Factors affecting sensitivity of group B streptococci to an exogenous murein hydrolase

Group B streptococci treated with cell wall synthesis inhibitors (penicillin or vancomycin) or by a variety of membrane-acting agents are sensitized to the lytic action of exogenous M1 muramidase. Muramidase without a sensitizing agent caused rupture of bacterial chains only, accompanied by the release of a small amount of cell wall peptidoglycan label and an increase of the number of colony-forming units. In combination with sensitizing agents the exogenous muramidase appeared to initiate hydrolysis of biosynthetically new peptidoglycan. Treatment of the cells with chloramphenicol or starvation for nutritionally required amino acids suppressed the rate of cell lysis and peptidoglycan hydrolysis during subsequent sensitization and muramidase treatment of the bacteria. Purified cell walls prepared from the amino acid starved cells were also hydrolyzed with a slower rate by muramidase. It is suggested that agents sensitizing the bacteria to the exogenous muramidase act by perturbing or removing some nonmurein components of the cell envelope which protect the peptidoglycan from the activity of exogenous enzyme. Agents increasing resistance against exogenous muramidase may also cause some alteration in peptidoglycan structure.     

Involvement of the relA gene in the autolysis of Escherichia coli induced by inhibitors of peptidoglycan biosynthesis

It is generally assumed that inhibitors of peptidoglycan biosynthesis do not kill nongrowing bacteria. An exceptional case is reported here. The addition of chloramphenicol to amino acid-deprived cultures of relA+ strains of Escherichia coli which were treated with beta-lactam antibiotics, D-cycloserine, or moenomycin resulted in lysis. This phenomenon is termed chloramphenicol-dependent lysis. To be effective, chloramphenicol had to be present at its minimum growth-inhibitory concentration (or higher). Analogs of chloramphenicol which did not bind to ribosomes were completely ineffective. Amino acid deprivation was actually not required to demonstrate chloramphenicol-dependent lysis, and cultures treated with growth-inhibitory levels of chloramphenicol alone were lysed when challenged with inhibitors of peptidoglycan synthesis. Peptidoglycan synthesis has been shown previously to be under stringent (relA+) control, and chloramphenicol is known to be an antagonist of stringent control. Thus, it is proposed that the mechanism of chloramphenicol-dependent lysis is based on the ability of chloramphenicol to relax peptidoglycan synthesis in nongrowing relA+ bacteria. This is also consistent with the observation that treatment of amino acid-deprived relA mutants with inhibitors of peptidoglycan synthesis resulted in lysis, i.e., without the mediation of chloramphenicol.     

Properties of cell wall peptidoglycan synthesized by amino acid deprived re1A mutants of Escherichia coli

Cell wall peptidoglycan synthesis in Escherichia coli is under stringent control. During amino acid deprivation, peptidoglycan synthesis is inhibited in re1A+ bacteria but not in re1A mutants. The relaxed synthesis of peptidoglycan by amino acid deprived re1A bacteria was inhibited by several beta-lactam antibiotics at concentrations which inhibited cell elongation in growing cultures suggesting that the transpeptidase activity of penicillin-binding protein (PBP-1B) was involved in this process. Structural studies on the peptidoglycan also indicated the involvement of transpeptidation in relaxed peptidoglycan synthesis. The peptidoglycan synthesized during amino acid deprivation was cross-linked to the existing cell wall peptidoglycan, and the degree of cross-linkage was the same as that of peptidoglycan synthesized by growing control cells. The relaxed synthesis of peptidoglycan was also inhibited by moenomycin, an inhibitor of the in vitro transglycosylase activities of PBPs, but the interpretation of this result depends on whether the transglycosylases are the sole targets of moenomycin in vivo. Most of the peptidoglycan lipoprotein synthesized by histidine-deprived re1A+ bacteria was in the free form as previously reported, possibly because of the restriction in peptidoglycan synthesis. In support of this proposal, most of the lipoprotein synthesized during histidine deprivation of re1A mutants was found to be covalently linked to peptidoglycan. Nevertheless, the peptidoglycan synthesized by amino acid deprived re1A bacteria was apparently deficient in bound lipoprotein as compared with peptidoglycan synthesized by normal growing control bacteria suggesting that the rate of lipoprotein synthesis during amino acid deprivation may be limiting.     

The effect of blood serum components on the growth, biochemical and biological properties of streptococcus

The components of cattle blood serum, added to the medium for the cultivation of group A streptococci, considerably decrease the period of adaptation and increase the balanced growth rate of streptococci, which is manifested by changes in the surface structures of the cell wall: the absence or modification of protein M. Streptococci grown under these conditions lose their capacity for phagocytosis, and from the cell walls obtained from these streptococci no surface protein M can be isolated by pepsin treatment. Nevertheless, the ratio of the main cell-wall components (proteins, polysaccharide and peptidoglycan), the amino acid composition, as well as the resistance of the cell walls to the action of trypsin and endo-N-acetylmuramidase are the same in M+ and Mx variants, that makes it possible to infer that the modification of protein M or the inhibition of its synthesis occurs during the growth of streptococci in the presence of blood serum components.     

Peptidoglycan composition and taxonomy of group D, E, and H streptococci, and Streptococcus mutans

The qualitative and quantitative composition of the peptidoglycan from the cell wall of groups D, E, and H streptococci, and Streptococcus mutans, was determined. In group D, S. faecalis and the closely related species S. liquefaciens and S. zymogenes were separated from S. faecium and the closely related species S. durans on the basis of their peptidoglycan composition. A relationship among S. bovis, S. equinus, and some strains of S. mutans was indicated by the presence in each of a similar peptidoglycan containing threonine. Threonine was released from the S. mutans polymer as a threonine-lysine dipeptide. Hydrolysis of the dipeptide at 100 C for 24 hr in 6 n HC1 was required to break the peptide bond. Motile group D streptococci possessed a peptidoglycan of the same composition as S. faecium. Group E and H strains were also similar in the composition of their peptidoglycan. The results demonstrate that peptidoglycan composition can be used to (i) aid in the division of members of an immunological group into subgroups, and (ii) indicate a relationship between members of the same genus which are not related on an immunological basis.     

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan.     

Mode of action of glycine on the biosynthesis of peptidoglycan

The mechanism of glycine action in growth inhibition was studied on eight different species of bacteria of various genera representing the four most common peptidoglycan types. To inhibit the growth of the different organisms to 80%, glycine concentrations from 0.05 to 1.33 M had to be applied. The inhibited cells showed morphological aberrations. It has been demonstrated that glycine is incorporated into the nucleotide-activated peptidoglycan precursors. The amount of incorporated glycine was equivalent to the decrease in the amount of alanine. With one exception glycine is also incorporated into the peptidoglycan. Studies on the primary structure of both the peptidoglycan precursors and the corresponding peptidoglycan have revealed that glycine can replace l-alanine in position 1 and d-alanine residues in positions 4 and 5 of the peptide subunit. Replacement of l-alanine in position 1 of the peptide subunit together with an accumulation of uridine diphosphate-muramic acid (UDP-MurNAc), indicating an inhibition of the UDP-MurNAc:l-Ala ligase, has been found in three bacteria (Staphylococcus aureus, Lactobacillus cellobiosus and L. plantarum). However, discrimination against precursors with glycine in position 1 in peptidoglycan synthesis has been observed only in S. aureus. Replacement of d-alanine residues was most common. It occurred in the peptidoglycan with one exception in all strains studied. In Corynebacterium sp., C. callunae, L. plantarum, and L. cellobiosus most of the d-alanine replacing glycine occurs C-terminal in position 4, and in C. insidiosum and S. aureus glycine is found C-terminal in position 5. It is suggested that the modified peptidoglycan precursors are accumulated by being poor substrates for some of the enzymes involved in peptidoglycan synthesis. Two mechanisms leading to a more loosely cross-linked peptidoglycan and to morphological changes of the cells are considered. First, the accumulation of glycine-containing precursors may lead to a disrupture of the normal balance between peptidoglycan synthesis and controlled enzymatic hydrolysis during growth. Second, the modified glycine-containing precursors may be incorporated. Since these are poor substrates in the transpeptidation reaction, a high percentage of muropeptides remains uncross-linked. The second mechanism may be the more significant in most cases.     

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity

Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface. Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation. Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F. In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis. However, their effect on the growth of S. sobrinus in the solid and liquid phase was notable. 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S. sobrinus in agar plates. Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.     

Effect of Streptococcus pyogenes on Tissue Cells

Human tissue cell lines from each of the three primary germinal sources, ectoderm (conjunctiva and carcinoma of the buccal mucosa), entoderm (intestine and liver), and mesoderm (heart and monocytes) were inoculated with group A Streptococcus pyogenes, Staphylococcus aureus, and group D streptococci and were then observed. In addition, the effect of these bacteria on mouse fibroblasts was studied. All of the cell lines appeared to be equally susceptible to damage, but damage to the cells by S. pyogenes occurred only when living, actively multiplying bacteria were in contact with the tissue cells. Streptococcal products in the form of "used" growth medium had no observable effect on the cells. Cytopathogenic effects were first noticed about the time one would expect the bacteria to have reached the end of the log phase of growth. No damage to the tissue cells was noted when group A streptococci were separated from the cells by membrane filter diffusion chambers or dialyzing membranes, but a membrane did not protect cells from deleterious effects of staphylococci or group D streptococci. Group A streptococci survived in the tissue culture medium, but multiplication did not occur unless living tissue cells were present.     

Biosynthesis of linkage units for teichoic acids in gram-positive bacteria: distribution of related enzymes and their specificities for UDP-sugars and lipid-linked intermediates.

The distribution and substrate specificities of enzymes involved in the formation of linkage units which contain N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) or glucose and join teichoic acid chains to peptidoglycan were studied among membrane systems obtained from the following two groups of gram-positive bacteria: group A, including Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, and Lactobacillus plantarum; group B, Bacillus coagulans. All the membrane preparations tested catalyzed the synthesis of N-acetylglucosaminyl pyrophosphorylpolyprenol (GlcNAc-PP-polyprenol). The enzymes transferring glycosyl residues to GlcNAc-PP-polyprenol were specific to either UDP-ManNAc (group A strains) or UDP-glucose (group B strains). In the synthesis of the disaccharide-bound lipids, GlcNAc-PP-dolichol could substitute for GlcNAc-PP-undecaprenol. ManNAc-GlcNAc-PP-undecaprenol, ManNAc-GlcNAc-PP-dolichol, Glc-GlcNAc-PP-undecaprenol, Glc-GlcNAc-PP-dolichol, and GlcNAc-GlcNAc-PP-undecaprenol were more or less efficiently converted to glycerol phosphate-containing lipid intermediates and polymers in the membrane systems of B. subtilis W23 and B. coagulans AHU 1366. However, GlcNAc-GlcNAc-PP-dolichol could not serve as an intermediate in either of these membrane systems. Further studies on the exchangeability of ManNAc-GlcNAc-PP-undecaprenol and Glc-GlcNAc-PP-undecaprenol revealed that in the membrane systems of S. aureus strains and other B. coagulans strains both disaccharide-inked lipids served almost equally as intermediates in the synthesis of polymers. In the membrane systems of other B. subtilis strains as well as B. licheniformis and B. pumilus strains, however, the replacement of ManNAc-GlcNAc-PP-undecaprenol by Glc-GlcNAc-PP-undecaprenol led to a great accumulation of (glycerol phosphate)-Glc-GlcNAc-PP-undecaprenol accompanied by a decrease in the formation of polymers.     

Septicaemic infections in pigs,caused by haemolytic streptococci of new Lancefield groups designated R,S and T

Pyogenic streptococci isolated from outbreaks and from sporadic infections in pigs and piglets were characterized by the almost unique combination of the properties of -haemolysis on horse blood agar and acid production from inulin. Three new serological groups were recognized, each with a single antigen different from those of any of the Lancefield groups. These antigens are polysaccharides located in the cell wall. About half the number of haemolytic streptococcal strains isolated from pigs were group R or group S streptococci, a few strains were group T streptococci, and the remaining strains were group L streptococci,S. equisimilis, group E streptococci, or group P streptococci, in this order of frequency. Only one out of about 150 haemolytic strains could not be identified serologically. Group R and group T streptococci differ from group S streptococci by acid production from raffinose. Infections with group R streptococci appeared to occur independently of age, whereas infections with group S streptococci were almost entirely confined to piglets.     

d-Alanylcardiolipin,a Major Component of the Unique Lipid Pattern of Vagococcus fluvialis

Motile group N streptococci, classified as Vagococcus fluvialis, have been isolated from cows’ udders, human and animal feces, river water, and seawater. They possess an unusual membrane lipid and fatty acid pattern. We isolated and characterized 13 polar lipids, 8 of them also found in other gram-positive bacteria: mono- and dihexosyldiacylglycerol, an acylated and a glycerophosphate-substituted derivative of the latter, cardiolipin, phosphatidylglycerol, d-alanylphosphatidylglycerol, and l-lysylphosphatidylglycerol. Besides them, we characterized two rare compounds, bis(acylglycero)phosphate and α-d-glucopyranosylcardiolipin, and two compounds so far not detected in nature, d-alanylbis(acylglycero)phosphate and d-alanylcardiolipin. The concomitant occurrence of four aminoacyl phospholipids in one organism is another unique finding. Substituted cardiolipins represent a novel lipid class: in vagococci, d-alanylcardiolipin is a major membrane lipid component, contributing 11 and 26 mol% of total lipids in the exponential and stationary phases of growth, respectively. The vagococcal lipids contain even-numbered straight-chain saturated and cis-monounsaturated fatty acids, but the cis-monoenic acids belong to the ω-9 series and not the ω-7 series, found in enterococci, lactococci, and streptococci.Chemical and molecular systematic studies have recently been done to clarify the phylogenetic relationship of the group N streptococci (42, 43). Nucleic acid hybridization studies and immunological relationships of superoxide dismutases demonstrated that “Streptococcus lactis” and its subspecies are closely related to each other but not to other streptococci, which led to the formation of a new genus, Lactococcus (43). When during this study the membrane lipids were investigated, group N streptococcus strain Kiel 48809 displayed a pattern that differed greatly from the lipid pattern of the S. lactis group. This strain, which had been isolated from a cow’s udder in Germany (19a), was motile and formed a group with other motile group N streptococci (NCDO 2497, NCDO 2498, and NCDO 2499) (43) which had been isolated in Japan from feces of humans and animals and from river water and seawater (20, 21). Although the motile strains possess the group N antigen, they are not genetically related to Lactococcus or to other streptococci examined. The polar-lipid patterns and their long-chain fatty acid compositions reinforced their distinctiveness and, along with genetic data, suggested that these strains may represent the nucleus of a new taxon (43). This was confirmed by 16S RNA sequence analyses which located the motile group N streptococci on a phylogenetic tree and led to their classification in a new genus, Vagococcus, as Vagococcus fluvialis sp. nov. (7). By using seven isolates, molecular characterization was done and evidence of a possible connection between V. fluvialis and human infections was provided (46).In this report, we describe the polar-lipid pattern of V. fluvialis, which was the same for all the four strains investigated. We isolated and characterized 13 polar lipids and found, in addition to the ubiquitous membrane lipids of gram-positive bacteria, rare and so-far-unknown structures. Of particular interest is d-alanylcardiolipin, which is a major component among the membrane lipids of vagococci. It is a novel representative of the lipid class of substituted cardiolipins. Other examples are α-d-glucopyranosylcardiolipin, found in this study and earlier in group B streptococci (10), and l-lysylcardiolipin, isolated from species of the genus Listeria (16a). So far, substituted cardiolipins have been found only in gram-positive bacteria.     

Induction of autolysis in nongrowing Escherichia coli

Unless relaxation of the stringent response is achieved, all nongrowing bacteria rapidly develop resistance to autolysis induced by a variety of agents, including all classes of cell wall synthesis inhibitors. We now describe inhibitors of cell wall synthesis which were unusual in that they could continue to effectively induce autolysis in relA+ Escherichia coli even after prolonged amino acid starvation. The process of cell wall degradation seems to be catalyzed by similar hydrolytic enzymes in nongrowing and growing cells, yet the activity of these new agents capable of inducing autolysis in the nongrowing relA+ cells did not involve relaxation of RNA or peptidoglycan synthesis. We propose that the suppression of autolysis characteristic of nongrowing cells can be bypassed by a novel mechanism of autolytic triggering which is independent of the relA locus.     

Effect of oleanolic acid-cyclodextrin inclusion compounds on dental caries by in vitro experiment and rat-caries model.

Earlier work in vitro showed that oleanolic acid (OA) was a potential inhibitor of insoluble glucan (ISG) synthesis from mutans streptococci (MS). In this study, two oleanolic acid-cyclodextrin inclusion compounds (OA-CDs), oleanolic acid-G1-beta-cyclodextrin (OA-G1-beta CD) and oleanolic acid-beta-cyclodextrin (OA-beta CD), were assayed for their effects on ISG synthesis from Streptococcus mutans MT8148R, and on the growth of oral bacteria. OA-beta CD inhibited ISG synthesis by 55.3 and 37.4% at 62.5 and 15.6 micrograms/ml of OA, respectively. Both OA-CDs inhibited the growth of MS, S. sanguis, and S. salivarius at 4 to 8 micrograms/ml of OA. The anticariogenic effect of the OA-beta CD was examined in a rat-caries model. Rats in the infected control groups showed the highest caries score. The infected treatment group B (0.5% OA in diet) showed lower scores than the control group. These results suggest that OA-beta CD is a potential anti-caries agent.     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

Streptococcus mutans and oral streptococci in dental plaque

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.     

Reactivity to bacterial peptidoglycans in a phagocytosis system. 1. The range of immunological specificity of Staphylococcus aureus peptidoglycan

The antigenic features of S. aureus peptidoglycan (PG) were studied in the reaction of stimulation of oxygen-dependent neutrophil metabolism, mediated by the IgG opsonins of normal human serum. The study was carried out at different taxonomic levels: the species (S. aureus), the genus (Staphylococcus), the family (Micrococcaceae), as well as in relation to remote taxons (organisms belonging to the families Streptococcaceae, Enterobacteriaceae, Neisseriaceae, to the genus Corynebacterium). All S. aureus strains were identical with respect to the specificity of their PG, essentially differing from other bacteria in this regard. After the removal of antibodies to different PG the effectiveness of PG opsonization decreased by 10.4-44.7%. Such decrease was most pronounced in experiments with the PG of streptococci (S. pyogenes, S. faecalis, S. salivarius) and Micrococcus luteus.     

Composition and properties of a group A streptococcal teichoic acid

Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.