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At least 19 recordsLinked to original sources
PubMed1976

Biosynthesis of peptidoglycan in Gaffkya homari. The incorporation of peptidoglycan into the cell wall and the direction of transpeptidation.

Wall membrane enzyme preparations from Gaffkya homari catalyze the formation of peptidoglycan from the precursor pairs: UDP-N-acetylglucosamine + UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla) and also from UDP-N-acetylglucosamine + UDP-N-acetylmuramyl-tetrapeptide (UDP-MurNAc-Ala-DGlu-Lys-DAla). Part of the reaction products is soluble in 2% sodium dodecylsfulfate whereas the other part is bound to pre-existing cell wall peptidoglycan. The incorporation into cell wall takes place by a transpeptidation reaction in which the D-alanyl-D-alanine sequences in the pre-existing cell wall function as donors and the epsilon-amino groups of the lysine residues in the newly synthesized peptidoglycan strands function as acceptors. Nepsilon-D-Alanyl-lysine linkages are formed. At saturating concentration of UDP-N-acetylglucosamine, the enzyme system exhibits similar apparent Km values (30--80 muM) for UDP-MurNAc-pentapeptide and UDP-MurNAc-tetrapeptide both for the formation of cell-wall bound peptidoglycan and total (i.e. soluble + cell-wall-bound) peptidoglycan. The V values are also in the same order of magnitude (270-650 pmol x min-1 x mg of protein -1). However, UDP-MurNAc-tetrapeptide was a slightly better substrate than UDP-MurNAc-pentapeptide for the formation of cell-wall-bound peptidoglucan. The synthesis of total and cell-wall-bound peptidoglycan from UDP-MurNAc-pentapeptide was competitively inhibited by UDP-MurNAc-tetrapeptide and vice versa. UDP-MurNAc-tripeptide and both UDP-Mur-NAc-pentapeptide and UDP-Mur-NAc-tetrapeptide in which the epsilon-amino group of the lysine residue was substituted by an acetyl group were utilized less efficiently than UDP-MurNAc-pentapeptide and UDP-MurNAc-tetrapeptide for the formation of soluble peptidoglycan; they were exceedingly poor substrates for the formation of cell-wall-bound peptidoglycan.

Acyltransferases↗
PubMed1977

The immunochemistry of peptidoglycan. Antibodies against a synthetic immunogen cross-reacting with an interpeptide bridge of peptidoglycan.

An albumin-peptide conjugate was synthesized, which carries pentaglycine peptides with C-terminal glycine residues as found in the interpeptide bridges of the peptidoglycan of many staphylococci. Immunization of rabbits with this synthetic immunogen yielded antisera containing predominantly antibodies against the peptide moiety of the conjugate. The quantitative precipitin and the Ouchterlony agar gel reaction with several synthetic protein-peptide-conjugates, immunoaffinity chromatography of the antisera on Sepharose-(Gly-Gly-Gly-Gly-Gly)n and hapten inhibition studies with several synthetic peptides and peptide derivatives demonstrated that the antibodies were highly specific for oligoglycine peptides with C-terminal glycine. These antibodies also reacted strongly with staphylococcal peptidoglycans with an interpeptide bridge composed of pentaglycine peptides or of pentaglycine peptides in which part of the glycine residues were replaced by L-serine. In contrast, all the peptidoglycans lacking interpeptide bridges composed of glycine residues gave no precipitin reaction at all. The final proof for identical determinant groups of albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the staphylococcal peptidoglycans applied in the precipitin reaction was furnished by double gel diffusion studies and by hapten inhibition of the precipitin reaction between antisera to albumin-(CH2CO-Gly-Gly-Gly-Gly-Gly)31 and the corresponding peptidoglycans. For rapid screening of the different peptidoglycans, a latex agglutination test was elaborated. Purified antibodies were adsorbed to latex particles, and the titers with the particular peptidoglycans were then determined. The test was highly sensitive, in that 10 nanograms of peptidoglycan could still be detected.

Amino Acids↗
PubMed1979

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 1. The incorporation of peptidoglycan into the cell wall.

Ether-treated cells of Pseudomonas aeruginosa catalyze the formation of crosslinked peptidoglycan from the two nucleotide precursors uridinediphospho-N-acetylglucosamine and uridinediphospho-N-acetylmuramyl-L-alanyl-D-gamma-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine. The main enzymatic reactions of biosynthesis were similar to those found in Escherichia coli. Part of the reaction products were soluble in 4% sodium dodecylsulfate whereas the other part was covalently bound to the preexisting cell wall peptidoglycan sacculus. The incorporation into cell wall is carried out by a transpeptidation reaction in which the nascent peptidoglycan functions mainly as the donor and the preexisting one as acceptor. The detergent-soluble peptidoglycan is composed of partially crosslinked peptidoglycan strands as well as low-molecular-weight peptidoglycan fragments. Pulse-chase biosynthesis experiments show that the detergent-soluble peptidoglycan is an intermediate that eventually becomes covalently bound to the wall. The DD-carboxypeptidase activity of P. aeruginosa is membrane-bound and does not hydrolyse C-terminal D-alanine residues from the L-lysine-containing nucleotide-precursor analogue. An LD-carboxypeptidase was also detected in P. aeruginosa.

Cell Wall↗
PubMed1975

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.

Acetylglucosamine↗
PubMed1976

Biosynthesis of peptidoglycan in Staphylococcus aureus: incorporation of the Nepsilon-Ala-Lys moiety into the peptide subunit of nascent peptidoglycan.

UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla was isolated from extracts of Staphylococcus aureus Copenhagen. This nucleotide accumulated in media deficient in glycine. To establish its role in peptidoglycan biosynthesis, the nucleotide-hexapeptide was compared with UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla in the reaction catalyzed by phospho-MurNAc-pentapeptide translocase and in the membrane-catalyzed nascent peptidoglycan-synthetizing system. In the exchange reaction catalyzed by the translocase, the Rmax and Rmax/Km are 1.79 muM/min and 4.47 X 10(-2)/min, respectively, for UDP-MurNAc-pentapeptide and 1.81 muM/min and 4.46 X 10(-2)/min, respectively, for UDP-Mur-NAc-hexapeptide. In the synthesis of nascent peptidoglycan, the Vmax is 1.8 muM/min X 10(-2) for both the nucleotide-hexapeptide and -pentapeptide. The Vmax/Km is 5.6 X 10(-4) and 4.3 X 10(-4)/min for the nucleotide-pentapeptide and -hexapeptide, respectively. Schleifer, Hammes, and Kandler (Adv. Microb. Physiol. in press) observed that growth of S. aureus Copenhagen on a glycine-poor medium results in a peptidoglycan structure in which 20% of the lysine residues are substituted at the epsilon-amino group by L-alanine residues that do not participate in interpeptide bridge information. The in vitro studies demonstrate that UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-Ala)-DAla-DAla is a possible precursor of the Nepsilon-Ala-Lys moiety.

Acetylglucosamine↗
PubMed1975

Coordinated incorporation of nascent peptidoglycan and teichoic acid into pneumococcal cell walls and conservation of peptidoglycan during growth.

Choline-containing pneumococcal cell wals are sensitive to autolysin, whereas ethanolamine-containing walls are not. Bacteria were labeled with radioactive peptidoglycan precursors while growing either in choline- or in ethanolaminecontaining media. Subsequently, the labeled cells were allowed to grow for four to five generations in nonradioactive medium supplemented with the alternative amino alcohol source (i.e. cells labeled in choline medium yields ethanolamine; cells labeled in ethanolamine medium yields choline). The autolysin sensitivity of the isotope label in cell walls prepared from such bacteria indicates that nascent peptidoglycan and teichoic acid units that are synthesized at the same time are attached to one another, incorporated into the cell surface at the cellular equator, and remain conserved during growth the division of the bacteria.

Alanine↗
PubMed1975

Endotoxin-like properties of the peptidoglycan.

Peptidoglycan is responsible for the endotoxin-like properties of the streptococcus cell wall. The pyrogenic response of rabbit to group A streptococcus peptidoglycan prepared by hot formamide or TCA is dose-dependent and is increased if the material is ultrasonically solubilized. The pyrogenicity can be eliminated by the antiserum to the peptidoglycan or by the degradation of the material by lysozyme. Peptidoglycans prepared from cell walls of group B and L streptococci, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pneumoniae produce fever effects comparable to the response after group A streptococcus peptidoglycan. Spirillum serpens and Escherichia coli contain in addition to endotoxin the peptidoglycan which is also pyrogenic. Repeated injections of bacterial peptidoglycan to rabbit result in tolerance to the fever effect. Cross-tolerance was recorded only exceptionally. Rabbits tolerant to endotoxin respond with a lower fever to S. aureus and group A streptococcus peptidoglycans. Intravenous administration of peptidoglycan to rabbit causes extensive alterations in the heart characterized by various stages of the degenerative and necrotic process. Local Shwartzman reaction can be elicited in rabbit by peptidoglycan used either as a preparative or as a provocative dose in combination with endotoxin, or it can be used for both doses. The results obtained with peptidoglycans prepared from various bacteria are fully comparable. Non-specific resistance of mice to infection induced by streptococcus cell walls was found to be dependent on the peptidoglycan activity; cell wall proteins and polysaccharide are inactive. These properties of peptidoglycan resemble those known from endotoxin studies. The data presented suggest the role of peptidoglycan in pathological reactions resulting from host-parasite interaction.

Animals↗
PubMed1978

The key role of peptidoglycan in the opsonization of Staphylococcus aureus.

In an effort to determine the staphylococcal cell surface component(s) of importance in opsonization, cell walls (peptidoglycan and teichoic acid) and peptidoglycan were isolated from Staphylococcus aureus strain H grown in [3H]glycine-containing broth. After incubation of the cell walls and peptidoglycan with various opsonic sources, uptake by human polymorphonuclear leukocytes was measured. The opsonic requirements for phagocytosis of cell walls and peptidoglycan were found to be similar to those of intact bacteria. Removal of teichoic acid from the cell wall did not affect opsonization. Likewise, a teichoic acid-deficient mutant strain of S. aureus H was opsonized in a manner similar to that of the parent strain. Immunoglobulin G functioned as the major heat-stable opsonic factor and both the classical and alternative pathways participated in opsonization. Kinetic studies revealed that opsonization of peptidoglycan, as well as C3-C9 consumption by peptidoglycan, proceeded at a slower rate via the alternative pathway (C2-deficient serum) than when the classical pathway was present (normal serum). The ability of peptidoglycan to activate C3-C9 was significantly reduced when normal and C2-deficient sera were preabsorbed with peptidoglycan at 2 degrees C suggesting that antibodies to peptidoglycan may be involved in activation of both the classical and alternative complement pathways. Thus, peptidoglycan appears to be the key cell wall component involved in staphylococcal opsonization, and it is suggested that host response to peptidoglycan, a major cell wall component of most gram-positive bacteria, may be related to the development of "natural immunity" to this group of microorganisms.

Cell Wall↗
PubMed1977

Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions.

Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.

Binding Sites↗
PubMed1976

Peptidoglycans synthesized by a membrane preparation of Micrococcus luteus.

By incubation of cell-free particulate preparations from Micrococcus luteus with nucleotidic precursors uridine 5'-diphosphate-N-acetylglucosamine and uridine 5'-diphosphate-N-acetylmuramic acid-L-Ala-D-iso-Glu-L-Lys-D-Ala-D-Ala, several types of peptidoglycans were obtained: soluble peptidoglycan, insoluble peptidoglycan bound to the membrane and solubilized by trypsin, and peptidoglycan, which remained insoluble after the action of trypsin. The structure of each type of peptidoglycan was studied by action of lytic enzymes and separation of the fragments on Sephadex. Soluble peptidoglycans consist of a mixture of un-cross-linked polymers of various molecular weights. Trypsin-solubilized peptidoglycans are also a mixture of polymers of various sizes. They contain a preponderance of un-cross-linked material and some bridges with dimer peptides. Insoluble peptidoglycans, after the action of trypsin, contain about 50% of un-cross-linked peptide residues; in the other moiety, peptide units are cross-linked by D-Ala leads to L-Lys and D-Ala leads to L-Ala bonds which characterize the natural peptidoglycan. Therefore, the cell-free particulate preparation possesses the whole enzymatic system necessary for synthesis of cross-linked peptidoglycan.

Amidohydrolases↗
PubMed1975

Interaction of peptidoglycans with anti-IgGs and with complement.

This report describes the interaction of peptidoglycan (Streptococcus group A, Staphylococcus epidermidis and Micrococcus lysodeikticus) with 2 serum mediator systems, namely with the anti-IgG system and with complement. The observation that the majority of rabbits hyperimmunized with A-variant streptococcal vaccine produced anti-group carbohydrate antisera containing anti-IgGs and antibodies directed to peptidoglycan suggested that the production of these 2 latter antibodies was related. This view was supported by the finding of a monoclonal 7S anti-IgG with antibody specificity for the pentapeptide of peptidoglycan as evidenced by inhibition of the coprecipitation of 7S anti-IgG with antigen-antibody complexes by the pentapeptide. Inhibition of the anti-idiotype reaction by the pentapeptide provided further evidence for the antibody specificity of 7S anti-IgG for peptidoglycan. When added to normal human sera all peptidoglycan preparations inhibited the hemolytic activity of the sera. Consumption of C3 in C2 deficient serum and consumption of C2 in normal serum indicated the activation of both known complement pathways. Activation of the classical pathway of complement was more efficient since 50 mug of peptidoglycan consumed approximately 70% of C2 per ml normal serum whereas more than 2 mg of the same preparations was required to inactivate 17-24% of C3 in C2 deficient sera. Each of the different peptidoglycan preparations consumed similar amounts of complement in all 20 sera tested. This finding suggested that activation of the classical complement pathway by peptidoglycan was not mediated by anti-peptidoglycan antibodies present in only 20-40% of normal human sera.

Animals↗
PubMed1977

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. I. Tolerance and effect of antibody in fever response, and heart damaging effect in rabbits.

Induced tolerance to the pyrogenic action of group A streptococcus peptidoglycan decreased after one week and was no longer detectable after the second week. However, one or two further doses of peptidoglycan rapidly restored the tolerance. The passive transfer of plasma from rabbits tolerant to streptococcus peptidoglycan to nontolerant animals failed to transfer tolerance. Antiserum to streptococcus peptidoglycan neutralized the pyrogenic effect of not only streptococcus but also staphylococcus and pneumococcus peptidoglycan; it did not influence the febrile response to endotoxin. Histopathologic changes in the rabbit heart produced by the intravenous injection of staphylococcus or pneumococcus peptidoglycans were similar and were characterized by various stages of degeneration and necrosis. The changes were less pronounced than after streptococcus peptidoglycan. Antiserum to streptococcus peptidoglycan had modest or no counteracting effect on the development of heart alterations after staphylococcus or pneumococcus peptidoglycan.

Animals↗
PubMed1976

Peptidoglycan biosynthesis in a thermosensitive division mutant of Escherichia coli.

Peptidoglycan biosynthesis during a bacterial division cycle was investigated in the thermosensitive division mutant Escherichia coli PAT 84 Synchronous cell division of this organism was initiated by a shift down from restrictive to permissive growth temperature. Cells harvested at different times after a shift down of temperature served as representatives of the various stages during cell division. These cells were made permeable to peptidoglycan nucleotide-sugar precursors by pretreatment with ether and were found capable of catalyzing the polymerization of externally added precursors as well as the covalent attachment of the newly synthesized peptidoglycan strands to those preexisting in the cell wall. Differences were observed in the rats of peptidoglycan synthesis and in the extent of peptide side-chain corss-linkage at the various stages of division. Nonseptate filaments, formed at the restrictive temperature, incorporated significantly more peptidoglycan which was more cross-linked than in normally dividing cells grown at the permissive temperature. Quantitative analyses of the carboxypeptidase and transpeptidase reactions in cells at different stages of division were performed and the inhibitory effect of a number of beta-lactam antibiotics was investigated. Of special significance was the finding that low doses of penicillin or growth at restrictive temperature, which did not affect transpeptidation, partially inhibited the carboxypeptidase activity. This inhibition was paralleled by an increase in incorporation of newly synthesized peptidoglycan into the preexisting cell wall. We therefore propose that carboxypeptidase activity regulates the availability of peptidoglycan precursor(s) for attachment to the preexisting peptidoglycan by transpepidation.

Amino Acids↗
PubMed1976

Immunochemical study of the peptidoglycan of gram-negative bacteria.

The specificity of antibodies directed against the peptidoglycan of gram-negative bacteria was studied. The peptidoglycans of Proteus vulgaris, Escherichia coli, Moraxella glucidolytica, Neisseria perflava, give identical precipitin reactions. By means of inhibition studies with various peptidoglycan subunits and synthetic peptides, it was shown that the antibodies are essentially directed against the peptide moiety of the peptidoglycan: L-Ala-D-Glu (L)-mesoA2pm-(L)-D-Ala, that the peptide reacts better with antibodies when it is not cross-linked, and that the C-terminal portion-meso-A2pm-D-Ala of the peptide is immunodominant. These results explain the immunological identity of the peptidoglycans of gram-negative bacteria, which possess the same peptide subunit. Only weak cross-reactivity was observed with the peptidoglycans of gram-positive bacteria (Streptococcus faecium, Micrococcus lysodeikticus, Corynebacterium poinsettiae) where meso-diaminopimelic acid is replaced by L-lysine or L-homoserine. However, the peptidoglycan of Bacillus megaterium which possesses the same peptide subunit as gram-negative bacteria, gives only a reaction of partial identity with these bacteria. This result suggests the presence on the peptidoglycan of gram-negative bacteria, of other undefined antigenic determinants.

Bacteria↗
PubMed1975

The thrombocytolytic activity of bacterial peptidoglycans.

The peptidoglycan of group A Streptococcus prepared by hot formamide or TCA extraction has an expressive ability to lyse rabbit blood platelets in vitro. Initial changes in the ultrastructure of platelets can be observed after 10 minutes incubation of rabbit platelets with 0.1 mug of Streptococcus peptidoglycan per ml. The submicroscopic alterations are characterized by changes of the shape and damage of the limiting membrane of the platelets. Larger amounts of peptidoglycan (10 mug/ml and more) cause total destruction of platelets; only remainders of the limiting membrane and free granulomers can be seen. Peptidoglycans of Streptococcus pneumoniae and Staphylococcus aureus exhibit a comparable effect on rabbit platelets. There are substantial differences in the sensitivity of blood platelets of different animal species to the streptococcal peptidoglycan. Rat platelets exhibit a similar thrombocytolytic effect after the Streptococcus peptidoglycan treatment as rabbit ones; however, the development of comparable changes in their submicroscopic structure needs a dose of peptidoglycan 10(2)-10(3) times higher. Platelets of guinea-pig, dog, cat, calf and human appear to be quite resistant under analogous conditions to as such a high dose of Streptococcus peptidoglycan as 500 mug/ml.

Animals↗
PubMed1978

Biological characteristics of peptidoglycans of group A streptococcus and some other bacterial species. II. Immunological mechanisms involved in thrombocytolysis.

Immunological mechanisms are involved in the thrombocytolytic activity of peptidoglycan of Group A streptococcus, Streptococcus pneumoniae and Staphylococcus aureus. Inactivation of particular components of complement (heating of blood serum to 56 degrees C,incubation with zymosan or NH4OH) inhibited the thrombocytolytic activity of group A streptococcus peptidoglycan. So did preincubation of Group A streptococcus peptidoglycan with homologous antipeptidoglycan antibody. On the other hand, antibody to Group A streptococcus peptidoglycan did not inhibit the thrombocytolytic effect of Streptococcus pneumoniae or Staphylococcus aureus peptidoglycan. Human platelets are resistant to peptidoglycans. They remain resistant in the presence of rabbit serum although rabbit platelets are highly sensitive to peptidoglycans. This suggests that, for the expression of the thrombocytolytic activity of bacterial peptidoglycan, specific receptors on the surface of platelets must be present in addition to serum factors.

Animals↗