The direction of glycan synthesis in a bacterial peptidoglycan

A cell-free membrane preparation from a poorly lytic mutant of Bacillus licheniformis was used to synthesize radioactive peptidoglycan. The product was apparently un-cross-linked. When UDP-N-acetyl[(14)C]glucosamine was used and the final peptidoglycan subjected to Smith degradation, no radioactive glycerol was found. On the other hand, when peptidoglycan labelled with meso-diamino[(14)C]pimelic acid was first hydrolysed in 0.1m-HCl at 60 degrees C for 2h and then subjected to alkaline conditions, radioactive lactyl-peptides were eliminated. The proportion of radioactive lactyl-peptide decreased with increasing time of incorporation. It is concluded that the glycan chains grow by extension at their reducing ends while remaining attached by some linkage labile to mild acid, such as a glycosyl link to undecaprenol pyrophosphate.     

Structural variation in the glycan strands of bacterial peptidoglycan

The normal, unmodified glycan strands of bacterial peptidoglycan consist of alternating residues of beta-1,4-linked N-acetylmuramic acid and N-acetylglucosamine. In many species the glycan strands become modified after their insertion into the cell wall. This review describes the structure of secondary modifications and of attachment sites of surface polymers in the glycan strands of peptidoglycan. It also provides an overview of the occurrence of these modifications in various bacterial species. Recently, enzymes responsible for the N-deacetylation, N-glycolylation and O-acetylation of the glycan strands were identified. The presence of these modifications affects the hydrolysis of peptidoglycan and its enlargement during cell growth. Glycan strands are frequently deacetylated and/or O-acetylated in pathogenic species. These alterations affect the recognition of bacteria by host factors, and contribute to the resistance of bacteria to host defence factors such as lysozyme.     

Production and characterization of peptide antibodies

Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist, including solid-phase peptide-carrier conjugation and peptide-carrier conjugation in solution. Upon immunization, adjuvants such as Al(OH)(3) are added together with the immunogenic peptide-carrier conjugate, which usually leads to high-titred antisera. Following immunization and peptide antibody purification, the antibodies are characterized based on their affinity or specificity. An efficient approach for characterization of peptide antibodies is epitope mapping using peptide based assays. This review describes standard solid-phase approaches for generation of peptide antibodies with special emphasis on peptide selection, generation of peptide conjugates for immunization and characterization of the resulting peptide antibodies.     

The immunochemistry of antibodies sharing concanavalin A's anti-mannosyl binding specificity.

Procedures were developed for the synthesis of the disaccharide hapten, p-isothiocyanatophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside, and for its conjugation to hemocyanin. The synthetic carbohydrate: protein antigen was then emulsified in complete Freund's adjuvant and injected into the footpads of New Zealand White rabbits. A population of the resulting anti-conjugate antibodies displayed some binding properties analogous to concanavalin A, the carbohydrate-binding protein of the jack bean. The antisera weakly percipitated mannans from Saccharomyces rouxii, S. cerevisiae, and an alpha-(1 leads to 3)-mannopyranosyl transferase-deficient mutant from Kluyveromyces lactis Y58a. These polysaccharides, possessing side chains containing terminal alpha-(1 leads to 2)-mannobiosyl residues, produce strong percipitation reactions with concanavalin A. In addition, various saccharides were tested for their ability to inhibit the interaction of anti-conjugate antisera with alpha-(1 leads to 2)-mannobiosyl-containing polymers. p-Nitrophenyl 2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside showed a strong complementarity for the binding sites of both the anti-conjugate antisera and concanavalin A. However, the antibody failed to bind a concanavalin A-reactive mouse fibrosarcoma or to stimulate mitogenesis of human peripheral lymphocytes.     

Formation of the glycan chains in the synthesis of bacterial peptidoglycan

The main structural features of bacterial peptidoglycan are linear glycan chains interlinked by short peptides. The glycan chains are composed of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), all linkages between sugars being beta,1-->4. On the outside of the cytoplasmic membrane, two types of activities are involved in the polymerization of the peptidoglycan monomer unit: glycosyltransferases that catalyze the formation of the linear glycan chains and transpeptidases that catalyze the formation of the peptide cross-bridges. Contrary to the transpeptidation step, for which there is an abundant literature that has been regularly reviewed, the transglycosylation step has been studied to a far lesser extent. The aim of the present review is to summarize and evaluate the molecular and cellullar data concerning the formation of the glycan chains in the synthesis of peptidoglycan. Early work concerned the use of various in vivo and in vitro systems for the study of the polymerization steps, the attachment of newly made material to preexisting peptidoglycan, and the mechanism of action of antibiotics. The synthesis of the glycan chains is catalyzed by the N-terminal glycosyltransferase module of class A high-molecular-mass penicillin-binding proteins and by nonpenicillin-binding monofunctional glycosyltransferases. The multiplicity of these activities in a given organism presumably reflects a variety of in vivo functions. The topological localization of the incorporation of nascent peptidoglycan into the cell wall has revealed that bacteria have at least two peptidoglycan-synthesizing systems: one for septation, the other one for elongation or cell wall thickening. Owing to its location on the outside of the cytoplasmic membrane and its specificity, the transglycosylation step is an interesting target for antibacterials. Glycopeptides and moenomycins are the best studied antibiotics known to interfere with this step. Their mode of action and structure-activity relationships have been extensively studied. Attempts to synthesize other specific transglycosylation inhibitors have recently been made.     

The action of lysozyme on peptidoglycan with N-unsubstituted glucosamine residues. Isolation of glycan fragments and their susceptibility to lysozyme.

1. A peptidoglycan preparation N-acetylated at about 30% of glucosamine residues was obtained by the treatment of the lysozyme-resistant cell wall paptidoglycan of Bacillus cereus with acetic anhydride at pH 7. Fractionation of dialyzable material resulting from lysozyme digestion of the glycan component of this peptidoglycan preparation yielded five oligosaccharides designated as S1 to S5 besides the disaccharide GlcNAc-MurAc. 2. Oligosaccharide S3, which accounted for about 30% of the disaccharide units recovered as disaccharides and oligosaccharides, was identified as GlcN-MurAc-GlcNAc-MurAc. Oligosaccharide S1, accounting for about 20% of the disaccharide units recovered, was characterized as GlcN-MurAc-GlcN-MurAc-GlcNAc-MurAc, while oligosaccharide S2, present in a smaller amount, as GlcNAc-MurAc-GlcN-MurAc-glcNAc-MurAc. Oligosaccharides S4, and S5, present in small amounts, were identified as GlcNAc-MurAc-GlcNAc-MurAc and MurAc-GlcNAc-MurAc, respectively. 3. Oligosaccharides S1, S3 and S5 proved to be completely insusceptible to lysozyme, whereas S2 was digsted by lysozyme to produce GlcNAc-MurAc and S3. S1 was found to act as a more potent inhibitor than S3 in lysozyme-catalyzed digestion of polysaccharides. 4. The results obtained show that the lysozyme-catalyzed hydrolysis of peptidoglycan oligosaccharides had an obligatory requirement for the N-acetyl group on the glucosamine residue located in subsite C in the enzyme-substrate complex.     

Theoretical studies on peptidoglycans. II. Conformations of the disaccharide–peptide subunit and the three-dimensional structure of peptidoglycan

Possible conformations of the disaccharide–peptide subunit of peptidoglycan (of Staphylococcus aureus or Micrococcus luteus) have been studied by an energy-minimization procedure. The favored conformation of the disaccharide N-acetyl-glucosaminyl-β(1–4)-N-acetylmuramic acid (NAG-NAM) is different from that of cellulose or chitin; this disagrees with the assumption of earlier workers. The disaccharide–peptide subunit favors three types of conformations, among which two are compact and the third is extended. All these conformations are stabilized by intramolecular hydrogen bonds. Based on these conformations of the subunit, two different models are proposed for the three-dimensional arrangement of peptidoglycan in the bacterial cell wall.     

Biosynthesis of peptidoglycan in Gaffkya homari: role of the peptide subunit of uridine diphosphate-N-acetylmuramyl-pentapeptide

The incorporation of N-acetylmuramyl (MurNAc)-peptides from nucleotide-activated precursors (reference: uridine diphosphate [UDP]MurNAc-Ala(1)-dGlu(2)-Lys(3)- dAla(4)-dAla(5)) with incomplete or modified peptide subunits into peptidoglycan was studied with membrane preparations from Gaffkya homari. The effectiveness of their utilization at low and high concentrations was compared on the basis of the values of V(max)/K(m) and V(max), respectively. At low concentration, replacement of alanine by glycine in position 5 has a small effect on the activity of the peptidoglycan synthesizing system, whereas it has a significantly larger effect in positions 1 and 4. The importance of d-alanine in position 4 at low substrate concentrations is also observed with the incomplete UDP-MurNAc-peptides. For UDP-MurNAc-tripeptide and -tetrapeptide, V(max)/K(m) is 0.06 and 0.55, respectively, of the value for the -pentapeptide. At high substrate concentration, replacement of d-alanine by glycine in either position 1 or 5 decreases the activity to 0.37 of the value for the reference nucleotide, whereas replacement in position 4 has a smaller effect (0.74). The profiles established from V(max) and V(max)/K(m) with UDP-MurNAc-tripeptide, -tetrapeptide, and -pentapeptide show good correlation. At low concentration the specificity profiles of phospho-MurNAc-pentapeptide translocase, catalyzing the initial membrane reaction, are similar to those for the peptidoglycan synthesizing system; at high concentration, however, the profiles differ. The translocase appears to provide a primary specificity barrier at high substrate concentration for UDP-MurNAc-Ala-dGlu-Lys-dAla-dAla and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla, and at low concentration for UDP-MurNAc-Ala-dGlu-Lys and UDP-MurNAc-Ala-dGlu-Lys-Gly-dAla. Moreover, it is suggested that an additional specificity barrier exists in the peptidoglycan synthesizing system for certain nucleotides. Thus, the cytoplasmic enzymes and the membrane-associated enzyme(s) cooperate to insure the formation of functioning peptidoglycan in this organism.     

Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping

Monoclonal antibodies (mAb) and a polyclonal antiserum were produced against a kainic acid receptor (KAR) purified from frog brain. Several of the mAb and the antiserum immunoprecipitated [3H]kainic acid binding activity from solubilized preparations of frog brain and labeled a group of proteins on immunoblots that migrated at Mr = 48,000. These results confirm that the ligand binding subunit of the frog brain KAR is contained in the Mr = 48,000 proteins. Immunoblots from different frog tissues demonstrated that the antibody reactivity was highly concentrated in the frog nervous system with no detectable immunoreactivity observed in non-neuronal tissues. The purified KAR was radioiodinated and subjected to two-dimensional gel electrophoresis and autoradiography. A series of proteins was detected at Mr = 48,000 with isoelectric points from 5.5 to 6.3. The anti-KAR mAb and the antiserum reacted with the same group of proteins from frog whole brain after separation by two-dimensional gel electrophoresis. Peptide maps of the 125I-labeled KAR separated by two-dimensional gel electrophoresis demonstrated that the group of proteins clustered at Mr = 48,000 is homologous. mAb KAR-B1 reacted on immunoblots with a protein in rat brain with a Mr = 99,000. This protein comigrated with an unreduced form of the KAR in frog brain. It was present in rat cerebral cortex, hippocampus, and cerebellum but was not detected in thalamus, globus pallidus, or brain stem, nor was it detected in rat non-neuronal tissues. The presence of the Mr = 99,000 immunoreactive polypeptide in discrete areas of rat brain suggests that this protein may be part of a mammalian KAR or a related receptor.     

Immunoelectron microscopic studies on the localization of peptidoglycan peptide subunit pentapeptides in bacterial cell walls

The peptide subunit pentapeptide H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH₂ of peptidoglycan was localized in the cell walls of several Gram-positive bacteria employing the indirect immunoferritin technique. Specific antibodies to the D-alanyl-D-alanine moiety of non-crosslinked peptide subunit pentapeptide were raised in rabbits by immunization with synthetic immunogen albumin-(CH₂CO-Gly-L-Ala-L-Ala-D-Ala-D-Ala-OH)₃₉. Specificity of these antibodies for the peptide subunit pentapeptide and not for the peptide subunit tetrapeptide was corroborated in a Farr-type radio-active hapten binding assay. Specificity of labelling with ferritin was established by immunoelectron microscopic controls, and by the excellent correlation between specific labelling of cells with ferritin and the particular peptidoglycan primary structure of bacterial strains investigated. Cells of Lactobacillus gasseri, Streptococcus pyogenes and Staphylococcus aureus revealing non-crosslinked peptide subunit pentapeptides in their peptidoglycans could specifically be labelled. Lactobacillus acidophilus and Bacillus subtilis, on the contrary, missing such pentapeptides, failed in labelling.The implication of this method to possibly localize the points of attack of penicillin or cycloserine is discussed.Abbreviations used meso-A₂pm meso-diaminopimelic acid - DSM Deutsche Sammlung für Mikroorganismen, Göttingen, FRG This paper is dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Specific antibodies to the N-termini of the interpeptide bridges of peptidoglycan

The synthetic peptides Gly₅--Ahx and l-Ala₃--Ahx, with structural similarity to the interpeptide bridge peptides of staphylococci or micrococci, respectively, were covalently linked to human serum albumin via their carboxylgroups. Antisera to these synthetic peptidyl-protein antigens contained fairly high amounts of antibodies with specificity to the N-terminal parts of the peptide chains attached to the carrier proteins. Antisera to (Gly₅--Ahx)₂₀-albumin gave, without exception, strong precipitin reactions in latex-agglutination with staphylococcal peptidoglycans. The antisera completely failed, however, in any reaction with peptidoglycans of micrococci or other bacteria which did not have these oligo-glycine peptides typical for staphylococci. On the contrary, antisera to (l-Ala₃--Ahx)₂₂-albumin strongly precipitated micrococcal peptidoglycans with oligo-l-alanine interpeptide bridges (e.g. Micrococcus varians, Micrococcus reseus), but showed no significant reaction with peptidoglycans of staphylococci or other bacteria lacking oligo-l-alanine interpeptide bridges.Abbreviations Use Ac acetyl- - -Ahx -amino caproic acid - ATCC American Type Culture Collection, Rockville, Md., U.S.A. - CCM Czechoslovak Collection of Microorganisms, Brno, CSSR - DSM Deutsche Sammlung für Mikroorganismen, München, FRG - IMRU Institute of Microbiology, Rutgers University, N.J., U.S.A. - Kiel Bundesanstalt für Milchforschung, Kiel, FRG - NPS o-nitrophenylsulphenyl- - -OMe methyl ester - -OSu succinimide ester - Z- benzyloxycarbonyl     

A radioactive hapten-binding assay for measuring antibodies to the pentapeptide determinant of peptidoglycan.

A major portion of the humoral immune response to peptidoglycans is directed against the non-cross-linked pentapeptide side chains of these ubiquitous bacterial antigens. At present, no specific and sensitive assay for pentapeptide antibody determination is available. Therefore, a radioimmunoassay has been developed which employs the synthetic pentapeptide hapten L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala, labeled by the active ester method of Bolton and Hunter to high specific activities (6.74 to 18.18 muCi/mug) with 125I, and used as a reagent for measuring pentapeptide antibody. A-variant streptococcal antisera, known to contain pentapeptide antibodies as shown by quantitative precipitation, would bind more than 95% of the radiolabeled hapten in contrast to 2 to 3% by preimmune rabbit sera. Specificity of the binding reaction was demonstrated by inhibition experiments imploying various synthetic oligopeptides related or unrelated to the pentapeptide in the radioimmunoassay. Binding curves established with serial dilutions of peptidoglycan antiserum were linear from 15 to 500 mug/ml of antibody permitting pentapeptide antibody measurement within this range. Comparative data on pentapeptide antibody determinations by quantitative precipitation and radioimmunoassay are given and the time course of the production of this antibody in 14 rabbits hyperimmunized with A-variant streptococcal vaccine is reported.     

The final step of peptidoglycan subunit assembly in Escherichia coli occurs in the cytoplasm.

The murG gene of Escherichia coli encodes the N-acetylglucosaminyltransferase responsible for the final step in the formation of the lipid-linked disaccharide-pentapeptide subunit of peptidoglycan. Using trypsin to probe maxicell spheroplasts, we show that this enzyme is peripherally associated with the inner face of the cytoplasmic membrane. Therefore, the peptidoglycan subunit is completely assembled before it traverses the cytoplasmic membrane.     

Quantum chemical studies on the conformational structure of bacterial peptidoglycan. I. MNDO calculations on the glycan moiety

Semi-empirical quantum chemical calculations at MNDO level of approximations have been carried out on the monosaccharide and disaccharide moiety of bacterial peptidoglycan to determine the energetically favoured conformation of their side groups and the relative orientations of sugar rings. The results have been compared with those obtained from empirical energy calculations. The MNDO results have also been discussed with available experimental data and suggest that a chitin-like structure is not favoured for the glycan moiety of peptidoglycan.     

Hemoglobins of the killifish Fundulus heteroclitus. Separation, characterization, and a model for the subunit compositions.

Polyacrylamide and starch gel electrophoresis of the hemoglobin of the killifish Fundulus heteroclitus reveal the presence of four clearly distinguishable components. These isohemoglobins, each tetramers consisting of alpha and beta chains, can be preparatively separated by ion exchange chromatography on DEAE-cellulose and are homogeneous according to isoelectric focusing in polyacrylamide gels. Oxygen equilibria of the isolated hemoglobin components (Hb I, Hb II, Hb III, and Hb IV) show only minor differences in the magnitude of the Bohr effect and in the effect of ATP on the binding of oxygen. Four different globin chains, alphaa, alphab, betaa, and betab, can be separated by ion exchange on CM-cellulose. Hb I is a homotetramer of alphab and betab chains, Hb IV consists of alphaa and betaa subunits, and components II and III are heterotetramers consisting of all four chains. The alpha and beta chains differ significantly in amino acid composition. A model suggesting the existence of 10 different isohemoglobins, 6 of which have stable intersubunit contacts, has been proposed to account for the qualitative and quantitative aspects of the electrophoretic behavior of the components. Separations of the isohemoglobins on DEAE-cellulose under slightly modified conditions provide additional support for the model.     

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.     

Structure of the peptide network of pneumococcal peptidoglycan

The peptide network of Streptococcus pneumoniae cell walls was solubilized using the pneumococcal autolytic amidase (N-acetylmuramoyl-L-alanine amidase, EC 3.5.1.28). The peptide material was fractionated into size classes by gel filtration followed by reverse-phase high-performance liquid chromatography which resolved the peptide population into over 40 fractions. About 40% of the lysines present participate in cross-links between stem peptides. The main components (3 monomers, 5 dimers, and 2 trimers), accounting for 77% of all the wall peptides, were purified. Their structures were determined using a combination of amino acid and end-group analysis, mass spectrometry, and gas-phase sequencing. Two different types of cross-links between stem peptides were found. In the most abundant type there is an alanylserine cross-bridge between the alanine in position 4 of the donor stem peptide and the lysine at position 3 of the acceptor peptide, as in type A3 peptidoglycan. In the second type of cross-link there is no intervening cross-bridge, as in the type A1 peptidoglycan of Gram-negative bacteria. The data indicate that pneumococcal peptidoglycan has a structural complexity comparable to that recently shown in some Gram-negative species.