Ultrastructural study of the reversion of protoplasts of Bacillus licheniformis to bacilli.

The reversion of protoplasts of Bacillus licheniformis 6346 His- on a medium containing 2.5% agar has been studied in sectioned material after reaction with a ferritin-conjugated antibody specific to the peptidoglycan isolated from the walls of the bacilli. Freeze etching has also been used. Fibrils of material reacting with the antibody have been detected emerging from isolated areas of the protoplasts after 3 h of incubation. This material gradually covers the cell and can eventually (at 6 h) be seen in freeze-etched preparations as a fringe of up to 400 nm around the cells and covering the surfaces with particles that can be removed by lysozyme. At later stages the wall begins to take on a compact, well-defined appearance that can be seen in sections; however, the cells are still grossly deformed. A transitory emergence, beyond the wall of long fibers of 6 nm in diameter, takes place after about 12 h of incubation. These fibers react with the conjugated antibody and after freeze etching show a regular banded structure. They are probably indentical with the fibers isolated elsewhere (Elliott et al., 1975) and shown to contain all the wall constituents (i.e., peptidoglycan, teichoic acid, and teichuronic acid). These fibers are not detectable in the final stages of reversion.     

Inhibitory protein controls the reversion of protoplasts and L forms of Bacillus subtilis to the walled state.

When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony. L bodies from such L colonies again plate as L-colony-forming units (CFU). However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 0.4% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies. The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B. subtilis. It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml. This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent. Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium. In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin. Trypsin also stimulates reversion in L colonies growing on soft agar. Latent RIF is activated by beta-mercaptoethanol. This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml. Comparison of the autolytic behavior of B. subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall. Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.     

Characterization of a novel linkage unit between ribitol teichoic acid and peptidoglycan in Listeria monocytogenes cell walls

The structure of the linkage unit between ribitol teichoic acid and peptidoglycan in the cell walls of Listeria monocytogenes EGD was studied. A teichoic-acid--glycopeptide preparation isolated from lysozyme digests of the cell walls of this strain contained mannosamine, glycerol, glucose and muramic acid 6-phosphate in an approximate molar ratio of 1:1:2:1, together with large amounts of glucosamine and other components of teichoic acid and glycopeptides. A teichoic-acid-linked sugar preparation, obtained by heating the cell walls at pH 2.5, also contained glucosamine, mannosamine, glycerol and glucose in an approximate molar ratio of 25:1:1:2. Part of the glucosamine residues were shown to be involved in the linkage unit. Thus, on mild alkaline hydrolysis, the teichoic-acid-linked sugar preparation gave a disaccharide characterized as N-acetylmannosaminyl(beta 1----4)-N-acetylglucosamine [ManNAc(beta 1----4)GlcNAc] in addition to the ribitol teichoic acid moiety, whereas the teichoic-acid - glycopeptide was separated into disaccharide-linked glycopeptide and the ribitol teichoic acid moiety by the same procedure. Furthermore, Smith degradation of the cell walls gave a characteristic fragment, EtO2-P-Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-ManNAc(beta 1----4)GlcNAc (where EtO2 = 1,2-ethylenediol and Gro = glycerol). The results lead to the conclusion that in the cell walls of this organism, the ribitol teichoic acid chain is linked to peptidoglycan through a novel linkage unit, Glc(beta 1----3)Glc(beta 1----1/3)Gro-P-(3/4)ManNAc-(beta 1----4)GlcNAc.     

Cell wall composition of Streptomyces roseoflavus var. roseofungini and its Nocardia-like variant

The composition of cell walls was comparatively studied in Streptomyces roseoflavus var. roseofungini 1128 and in its variant 1-68. In the logarithmic phase of growth, the content of teichoic acid in the cell wall of the parent culture was four times as high as in the cell wall of the variant. The cell walls of the parent culture contained 5 to 7 times more O-lysyl residues not only due to a higher content of teichoic acid in the walls but also owing to a lower content of lysyl groups in the teichoic acid of the variant. An additional polysaccharide comprising galactose and glucosamine was found in the cell wall of the variant but not in the parent strain. The peptidoglycan of the both cultures had a structure typical of Streptomyces spp.; its content in the cell walls of the two cultures was identical (ca. 50% of the dry cell wall biomass weight). The results are discussed in connection with the peculiarities of the variant hyphal septation.     

Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis

It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264. Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof. Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed. The experiments were performed by incubating B. subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264. The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Study of the formation of N-glycolylmuramic acid from Nocardia asteroides (author's transl)]

Nocardia asteroides was grown in Sauton medium containing sodium [carboxy-14C]acetate. The biosynthesis of the peptidoglycan was inhibited by adding penicillin or phosphonomycin to the growth medium. These antibiotics give an accumulation of radioactive nucleotidic precursors of the peptidoglycan. In the presence of penicillin, there was an accumulation of uridine diphosphate-N-glycolylmuramyl peptide (UDP-MurNGlyc peptide) and of a mixture of uridine diphosphate-N-acetyl and N-glycolylmuramic acid (UDP-MurNAc) and UDP-MurNGlyc). In the presence of phosphonomycin, the biosynthesis of muramic acid was blocked and there was an accumulation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-N-glycolyglucosamine (UDP-GlcNGlyc). Thus the formation of a N-glycolyl group can be performed upon the neucleotidic derivatives of glucosamine and muramic acid. However in the peptidoglycan synthesized in vivo in the absence of antibiotic, only muramic acid was glycolyated. So, glycolylation seems to take place essentially on UDP-MurNAc. When the binding of peptide chain to muramic acid is achieved, all the muramic acid is glycolylated, then the polymerisation of glycan and peptidoglycan units by the mean of particulate enzymes is carried out on the N-glycolylated derivative of muramic acid. A cell-free preparation from Nocardia asteroides was obtained which can hydroxylate the acetyl group of UDP-MurNAc. The activity was localised in the soluble fraction. This system acts as a hydroxylase and requires the presence of NADPH.     

Chemical Composition of the Cell Walls of Bacillus stearothermophilus

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.     

The isolation of structural components present in the cell wall of Bacillus licheniformis N.C.T.C. 6346

1. Four of the known components of wall preparations of vegative cells of Bacillus licheniformis N.C.T.C. 6346 have been isolated free of each other after successive treatments of the walls with trichloroacetic acid and lysozyme: (a) a mucopeptide consisting of glucosamine, muramic acid, alphain-diaminopimelic acid, glutamic acid and alanine in the molar proportions 1.0:0.8:1.0:1.2:1.7; (b) an insoluble protein; (c) teichoic acid containing phosphorus and glucose in equimolar amounts; (d) teichuronic acid containing equimolar amounts of N-acetylgalactosamine and glucuronic acid, as found by Janczura, Perkins & Rogers (1961). 2. Evidence has been obtained for the presence in the soluble fraction obtained by lysozyme treatment of whole walls of a stable covalent complex of the teichoic acid and the mucopeptide components. 3. The molar ratio of phosphorus to glucose in the teichoic acid present in intact walls or the soluble fractions obtained by extraction of the walls with lysozyme or trichloroacetic acid is 1.0:0.25, in contrast with values of about unity obtained for the purified teichoic acid. 4. Intact walls have been shown to contain polyribitol phosphate chains bearing different amounts of glucose substituents. 5. Trichloroacetic acid extracts of walls also contain polyribitol phosphate compounds of different chain lengths. Dialysis of trichloroacetic acid extracts removes the short chains of polyribitol phosphate that have been found to carry only very low amounts of glucose side chains. By contrast, the longer chains present in the non-diffusible fraction contain phosphorus and glucose in almost equimolar amounts.     

Isolation and Chemical Structure of the Peptidoglycan of Spirillum serpens Cell Walls

The peptidoglycan layer of Spirillum serpens cell walls was isolated from intact cells after treatment with sodium dodecylsulfate and digestion with Pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid, and meso-diaminopimelic acid in the approximate molar ratio of 1:1:2:1:1. Aspartic acid and glycine were the only other amino acids found in significant quantities. N-terminal amino acid analyses of the tetrapeptide amino acids in the peptidoglycan revealed that 54% of the diaminopimelic acid molecules are involved in cross-linkage between tetrapeptides. This amount of cross-linkage is greater than that found in the peptidoglycan of previously studied cell walls of gram-negative bacteria. The polysaccharide backbone was isolated, after myxobacter AL-1 enzyme digestion of the peptidoglycan, by fractionation with ECTEOLA-cellulose and Sephadex G-100. An average length of 99 hexosamines for the polysaccharide chains was found (ratio of total hexosamines to reducing end groups).     

Insertion and fate of the cell wall in Bacillus subtilis

Cell wall assembly was studied in autolysin-deficient and -sufficient strains of Bacillus subtilis. Two independent probes, one for peptidoglycan and the other for surface-accessible teichoic acid, were employed to monitor cell surface changes during growth. Cell walls were specifically labeled with N-acetyl-D-[3H]glucosamine, and after growth, autoradiographs were prepared for both cell types. The locations of silver grains revealed that label was progressively lost from numerous sites on the cell cylinders, whereas label was retained on the cell poles, even after several generations. In the autolysin-deficient and chain-forming strain, it was found that the distance between densely labeled poles approximately doubled after each generation of growth. In the autolysin-sufficient strain, it was found that the numbers of labeled cell poles remained nearly constant for several generations, supporting the premise that completed septa and poles are largely conserved during growth. Fluorescein-conjugated concanavalin A was also used to determine the distribution of alpha-D-glucosylated teichoic acid on the surfaces of growing cells. Strains with temperature-sensitive phosphoglucomutase were used because in these mutants, glycosylation of cell wall teichoic acids can be controlled by temperature shifts. When the bacteria were grown at 45 degrees C, which stops the glucosylation of teichoic acid, the cells gradually lost their ability to bind concanavalin A on their cylindrical surfaces, but they retained concanavalin A-reactive sites on their poles. Discrete areas on the cylinder, defined by the binding of fluorescent concanavalin A, were absent when the synthesis of glucosylated teichoic acid was inhibited during growth for several generations at the nonpermissive temperature. When the mutant was shifted from a nonpermissive to a permissive temperature, all areas of the cylinder became able to bind the labeled concanavalin A after about one-half generation. Old cell poles were able to bind the lectin after nearly one generation at the permissive temperature, showing that new wall synthesis does occur in the cell poles, although it occurs slowly. These data, based on both qualitative and quantitative experiments, support a model for cell wall assembly in B. subtilis, in which cylinders elongate by inside-to-outside growth, with degradation of the stress-bearing old wall in wild-type organisms. Loss of wall material, by turnover, from many sites on the cylinder may be necessary for intercalation of new wall and normal length extension. Poles tend to retain their wall components during division and are turned over much more slowly.     

Binding of Polysaccharide to Peptidoglycan in Cell Wall of Streptomyces roseochromogenes

The polysaccharide-peptidoglycan complex, which was prepared with lysozyme from Streptomyces roseochromogenes IAM53 cell walls, was hydrolyzed with lytic enzyme of Flavo-bacterium to separate polysaccharide. The enzymatically prepared polysaccharide (100 mg) contained 500 μmoles of hexoses, 40 μmoles of hexosamines and 31 μmoles of phosphate. Hexoses consisted of mannose and galactose in a molar ratio of 5 to 1. Hexosamines consisted of equimolar glucosamine and muramic acid, a half of which was identified as muramic acid 6-phosphate. The reducing end of the polysaccharide was muramic acid. The polysaccharide extracted with trichloroacetic acid contained no muramic acid-phosphate. So the polysaccharide moiety of S. roseochromogenes cell walls must be linked covalently to 6-position of muramic acid in peptidoglycan through phosphate,     

The formation and regeneration of mycelial protoplasts in hyper lignolytic fungus,strain IZU-154

Over 2 × 10⁷/ml protoplasts were obtained from mycelia of hyper lignolytic fungus (nomenclatured as strain IZU-154) by treatment with the lytic enzyme NovoZym 234 in the presence of 0.05 M maleic acid buffer (pH 5.6) containing 0.6 M MgSO₄. The protoplasts regenerated at more than 10% of frequency on solid 2% agar medium containing 0.6 M sucrose as an osmotic stabilizer overlaid with 0.5% agar containing the stabilizer. In the determination of the lignolytic activities of 50 regenerants from protoplasts, 2 strains which degraded more than 56% of the lignin during incubation for 30 d and showed activity higher than the parent were found. The regeneration from protoplasts of this fungus was suggested to be useful for the breeding of strains having higher lignolytic activity than this fungus.     

Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H.

1. In addition to poly(ribitol phosphate) the walls of a bacteriophage-resistant mutant of Staphylococcus aureus H contain glycerol phosphate residues that are not removed on digestion with trypsin or extraction with phenol. 2. The glycerol phosphate is present in a chain, containing three or four glycerol phosphate residues, which is covalently attached to the peptidoglycan through a phosphodiester linkage to muramic acid; this linkage is readily hydrolysed by dilute alkali. 3. The degradative studies described suggest that the poly(ribitol phosphate) chains of the wall teichoic acid may be attached to the wall by linkage to this glycerol phosphate oligomer.     

The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.     

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.     

Structure of the cell wall of Bacillus stearothermophiluys: mode of action of a thermophilic bacteriophage lytic enzyme

The mode of action of a bacteriophage lytic enzyme on cell walls of Bacillus stearothermophilus (NCA 1503-4R) has been investigated. The enzyme is an endopeptidase which catalyzes the hydrolysis of the l-alanyl-d-glutamyl linkage in peptide subunits of the cell wall peptidoglycan. Preliminary studies on the soluble components in lytic cell wall digests indicate that the glycan moiety is composed of alternating glucosamine and muramic acid; one half of the muramic acid residues contain the tripeptide, l-alanyl-d-glutamyldiaminopimelic acid, and the remaining residues contain the tetrapeptide, l-alanyl-d-glutamyldiaminopimeyl-d-alanine. Almost one half of the peptide subunits are involved in cross-linkages of chemotype I. A structure for the cell wall peptidoglycan is proposed in the light of these findings.     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Electron-microscopic and Physical Study

Protoplasts of Bacillus subtilis plated on SD medium form L colonies in quantitative yield and propagate in the L form indefinitely. L bodies or protoplasts placed in 25% gelatin medium form bacillary colonies. Details of the reversion of naked bodies to the walled form are reported. In 25% gelatin medium, reversion begins earlier (about 50% reversion in 4 hr) than the multiplication of bacilli. Thus, virtually all the observed bacillary forms are themselves revertants and not the offspring of a few growing clones. The optimal temperature for reversion is 26 C in 25% gelatin. When cells reverting at 26 C are warmed to 40 C for 3 min, reversion is delayed markedly, whereas viability is unaffected. For electron microscopy, a dense protoplast inoculum was placed on a gelatin surface, incubated, and then fixed in situ. There was no multiplication, but crowding delayed reversion markedly. Successive events of reversion are as follows. The loose nucleoid of the protoplasts condenses in response to the gelatin medium and condenses further and further as reversion proceeds. A thin coat of wall develops around the bodies of various sizes and shapes and then increases uniformly in thickness until a wall of normal aspect is formed. Rod-shaped cells grow out from these bodies-sometimes in several directions at once. A few mesosomes begin to appear only after a thin coat of wall has been formed. These are dense, atypical structures compartmented by membranes. They are located at the cell periphery and do not seem to be in contact with the nucleoids. Quantitative estimates showed that only 20 to 25% of revertant cells or cells grown on gelatin contain even a single mesosome. The others have no mesosome at all. Mesosomes thus do not appear to play a significant role in reversion, and normal mesosome functions must presumably be performed elsewhere in the cell in gelatin-grown bacilli. The role of cell wall, its synthesis, and its chemical nature in successive steps in reversion are discussed.     

Chemical analysis of the outer cyst wall and inclusion material ofBdellovibrio bdellocysts

The outer cyst wall and inclusion material fromBdellovibrio bdellocysts were isolated and their chemical composition was determined. The outer cyst wall is primarily peptidoglycan containing glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid. The cyst walls are resistant to lysozyme, but are rendered sensitive following deacylation and N-acetylation. Isolated inclusions were degraded quantitatively to glucose by HCl and by amyloglucosidase, whereas α-amylase degraded the polymer only partially with the release of reducing groups. The inclusion material is therefore an amylopectin-like polysaccharide, being a polyglucose containing both α-1,4 and α-1,6 linkages.     

Primary structure of the peptidoglycan from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714

A peptidoglycan fraction free of non-peptidoglycan components was isolated from the unicellular cyanobacterium Synechocystis sp. strain PCC 6714. Hydrofluoric acid treatment (48%, 0 degrees C, 48 h) cleaved off from the peptidoglycan non-peptidoglycan glucosamine, mannosamine, and mannose. The purified peptidoglycan consists of N-acetyl muramic acid, N-acetyl glucosamine, L-alanine, D-alanine, D-glutamic acid, and meso-diaminopimelic acid in approximately equimolar amounts. At least partial amidation of carboxy groups in the peptide subunits is indicated. Peptide analyses and 2,4-dinitrophenyl studies of partial acid hydrolysates revealed the structure of the Synechocystis sp. strain PCC 6714 peptidoglycan to belong to the A1 gamma type (direct cross-linkage) of peptidoglycan classification. The degree of cross-linkage is about 56% and thus is in the range of that found in gram-positive bacteria. Some of the peptide units are present as tripeptides lacking the carboxy-terminal D-alanine.