shown in Fig. 17c. 74 MICROBIAL CELL WALLS ENZYMIC DEGRADATION AND BIOSYNTHESIS 75 Fig. 17. Effects of penicillin on cell-wall structure, (a) Thin section of untreated cells of Staphylococcus aureus (x 36,000); (b) effects of ex- posing Staphylococcus aureus to penicillin for three hours (x41,500). From the study of Murray, Francombe, and Mayall (Ref. 57). (c) Vibrio metchnikovi "protoplasts" prepared by growth in the presence of penicillin. The weakened wall from the right-hand "protoplast" became detached during preparation for electron microscopy (x 12,500). M. R. J. Salton, unpublished. Since the early investigations of Park and Johnson -^ and Park,29 a number of uridine nucleotides containing typical wall components has been isolated from untreated cells as well as from organisms whose giowth has been inhibited by antibiotics or deprivation of specific amino acids. Baddiley et al.*^^ isolated cytidine diphosphoribitol and cytidine di- 76 MICROBIAL CELL WALLS phosphoglycerol from normal cells of Lactobacillus arabi- nosus, and the search for the biochemical functions of these nucleotides led them to the discovery of the cell-wall teichoic acids. Cell-wall nucleotide intermediates have been found in both Gram-positive and Gram-negative bacteria, and some of the nucleotides so far identified are listed in Table 23. Nucleotide accumulation also occurs with the antibiotics bacitracin ^^ and novobiocin,*^^ and recent studies by two groups of workers have shown that 5-fluorouracil induces nucleotides to accumulate. '^-^^ xhe precise manner in which penicillin brings about the accumulation of the vari- TABLE 23 Nucleotides Identified as Probable Cell-Wall Intermediates in Various Bacteria Organism Inhibitor Nucleotide Staphylococcus aureus Escherichia coli (DAP-depend- ent mutant) Streptococcus (Group A) Lactobacillus arabinosus Penicillin UDP-AG-lact-ala-glu-lys-ala-ala UDP-AG-lact-ala UDP-AG-lact * UDP-AG-lact-ala-glu-lys jUDP-AG-lact-ala-glu I UDP-AG-lact-ala Gentian violet CDP-ribitol None UDP-AG-Iact-ala-glu-DAP-ala-ala DAP-depriva- UDP-AG-Iact-ala-glu tion None UDP-AG-lact Oxamycin Lysine-dep- rivation None CDP-ribitol * UDP-AG-lact = uridine diphospho-N-acetylmuramic acid. References 28-30, 35, 61-70. ENZYMIC DEGRADATION AND BIOSYNTHESIS 77 ous nucleotides is not known. At least the probable mecha- nism of the antibiotic action of oxamycin (o-cycloserine) has been amenable to study almost at the level of a single enzyme system. Direct evidence for the inhibition of wall synthesis came from the studies of Shockman '* using Strep- tococcus faecalis and from Strominger, Threnn, and Scott ^^ with Staphylococcus aureus. Both investigations led to the conclusion that oxamycin was acting as a competitive an- tagonist of the incorporation of D-alanine into wall. Strom- inger 3^ has pointed out the close structural relationship of oxamycin to D-alanine as shown below: H H H H II II H— C C— NH2 H— C C— NHo II II 0. .C=0 H .C=0 Oxamycin D-alanine Nucleotides isolated from oxamycin-inhibited cells have given some further evidence of the sequence of the build- ing up of the wall peptide, and Strominger, Threnn, and Scott 63 have shown (Table 24) that the nucleotide accumu- lation induced by oxamycin can be antagonized by D-ala- nine. Strominger ^^ has thus suggested that oxamycin in- hibits the enzymic reaction involved in the addition of D-alanine to the nucleotide UDP-AG-lact-ala-glu-lys. It is curious that none of the cell-wall intermediates so far isolated from Staphylococcus aureus contains either gly- cine or N-acetylglucosamine, the other two major cell-wall constituents. It may well be that the peptides attached to the nucleotides isolated up to the present time are far from complete, despite the remarkable similarity in their amino 78 MICROBIAL CELL WALLS TABLE 24 Antagonism by D-alanine of Uridine Nucleotide Accumulation Induced by Oxamycin Antagonist Added Experiment 1 Experiment 2 None 41.4 30.0 D-alanine (500 /ug/ml) 17.0 12.1 D-alanine (5000 /ag/ml) 4.5 6.9 L-alanine (5000 fig/ml) 41.5 32.2 DL-alanyl-DL-alanine (5000 /xg/ml) 33.5 D-serine (5000 /xg/ml) 34.2 In Experiment 1 oxamycin (75 ^g/nil) and possible antagonists were added together at 0 time. In Experiment 2 oxamycin (75 /Ag/ml) was added at 0 time. At 45 minutes, 20.4 ^M of nucleotide had accumulated. At this time possible antagonists were added and incubation was continued for 45 minutes longer. Data are expressed as /xmoles of uridine nucleotide per liter of culture at half-maximal growth. Reference 63. acid composition to that of the cell-wall mucopeptide ^^ and even very close agreement in the proportions of the d- and L-alanine,*^^ as shown in Table 25. Of course, an alternative explanation of the presence of glycine in a "special" peptide or structure would equally well explain its absence from the nucleotides. Enzyrjiic Synthesis of Wall-Precursor Nucleotides. Con- ditions leading to the formation of a uridine nucleotide have been investigated for only one of the cell-wall inter- mediates. Ito and Strominger "'^ have found that an en- zyme from Staphylococcus aureus purified about 500-fold, will catalyze the formation of UDP-AG-lact-ala-glu-lys under the conditions summarized in Table 26. ENZYMIC DEGRADATION AND BIOSYNTHESIS 79 TABLE 25 Optical Configuration of Alanine Samples Obtained from a Uridine Nucleotide and from the Cell Wall of Staphylococcus aureus Alanine Samples % L-alanine % D-alanine 1. UDP-AG-lact-peptide from novo- biocin-inhibited Staphylococcus aureus (strain Copenhagen) 32.1 68.0 2. UDP-AG-lact-peptide from peni- cillin-inhibited Staphylococcus aureus (strain H) 33.7 65.6 3. Cell wall, prep. 1 27.3 65.4 4. Cell wall, prep. 2 33.4 66.2 5. Cell wall, prep. 3 34.0 65.7 Reference 65. TABLE 26 Requirements for the Enzymatic Synthesis of UDP-GNAc-Lactyl-(L)Ala-(D)Glu-(L)Lys Experiment 1: Experiment 2: System Cpm Nucleotide Added Cpm Complete 2620 UDP-GNAc-lactyl-ala-glu 5080 -UDP-GNAc-lactyl-ala-glu 0 UDP-GNAc-lactyl-ala 0 -ATP 0 UDP-GNAc-lactyl-ala-glu-lys 6 -Mg+ + 0 UDP-GNAc-lactyl-ala-glu-lys ala-ala 0 In Experiment 1 various components were omitted from the complete incubation mixture, which contained buffer, MgClg, Ci4-lysine, ATP, UDP-GNAc-lactyl-(L)ala-(D)glu, and purified en- zyme. In Experiment 2 other uridine nucleotides were substi- tuted for UDP-GNAc-lactyl-(L)ala-(D)glu. Data are expressed as cpm of Ci4-lysine incorporated into nucleotide, measured as char- coal-adsorbable radioactivity. Reference 76. 80 MICROBIAL CELL WALLS Pathways for Cell-Wall Biosynthesis The evidence that the uridine and cytidine nucleotides containing a number of typical cell-wall compounds are indeed cell-wall intermediates is most convincing when their compositions are compared with cell walls. Yet the hard fact remains that convincing incorporation or transfer of the muramic-acid-peptide moiety of the nucleotide to the wall has not been demonstrated. This, of course, has prompted the sceptics to say "I told you so!" However, the problem of getting such a nucleotide through the existing wall into the right part of the membrane and close to ac- ceptor sites on the wall must be a tremendous one. Stromin- ger has also clearly pointed out that very low levels of transfer w^ould not be surprising if one attempted to assess the probable number of acceptor sites on the wall. "If it is assumed that intact organisms contain of the order of 1000 acceptor sites per cell, then all the cell walls obtained from a liter of culture containing 10^ cell per milliliter would contain only lO^^ acceptor sites or 0.01 ^^M of acceptor per liter of culture." ^^ This problem of experimental demon- stration of the transfer of the obvious intermediates into wall is a difficult one and indeed seems to be general to the whole problem of the synthesis of large polymers including cellulose."^^ However, some ideas of the mechanisms of wall biosynthesis are emerging from pioneer work of Strominger and his colleagues. The biochemical unity of life so admirably discussed by the late Kluyver and Van Niel in their Prather Lectures ^^ prompts me to be optimistic and believe that some of the pathways for wall synthesis recently suggested by Stromin- ger 35 will become established as general mechanisms for the synthesis of these most interesting heteropolymers. These schemes, as Park ^^ has also pointed out, involve a transfer ENZYMIC DEGRADATION AND BIOSYNTHESIS 81 mechanism of transglycosylation, commonly encountered in the biosynthesis of many compounds, including the struc- tural polymer chitin. Thus the basis of these reactions in- volving uridine and cytidine nucleotides (the only two classes so far implicated in bacterial wall synthesis) would be analogous to that found for chitin synthesis by Glaser and Brown.^^ Nucleotide — wall component -|- acceptor (wall) -» acceptor — wall component -|- nucleotide diphosphate Two major pathways for biosynthesis of part of the bac- terial wall have been suggested by Strominger ^s- 65 and are presented in Figs. 18 and 19. For those organisms possess- ing an amino sugar backbone, a further part of the biosyn- thetic scheme can be suggested, since some of the possible intermediates are already known (UDP-AG-lact, UDP-AG, muramic acid-6 phosphate ^^). This hypothetical pathway illustrated in Fig. 20 could be envisaged as being integrated with the other pathways (Figs. 18, 19), thereby adding the muramic acid-peptide residue to an amino sugar backbone already built on to the cell-wall acceptor. Epideictic In the period of the last ten years a new class of structural heteropolymers has been discovered in bacterial cell walls and in at least some of the related blue-green algae. We are just beginning to understand some of the properties and structures of these mucopeptide and mucopolysaccharide substances, and some exciting details of the biosynthesis of the major structural component of microbial cells are be- ginning to emerge. It is perhaps fortunate for mankind that nature saw fit to encase bacteria in a wall containing amino sugar and amino acid structures not normally en- 82 MICROBIAL CELL WALLS r2 ^ 13 1 cc q" o s _>. c o .i; Sh 1 CO >> o 1 ^— s c 3 Jh 3 ►J 'S -a 'bJD 1 Oi >> li 'G 1 1 s s ^ I x^ri o/ _2> X 1 J o < < < z 2 Z o o o Ph Ph fe Q Q Q ::) ;^ ;d o I Ph Q ^ 3 q; « u t^ 2 s; O o o ri O ?N OJ -Gh ^ « ■5 S c o U 4; < •? C . o 00 i^ "" on ENZYMIC DEGRADATION AND BIOSYNTHESIS 83 / <: / z / o / Pu / Q ^ A 1 1 \ PL, c a c ■" be