Production and characterization of a monoclonal antibody to the O-acetylated peptidoglycan of Proteus mirabilis.

A monoclonal antibody (PmPG5-3) specific for the O-acetylated peptidoglycan of Proteus mirabilis 19 was produced by an NS-1 myeloma cell line and purified from ascites fluid by a combination of ammonium sulfate precipitation and affinity chromatography. The monoclonal antibody (an immunoglobulin M) was characterized by a competition enzyme-linked immunosorbent assay to be equally specific for both insoluble and soluble O-acetylated peptidoglycan but weakly recognized chemically de-O-acetylated P. mirabilis peptidoglycan, the non-O-acetylated peptidoglycans from Escherichia coli and Bacillus subtilis, and the peptidoglycan monosaccharide precursors N-acetylglucosamine and N-acetylmuramic acid dipeptide. The monoclonal antibody did not react with D-alanine or lipopolysaccharide isolated from P. mirabilis. Based on this evidence, the binding epitope on the P. mirabilis peptidoglycan is predicted to be linear and to comprise the glycan backbone, including both the N- and O-acetyl moieties. Monoclonal antibody PmPG5-3 was used to localize the O acetylation of the P. mirabilis peptidoglycan by immunoelectron microscopy. Murein sacculi of P. mirabilis were heavily and randomly labelled with the immunogold, whereas very little labelling and no labelling were observed on the sacculi isolated from de-O-acetylated P. mirabilis and E. coli, respectively. Based on the apparent pattern of immunogold labelling, a physiological role for peptidoglycan O acetylation in P. mirabilis is proposed.     

Binding of S-layer homology modules from Clostridium thermocellum SdbA to peptidoglycans

S-layer homology (SLH) module polypeptides were derived from Clostridium josui xylanase Xyn10A, Clostridium stercorarium xylanase Xyn10B, and Clostridium thermocellum scafoldin dockerin binding protein SdbA as rXyn10A-SLH, rXyn10B-SLH, and rSdbA-SLH, respectively. Their binding specificities were investigated using various cell wall preparations. rXyn10A-SLH and rXyn10B-SLH bound to native peptidoglycan-containing sacculi consisting of peptidoglycan and secondary cell wall polymers (SCWP) prepared from these bacteria but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWP, suggesting that SCWP are responsible for binding with SLH modules. In contrast, rSdbA-SLH interacted with HF-EPCS, suggesting that this polypeptide had an affinity for peptidoglycans but not for SCWP. The affinity of rSdbA-SLH for peptidoglycans was confirmed by a binding assay using a peptidoglycan fraction prepared from Escherichia coli cells. The SLH modules of SdbA must be useful for cell surface engineering in bacteria that do not contain SCWP.     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

The extraction of cell walls of Pseudomonas aeruginosa with aqueous phenol. The insoluble residue and material from the aqueous layers

1. The insoluble residue and material present in the aqueous layers resulting from treatment of cell walls of Pseudomonas aeruginosa with aqueous phenol were examined. 2. The products (fractions AqI and AqII) isolated from the aqueous layers from the first and second extractions respectively account for approx. 25% and 12% of the cell wall and consist of both lipopolysaccharide and muropeptide. 3. The lipid part of the lipopolysaccharide is qualitatively similar to the corresponding material (lipid A) from other Gram-negative organisms, as is the polysaccharide part. 4. The insoluble residue (fraction R) contains sacculi, which also occur in fraction AqII. On hydrolysis, the sacculi yield glucosamine, muramic acid, alanine, glutamic acid and 2,6-diaminopimelic acid, together with small amounts of lysine, and they are therefore similar to the murein sacculi of other Gram-negative organisms. Fraction R also contains substantial amounts of protein, which differs from that obtained from the phenol layer. 5. The possible association or aggregation of lipopolysaccharide, murein and murein sacculi is discussed.     

Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 10(7) N/m(2) was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intact E. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927-953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301-366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.     

Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.     

Elasticity of the sacculus of Escherichia coli.

Preparations of purified peptidoglycan of Escherichia coli (i.e., sacculi) were studied by low-angle laser light scattering. Control experiments and theoretical calculations based on the Rayleigh-Gans theory showed that the mean sacculus surface area could be accurately inferred from measurements with our apparatus by using computer routines developed previously. Large changes in the mean saccular surface area resulted from alterations in the stress caused by varying the net charge on the sacculi. The net charge was affected by altering the suspending medium pH, causing carboxyl and amino groups in the peptidoglycan to gain or lose protons, or by acetylation or succinylation of the amino groups. A preponderance of either plus or minus charges caused an expansion of the mean sacculus surface area. The largest increase in area probably represents the elastic limit of the peptidoglycan and was 300% above the area of isoionic sacculi. This degree of expansion is consistent with possible conformations of the intact peptidoglycan structure without necessitating rupture of the wall fabric. Our findings concerning saccular elasticity provide support for the surface stress theory. It provides a mechanism so that bacteria can grow and divide while maintaining turgor pressure, without the necessity of having and using proteins to do the mechanical work.     

Different binding specificities of S-layer homology modules from Clostridium thermocellum AncA, Slp1, and Slp2

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Extracellular and cellular distribution of muramidase-2 and muramidase-1 of Enterococcus hirae ATCC 9790.

A substantial portion of the second peptidoglycan hydrolase (muramidase-2) activity of Enterococcus hirae ATCC 9790 (formerly Streptococcus faecium) is present in the supernatant culture medium. In contrast, nearly all muramidase-1 activity is associated with cells in the latent, proteinase-activatable form. Muramidase-2 activity is produced and secreted throughout growth, with maximal levels attained at or near the end of exponential growth in a rich organic medium. Muramidase-2 activity in the culture medium remained high even during overnight incubations in the absence of proteinase inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of supernatant culture medium concentrated by 60% saturated ammonium sulfate precipitation showed the presence of several Coomassie blue-staining bands. One intensely staining protein band, at about 71 kDa, selectively adsorbed to the insoluble peptidoglycan fraction of cell walls of E. hirae, retained muramidase-2 activity, and reacted in Western immunoblots with monoclonal antibodies to muramidase-2. The mobility of extracellular muramidase-2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was indistinguishable from that of muramidase-2 extracted with 6 M guanidine hydrochloride from intact bacteria. Muramidase-2 appears to have only a limited number of binding sites on the peptidoglycan of E. hirae cell walls but binds with high affinity. Although high levels of muramidase-2 activity were present in supernatants of stationary-phase cultures, the bacteria were resistant to autolysis. Thus it appears that the peptidoglycan in walls of intact cells of E. hirae is somehow protected from the hydrolytic action of extracellular muramidase-2.     

Different Binding Specificities of S-Layer Homology Modules from Clostridium thermocellum AncA,Slp1, and Slp2

S-layer homology (SLH) module polypeptides were derived from Clostridium thermocellum S-layer proteins Slp1 and Slp2 and cellulosome anchoring protein AncA as rSlp1-SLH, rSlp2-SLH, and rAncA-SLH respectively. Their binding specificities were investigated using C. thermocellum cell-wall preparations. rAncA-SLH associated with native peptidoglycan-containing sacculi from C. thermocellum, including both peptidoglycan and secondary cell wall polymers (SCWP), but not to hydrofluoric acid-extracted peptidoglycan-containing sacculi (HF-EPCS) lacking SCWPs, suggesting that SCWPs are responsible for binding with SLH modules of AncA. On the other hand, rSlp1-SLH and rSlp2-SLH associated with HF-EPCS, suggesting that these polypeptides had an affinity for peptidoglycan. A binding assay using a peptidoglycan fraction prepared from Escherichia coli cells definitely confirmed that rSlp1-SLH and rSlp2-SLH specifically interacted with peptidoglycan but not with SCWP.     

The peptidoglycan sacculus of Myxococcus xanthus has unusual structural features and is degraded during glycerol-induced myxospore development

Upon nutrient limitation cells of the swarming soil bacterium Myxococcus xanthus form a multicellular fruiting body in which a fraction of the cells develop into myxospores. Spore development includes the transition from a rod-shaped vegetative cell to a spherical myxospore and so is expected to be accompanied by changes in the bacterial cell envelope. Peptidoglycan is the shape-determining structure in the cell envelope of most bacteria, including myxobacteria. We analyzed the composition of peptidoglycan isolated from M. xanthus. While the basic structural elements of peptidoglycan in myxobacteria were identical to those in other gram-negative bacteria, the peptidoglycan of M. xanthus had unique structural features. meso- or LL-diaminopimelic acid was present in the stem peptides, and a new modification of N-acetylmuramic acid was detected in a fraction of the muropeptides. Peptidoglycan formed a continuous, bag-shaped sacculus in vegetative cells. The sacculus was degraded during the transition from vegetative cells to glycerol-induced myxospores. The spherical, bag-shaped coats isolated from glycerol-induced spores contained no detectable muropeptides, but they contained small amounts of N-acetylmuramic acid and meso-diaminopimelic acid.     

Properties of cell wall-associated DD-carboxypeptidase of Enterococcus hirae (Streptococcus faecium) ATCC 9790 extracted with alkali.

DD-Carboxypeptidase (DD-CPase) activity of Enterococcus hirae (Streptococcus faecium) ATCC 9790 was extracted from intact bacteria and from the insoluble residue (crude cell wall fraction) of mechanically disrupted bacteria by a brief treatment at pH 10.0 (10 mM glycine-NaOH) at 0 degrees C or by extraction with any of several detergents. Extractions with high salt concentrations failed to remove DD-CPase activity from the crude wall fraction. In contrast to N-acetylmuramoylhydrolase (both muramidase 2 and muramidase 1) activities, DD-CPase activity failed to bind to insoluble cell walls or peptidoglycan matrices. Thus, whereas muramidase 1 and muramidase 2 activities can be considered to be cell wall proteins, the bulk of the data are consistent with the interpretation that the DD-CPase of this species is a membrane protein that is sometimes found in the cell wall fraction, presumably because of hydrophobic interactions with other proteins and cell wall polymers. The binding of [14C]penicillin to penicillin-binding protein 6 (43 kilodaltons) was proportional to DD-CPase activity. Kinetic parameters were also consistent with the presence of only one DD-CPase (penicillin-binding protein 6) in E. hirae.     

Peptidoglycan-associated polypeptides of Mycobacterium tuberculosis.

Important protein-based immunoreactivities have long been associated with the cell wall core of mycobacteria. In order to explore the molecular basis of such activities, purified cell walls of Mycobacterium tuberculosis were extracted with sodium dodecyl sulfate to produce an insoluble residue composed of the mycolylarabinogalactan-peptidoglycan complex and about 2% of unextractable protein. Treatment of the product from an avirulent strain of M. tuberculosis with trifluoromethanesulfonic acid released a single polypeptide with a molecular size of 23 kilodaltons, accounting for all of the insoluble cell wall protein. Extensive purification and then analysis of the 23-kilodalton protein demonstrated the absence of diaminopimelic acid, muramic acid, or other peptidoglycan components, pointing to either a novel linkage between protein and peptidoglycan or a noncovalent but tenacious association. The released 23-kilodalton protein showed amino acid homology and other similarities to the outer membrane protein OmpF of Escherichia coli. Although a similar product was released in small quantities from cell walls of the virulent M. tuberculosis Erdman and H37Rv by lysozyme treatment, the cell walls of virulent bacilli were dominated by the presence of poly-alpha-L-glutamine, accounting for as much as 10% of their weight. The poly-alpha-L-glutamine was successfully separated from the cell wall proper, demonstrating again the absence of a covalent association between peptidoglycan and the polymer. The antigenicity of these products is demonstrated, and their roles vis-a-vis analogous polypeptides from other bacteria in immunogenicity, pathogenicity, and bacterial physiology are discussed.     

Structure of the rigid-layer of Rhizobium cell wall. III. Electron microscopic evidence for the cellulose microfibrils association with peptidoglycan sacculi

The morphology of peptidoglycan layer of Rhizobium cell wall was examined by transmission electron microscopy. Peptidoglycans were isolated from intact cells after treatment with sodium dodecyl sulfate, extraction with aqueous 45% phenol and then with a mixture of chloroform-methanol. Finally rigid layers were digested with trypsin and chymotrypsin. The results indicate the presence of lump or bar-like structures on the surface of the cell shaped peptidoglycan sacculi. Evidence is provided suggesting that the cellulose microfibrils arise directly from these excrescences found on the peptidoglycan surface. Digestion with cellulase removed all cellulose microfibrils whereas the lumps and bars remained as an integral part of the Rhizobium peptidoglycan.     

Induction kinetics and cell surface distribution of Escherichia coli lipoprotein under lac promoter control.

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the lpp gene under control of the lac promoter. Free lipoprotein appeared rapidly after induction with isopropyl-beta-D-thiogalactopyranoside and reached a steady-state level after 30 min. The newly induced lipoprotein was slowly bound to the peptidoglycan layer. Immunological methods were developed to detect lipoprotein accessible at the cell surface after various pretreatments as well as peptidoglycan-bound lipoprotein at the surface of isolated peptidoglycan sacculi with specific antibodies in combination with 125I-protein A. With these methods an increase in lipoprotein molecules at the cell surface and bound to the peptidoglycan sacculus could be detected following induction. The topology of newly synthesized lipoprotein was examined in thin sections as well as at the cell surface and the surface of the peptidoglycan sacculus with immunoelectron microscopy. Ultrathin cell sections, whole cells, and isolated peptidoglycan sacculi showed lipoprotein distributed homogeneously over the entire surface.     

Identification of the namH gene, encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan

The peptidoglycan of most bacteria consists of a repeating disaccharide unit of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. However, the muramic acid moieties of the mycobacterial peptidoglycan are N-glycolylated, not N-acetylated. This is a rare modification seen only in the peptidoglycan of mycobacteria and five other closely related genera of bacteria. The N-glycolylation of sialic acids is a unique carbohydrate modification that has been studied extensively in eukaryotes. However, the significance of the N-glycolylation of bacterial peptidoglycan is unknown. The goal of this project was to identify the gene encoding the hydroxylase responsible for the N-glycolylation of the mycobacterial peptidoglycan. We developed a novel assay for the mycobacterial UDP-N-acetylmuramic acid hydroxylation reaction and demonstrated that Mycobacterium smegmatis has an enzyme activity that can convert UDP-N-acetylmuramic acid to UDP-N-glycolylmuramic acid. We identified the gene namH encoding the mycobacterial UDP-N-acetylmuramic acid hydroxylase by computer data base searching and motif comparisons with the eukaryotic enzymes responsible for the N-glycolyation of sialic acids. The namH gene is not essential for in vitro growth as we were successful in deleting the gene in M. smegmatis. The M. smegmatis mutant is devoid of UDP-N-acetylmuramic acid hydroxylase activity and synthesizes only N-acetylated muropeptide precursors. Furthermore, the mutant exhibits increased susceptibility to beta-lactam antibiotics and lysozyme. Our studies suggest that the N-glycolylation of mycobacterial peptidoglycan may play a role in lysozyme resistance or may contribute to the structural stability of the cell wall architecture.     

Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli.

In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine. The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used. Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid. Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi. For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed. The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values. Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis. The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides. HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi.     

Novel peptidoglycans in Caulobacter and Asticcacaulis spp.

Peptidoglycan sacculi free of poly-beta-hydroxybutyric acid were prepared from whole cells of four species of Caulobacter and two species of Asticcacaluis and from morphological mutants of Caulobacter crescentus and Caulobacter leidyi. Acid hydrolysates of the sacculi were analyzed quantitatively, and each of the hydrolysates was found to contain significant amounts of only five ninhydrin-reactive compounds: alanine, glutamic acid, alpha , omega-diaminopimelic acid, muramic acid, and glucosamine. Four types of peptidoglycans were distinguishable on the basis of the molar ratios among these five compounds. The respective ratios were as follows: in C. leidyi, 2:1:1:1:0.8; in Asticcacaulis biprosthecum, 1.7:1.6:1.1:0.7; in the cells of the remaining species, 2:1:1:1.2:0.8; and in stalks shed by the abscission mutant 2NY66, 2:1:1:1:1.67. Thus, in addition to some species differences among these caulobacters, it was found that the peptidoglycan sacculus of the stalked C. crescentus cell is chemically differentiated; the cellular peptidoglycan is richer in muramic acid than is the peptidoglycan of typical gram-negative bacteria, and the peptidoglycan of the stalk is correspondingly rich in glucosamine. Empirical formulas for the repeating units of the peptidoglycans have been inferred on the basis of the molar ratios of their amino components.     

Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus

The capsular polysaccharide of group B Streptococcus is a key virulence factor and an important target for protective immune responses. Until now, the nature of the attachment between the capsular polysaccharide and the bacterial cell has been poorly defined. We isolated insoluble cell wall fragments from lysates of type III group B Streptococcus and showed that the complexes contained both capsular polysaccharide and group B carbohydrate covalently bound to peptidoglycan. Treatment with the endo-N-acetylmuramidase mutanolysin released soluble complexes of capsular polysaccharide linked to group B carbohydrate by peptidoglycan fragments. Capsular polysaccharide could be enzymatically cleaved from group B carbohydrate by treatment of the soluble complexes with beta-N-acetylglucosaminidase, which catalyzes hydrolysis of the beta-D-GlcNAc(1-->4)beta-D-MurNAc subunit produced by mutanolysin digestion of peptidoglycan. Evidence from gas chromatography/mass spectrometry and (31)P NMR analysis of the separated polysaccharides supports a model of the group B Streptococcus cell surface in which the group B carbohydrate and the capsular polysaccharide are independently linked to the glycan backbone of cell wall peptidoglycan; group B carbohydrate is linked to N-acetylmuramic acid, and capsular polysaccharide is linked via a phosphodiester bond and an oligosaccharide linker to N-acetylglucosamine.