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PubMed2005

Staphylococcus aureus peptidoglycan is a toll-like receptor 2 activator: a reevaluation.

Since the ability of peptidoglycan (PGN) to activate Toll-like receptor 2 (TLR2) was recently questioned, we reevaluated activation of TLR2 by PGN. Polymeric soluble or insoluble Staphylococcus aureus PGN, repurified by sodium dodecyl sulfate or phenol extraction, activated TLR2 at 0.1 to 1 or 10 mug/ml, respectively, and induced tumor necrosis factor alpha production. The TLR2 activation by PGN, but not by lipoteichoic acid, was abolished by muramidase digestion. We conclude that polymeric S. aureus PGN is a TLR2 activator.

Humans↗
PubMed1980

Structure of the regular surface layer of Sporosarcina ureae.

Optical diffraction and computer image processing of electron micrographs were employed to analyze the structure of the regular surface layer of Sporosarcina ureae at high resolution. Negatively stained preparations of regular surface layer fragments showed two types of tetragonal pattern, each having p4 symmetry in projection with a = 12.8 nm. Although the two patterns differed greatly in overall appearance, both had a common pattern of areas of high stain density which we interpret as arising from gaps or holes in the structure. We speculate that these holes may be related to a protective role of the regular surface layer, whereby hostile environmental agents (such as muramidases) larger than about 2 nm would be screened from the underlying layers of the bacterial surface, while the free passage of nutrients and waste products into and out of the cell would still be allowed.

Bacillaceae↗
PubMed1985

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.

Amino Acids↗
PubMed1985

Analysis of Neisseria gonorrhoeae peptidoglycan by reverse-phase, high-pressure liquid chromatography.

The muramidase digest of peptidoglycan from Neisseria gonorrhoeae was isolated and analyzed by the use of a reverse-phase, high-pressure liquid chromatography system. As was found previously in the case of Escherichia coli, gonococci peptidoglycan is also composed of a greater number of muropeptides than can be resolved with thin-layer chromatography systems. Preliminary classification of the muropeptide components into subclasses based on O-acetyl modification and degree of cross-linkage was achieved. Examination of a penicillin-susceptible strain and a highly resistant strain with two penicillin-binding protein alterations synthesized distinctly different peptidoglycan structures, as revealed by this technique.

Bacterial Proteins↗
PubMed1987

Identification and preliminary characterization of a Streptococcus sanguis fibrillar glycoprotein.

Cell surface fibrils could be released from Streptococcus sanguis 12 but not from strains 12na or N by freeze-thawing followed by brief homogenization. Fibrils were isolated from the homogenate by ultracentrifugation or ammonium sulfate precipitation. Electron microscopy demonstrated the presence of dense masses of aggregated fibrils in these preparations. Under nondenaturing conditions, no proteins were seen in polyacrylamide gel electrophoresis (PAGE). Sodium dodecyl sulfate (SDS)-PAGE analysis revealed a single band stained with Coomassie blue and periodic acid Schiff stain with a molecular weight in excess of 300,000. The protein has been given the name long-fibril protein (LFP). The molecule was susceptible to digestion with subtilisin, pronase, papain, and trypsin, but was unaffected by chymotrypsin or muramidases. Attempts to dissociate the protein into smaller subunits with urea, guanidine, sodium thiocyanate, and HCl were unsuccessful. Gel filtration on a column of Sephacryl S-400 in the presence of 2% SDS resulted in elution of the protein at the void volume. Antibody raised against the LFP excised from an SDS-PAGE gel reacted with long fibrils on the surface of strain 12 and with isolated fibrils by an immunogold labeling technique. Monoclonal antibody reactive with LFP in SDS-PAGE also reacted with fibrils present on the cell. Antisera raised against the fibrils inhibited adherence to saliva-coated hydroxyapatite.

Amino Acids↗
PubMed1987

Two distinct transpeptidation reactions during murein synthesis in Escherichia coli.

Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.

Amdinocillin↗
PubMed1991

femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.

Acetylglucosamine↗
PubMed1993

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.

Amino Acids↗
PubMed1995

Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection.

F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection.

Amino Acid Sequence↗
PubMed1996

Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the delta-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid delta-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% delta-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulation-specific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid delta-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of delta-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed.

Bacillus subtilis↗
PubMed1996

Analysis of the peptidoglycan structure of Bacillus subtilis endospores.

Peptidoglycan was prepared from purified Bacillus subtilis spores of wild-type and several mutant strains. Digestion with muramidase resulted in cleavage of the glycosidic bonds adjacent to muramic acid replaced by peptide or alanine side chains but not the bonds adjacent to muramic lactam. Reduction of the resulting muropeptides allowed their separation by reversed-phase high-pressure liquid chromatography. The structures of 20 muropeptides were determined by amino acid and amino sugar analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In wild-type spores, 50% of the muramic acid had been converted to the lactam and 75% of these lactam residues were spaced regularly at every second muramic acid position in the glycan chains. Single L-alanine side chains were found on 25% of the muramic acid residues. The remaining 25% of the muramic acid had tetrapeptide or tripeptide side chains, and 11% of the diaminopimelic acid in these side chains was involved in peptide cross-links. Analysis of spore peptidoglycan produced by a number of mutants lacking proteins involved in cell wall metabolism revealed structural changes. The most significant changes were in the spores of a dacB mutant which lacks the sporulation-specific penicillin-binding protein 5*. In these spores, only 46% of the muramic acid was in the lactam form, 12% had L-alanine side chains, and 42% had peptide side chains containing diaminopimelic acid, 29% of which was involved in cross-links.

Acetylglucosamine↗
PubMed1998

Acquisition of five high-Mr penicillin-binding protein variants during transfer of high-level beta-lactam resistance from Streptococcus mitis to Streptococcus pneumoniae.

Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in beta-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of beta-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzyl-penicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 microg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level beta-lactam resistance by interspecies gene transfer.

Amino Acid Sequence↗
PubMed1998

Generation and properties of a Streptococcus pneumoniae mutant which does not require choline or analogs for growth.

A mutant (JY2190) of Streptococcus pneumoniae Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (N-acetylmuramyl-L-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86-93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.

Carbohydrate Sequence↗
PubMed2003

Substrate specificity classes and the recognition signal for Salmonella type III flagellar export.

Most flagellar proteins of Salmonella are exported to their assembly destination via a specialized apparatus. This apparatus is a member of the type III superfamily, which is widely used for secretion of virulence factors by pathogenic bacteria. Extensive studies have been carried out on the export of several of the flagellar proteins, most notably the hook protein (FlgE), the hook-capping protein (FlgD), and the filament protein flagellin (FliC). This has led to the concept of two export specificity classes, the rod/hook type and the filament type. However, little direct experimental evidence has been available on the export properties of the basal-body rod proteins (FlgB, FlgC, FlgF, and FlgG), the putative MS ring-rod junction protein (FliE), or the muramidase and putative rod-capping protein (FlgJ). In this study, we have measured the amounts of these proteins exported before and after hook completion. Their amounts in the culture supernatant from a flgE mutant (which is still at the hook-type specificity stage) were much higher than those from a flgK mutant (which has advanced to the filament-type specificity stage), placing them in the same class as the hook-type proteins. Overproduction of FliE, FlgB, FlgC, FlgF, FlgG, or FlgJ caused inhibition of the motility of wild-type cells and inhibition of the export of the hook-capping protein FlgD. We also examined the question of whether export and translation are linked and found that all substrates tested could be exported after protein synthesis had been blocked by spectinomycin or chloramphenicol. We conclude that the amino acid sequence of these proteins suffices to mediate their recognition and export.

Bacterial Outer Membrane Proteins↗
PubMed1965

PURIFICATION OF STAPHYLOCOCCAL BETA-HEMOLYSIN AND ITS ACTION ON STAPHYLOCOCCAL AND STREPTOCOCCAL CELL WALLS.

Chesbro, William R. (University of New Hampshire, Durham), Fred P. Heydrick, Roland Martineau, and Gail N. Perkins. Purification of staphylococcal beta-hemolysin and its action on staphylococcal and streptococcal cell walls. J. Bacteriol. 89:378-389. 1965.-After growth of bovine-derived strains of Staphylococcus aureus in a completely dialyzable medium, the beta-hemolysin in the culture supernatant fluids was purified by gradient-elution chromatography on cellulose phosphate. The purified hemolysin contained two components, demonstrable by immunodiffusion or electrophoresis, but was free from alpha-hemolysin, coagulase, Delta-hemolysin, enterotoxins A and B, glucuronidase, hyaluronidase, lipase, muramidase, Panton-Valentine leukocidin, phosphatase, and protease. The hemolysin was heat-labile and sulfhydryl-dependent, and the preparation was leukocidal for guinea pig macrophages. When rabbit red blood cell (RBC) stroma and staphylococcal or enterococcal cell walls were treated with the purified hemolysin, it liberated mucopolysaccharides from the rabbit RBC stroma, polysaccharides and mucopolysaccharides (or mucopeptides) from the staphyloccoal cell walls, and rhamnose, glucose, an unidentified monosaccharide, N-acetylglucosamine, and at least two polysaccharides from the enterococcal cell walls. The hemolytic and cell-wall degradative activities had similar thermal inactivation kinetics, pH optima, sedimentation coefficients, and chromatographic and electrophoretic mobilities; both required Mg and were inhibited by thiol-inactivating agents. Consequently, it seems likely that both activities are expressions of the same enzyme.

Animals↗
PubMed1968

Peptidoglycan of Myxococcus xanthus: structure and relation to morphogenesis.

The chemical nature and distribution of the peptidoglycan in Myxococcus xanthus at various stages of the cellular life cycle were investigated. Vegetative cells and microcysts contained approximately 0.6% by weight of peptidoglycan. The overall composition of the peptidoglycan was similar in both cell types and was approximately 1 glutamic acid, 1 diaminopimelic acid, 1.7 alanine, 0.75 N-acetylglucosamine, and 0.75 N-acetylmuramic acid. (We have assumed that all the hexosamines are N-acetylated.) The sizes of the subunits (estimated by gel filtration) solubilized by muramidases were considerably larger (tetramer and oligomer) in the microcysts than in the vegetative cells (mostly dimer). There was a transient decrease in cross-linking (measured as an increase in the amount of free amino group of diaminopimelic acid) during the stage of microcyst formation when the cells converted from ovoids to spheres. At the same time, there occurred a large and rapid increase in a galactosamine derivative which may have reflected the synthesis of capsular material. Immediately prior to this period of morphogenesis, the cells became resistant to penicillin but remained sensitive to d-cycloserine. The walls of vegetative cells were completely disaggregated by trypsin and sodium lauryl sulfate, suggesting a discontinuous peptidoglycan layer. This was no longer apparent after the ovoid-sphere stage of microcyst formation. The relationship to morphogenesis of the chemical changes in the cell wall is discussed.

Amino Acids↗
PubMed1997

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.

Bacterial Typing Techniques↗
PubMed1999

Relatedness of Streptococcus suis serotype 2 isolates from different geographic origins as evaluated by molecular fingerprinting and phenotyping.

The genetic diversity of 88 Streptococcus suis serotype 2 isolates which were recovered from various countries was examined by randomly amplified polymorphic DNA (RAPD) analysis with three primers. This bacterial collection included 80 isolates of porcine origin and 8 of human origin. This investigation allowed the identification of 23 RAPD types containing 1 to 30 isolates originating from one to six countries. Common RAPD patterns were found between human and pig isolates. The isolates were also tested for the production of virulent factors such as hemolysin, muramidase-released protein (MRP), and extracellular factor (EF). All isolates exhibiting the virulent phenotype hemolysin+ MRP+ EF+ clearly clustered on the basis of fingerprinting by RAPD analysis. In a similar way, most of isolates with the hemolysin- MRP- EF- phenotype were assigned to one RAPD cluster. Therefore, RAPD clusters are more related to the phenotype defined with hemolysin, MRP, and EF than to the geographic origin of the isolates. These data indicate that RAPD analysis used in conjunction with phenotypic methods provides a reliable method for the assessment of the clonal relationship between S. suis isolates responsible for infections in pigs or humans, especially for those exhibiting the classic "virulent" phenotype hemolysin+ MRP+ EF+.

Animals↗