Diaminopimelic Acid Residues Research Articles

Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.

Synthesis of 2,6‐Diaminopimelic Acid (DAP) and Its Analogues

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

PGRP-SB1: An N-acetylmuramoyl l-alanine amidase with antibacterial activity

The peptidoglycan recognition protein (PGRP) family is conserved from insects to mammals and is involved in immune regulation and bacterial clearance. They form at least three functional classes; receptors required for immune gene expression; amidases that degrade peptidoglycan and scavenge the tissues from immune-stimulating peptidoglycan; and as proteins with antibacterial activity. We here report that PGRP-SB1 is an N-acetylmuramoyl l-alanine amidase, which (in contrast to the previously described PGRP-amidases) shows antibacterial activity. PGRP-SB1 is highly active against peptidoglycans that have a diaminopimelic acid (DAP) residue in the cross-linking peptide, but lack activity to most lysine-containing peptidoglycans. The antibacterial activity is pronounced against Bacillus megaterium with an LD 50 of 1.5 μg ml −1. The bactericidal effect of PGRP-SB1 is dependent on its enzymatic activity, as the zinc co-factor is essential. The bactericidal mode of action is thus different from non-enzymatic vertebrate PGRPs that have been reported to be antibacterial.

Cloning, expression and characterization of the Streptococcus pyogenes murE gene encoding a UDP-MurNAc- l-alanyl- d-glutamate: l-lysine ligase

The l-lysine-adding enzyme encoded by the murE gene catalyzes the ATP-dependent formation of UDP-MurNAc- l-alanyl- d-glutamyl- l-lysine (UDP-MurNAc-tripeptide). MurE has been cloned from Streptococcus pyogenes ( Spy) and expressed as a glutathione- S-transferase (GST)/polyhistidine (His 12) fusion in Escherichia coli ( Eco). Initial velocity studies show that the fusion enzyme has values of k cat=9 s −1 and of K m (ATP) = 125 μM, K m ( l-lysine) = 122 μM and K m (UDP-MurNAc-dipeptide) = 20.5 μM, at 23 °C. Spy murE is expressed using a new plasmid developed for the expression of GST-HIS 12 tagged proteins in Eco. Identification and purification of the UDP-MurNAc- l-alanyl- d-glutamyl- l-lysine product of the GST-HIS 12- Spy MurE enzyme is also described. Surprisingly, this product is a substrate for Eco MurF, an enzyme that normally handles a UDP-MurNAc-tripeptide that contains a diaminopimelic acid residue instead of l-lysine.

The three-for-one model for gram-negative wall growth: a problem and a possible solution.

The murein wall in Gram-negative bacteria is so thin that the mechanism of growth is necessarily complicated. From analytical data of murein components, Höltje suggested a model for the growth mechanism that would lead to safe wall enlargement. The model depended on the formation of trimers of peptidoglycan disaccharides linked via their pentapeptides. In the 'three-for-one' model three oligopeptidoglycan chains are linked to each other in the usual linkages between the carboxyl group of D-alanine residues and the epsilon-amino group of diaminopimelic acid residues; these are designated 'tail-to-tail' linkages. This three-chained raft is then linked to the stress-bearing wall via the formation of trimers, defined as three peptide chains linked together by tail-to-tail linkages. Then by autolyzing the oldest bonds in each trimer, the old chain is excised and the raft becomes part of the stress-bearing wall and the wall is enlarged. There is a problem with the three-for-one model in that it demands a precise fitting of the prefabricated raft of three crosslinked chains to a stress-bearing chain in the wall fabric to allow the series of trimer linkages to form. Because the wall, when bearing stress, must be pulled into a 'honeycomb' structure, the end-to-end distance would be shortened. The possibility is raised here that the glycan chains in the stress-bearing wall are stretched to a sufficient degree by the cell's turgor pressure to compensate for its zig-zag structure; this could allow the model to function. A calculation is presented that assumes that the area of the pores in the fabric, called tessera, is maximized by the cell's turgor pressure. In this case the glycan chain must stretch 10% (and the end-to-end distance of peptide strands stretch 28%) so that the end-to-end distance of a glycan chain in the stress-bearing wall and the unstretched nascent wall can be the same and permit indefinite stable growth.

The three-for-one model for Gram-negative wall growth: a problem and a possible solution

The murein wall in Gram-negative bacteria is so thin that the mechanism of growth is necessarily complicated. From analytical data of murein components, Höltje suggested a model for the growth mechanism that would lead to safe wall enlargement. The model depended on the formation of trimers of peptidoglycan disaccharides linked via their pentapeptides. In the `three-for-one' model three oligopeptidoglycan chains are linked to each other in the usual linkages between the carboxyl group of d-alanine residues and the ϵ-amino group of diaminopimelic acid residues; these are designated `tail-to-tail' linkages. This three-chained raft is then linked to the stress-bearing wall via the formation of trimers, defined as three peptide chains linked together by tail-to-tail linkages. Then by autolyzing the oldest bonds in each trimer, the old chain is excised and the raft becomes part of the stress-bearing wall and the wall is enlarged. There is a problem with the three-for-one model in that it demands a precise fitting of the prefabricated raft of three crosslinked chains to a stress-bearing chain in the wall fabric to allow the series of trimer linkages to form. Because the wall, when bearing stress, must be pulled into a `honeycomb' structure, the end-to-end distance would be shortened. The possibility is raised here that the glycan chains in the stress-bearing wall are stretched to a sufficient degree by the cell's turgor pressure to compensate for its zig-zag structure; this could allow the model to function. A calculation is presented that assumes that the area of the pores in the fabric, called tessera, is maximized by the cell's turgor pressure. In this case the glycan chain must stretch 10% (and the end-to-end distance of peptide strands stretch 28%) so that the end-to-end distance of a glycan chain in the stress-bearing wall and the unstretched nascent wall can be the same and permit indefinite stable growth.

Composition of the peptidoglycan of Haemophilus influenzae

The composition of the peptidoglycan of Haemophilus influenzae was determined by analyzing glycopeptides generated by M1 muramidase hydrolysis using high pressure liquid chromatography, fast atom bombardment mass spectrometry, and fast atom bombardment collisionally activated dissociation tandem mass spectrometry, and amino acid analysis. The structures of 17 glycopeptides, representing 96% of the total peptidoglycan, were ascertained. Fifteen glycopeptides resembled species described for Escherichia coli peptidoglycan (Glauner, B., and Schwarz, U. (1983) The Target of Penicillin (Hackenbeck, R., ed), Walter de Gruyter, Berlin pp. 29-34) as compared with 9 in common with Bordetella pertussis (Tuomanen, E., Schwartz, J., Sande, S., Light, K., and Gage, D. (1989) J. Biol. Chem. 264, 11093-11098). Substitutions for L-alanine in the fourth position of the stem peptide included glycine, aspartic acid, and serine. The peptidoglycan was 27% cross-linked, 2% of which formed between diaminopimelic acid residues. No species was identified containing lysyl-arginine residues characteristic of lipoprotein. The peptidoglycan of non-beta-lactamase-mediated antibiotic-resistant H. influenzae differed from that of sensitive strains by an increase in the amount of disaccharide tripeptides and a decrease in 1,6-anhydro dimers. Both changes were transformable properties that changed in a stepwise fashion in parallel with the degree of antibiotic resistance.

Evidence for the Presence of N-unsubstituted Glucosamine Residues in the Peptidoglycan of Sarcobium cytolyticum — an Intracellular Bacterial Parasite of Amoebae

Summary The chemical structure of peptidoglycan of the cell wall of Sarcobium cytolyticum , an intracellular bacterial parasite of small amoebae, was investigated. The bag-shaped peptidoglycan was isolated from intact cells by a procedure involving treatments with sodium dodecyl sulfate and digestion with pronase. The final preparation was virtually free from proteins and neutral sugars. Hydrolysis of peptidoglycan with 4 N HCl at 100°C for 12 hours yielded, in addition to glucosamine, muramic acid, alanine, diaminopimelic acid, glutamic acid and glycine (molar ratiios 0.4 : 0.4 : 1.4 : 1 : 1 : 0.3), significant amounts of a disaccharide composed of glucosaminyl-β (1–4) muramic acid. Peptidoglycan peracetylated with acetic anhydride produced on acid hydrolysis only monomeric subunits represented by glucosamine, muramic acid, alanine, diaminopimelic acid, glutamic acid and glycine in a molar ratio of 1 : 1 : 1.4 : 1 : 1 : 0.3. Estimation of free NH 2 groups in the native peptidoglycan by dinitrophenylation revealed that about 80% of the glucosamine residues were not substituted by acetyl groups and that only 44% of the diaminopimelic acid residues were involved in cross-linkage of peptide subunits. N-acetylation of peptidoglycan did not completely restore its sensitivity to the hydrolytic action of egg white lysozyme.

Incorporation of bacterial peptidoglycan constituents into macrophage lipids during phagocytosis.

It has previously been established that several glycopeptides of peptidoglycan origin are formed as a result of processing of Bacillus subtilis cell walls by the macrophage-like cell line RAW264. Although the formation of these glycopeptides could account for the humoral immune responses characteristic of bacterial peptidoglycans, their formation does not account for the cellular-mediated immune responses observed for water-in-oil emulsions of peptidoglycan or for lipophilic derivatives of glycopeptide fragments thereof. Therefore, the processing of peptidoglycan by macrophages was reexamined to establish whether the lipophilic derivative of any peptidoglycan-derived glycopeptide was formed. The experiments were performed by incubating B. subtilis cell walls radiolabeled in muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid residues in the presence of the macrophage-like cell line RAW264. The crude lipid fraction derived from the macrophages was further fractionated and analyzed, revealing the presence of two lipophilic glycopeptides that contained glucosamine, muramic acid, and alanine of bacterial origin.

Bacterial peptidoglycans as modulators of sleep. I. Anhydro forms of muramyl peptides enhance somnogenic potency

Chemically defined muramyl peptides (MPs), derived primarily from enzymatic digest of Neisseria gonorrhoeae peptidoglycan, were used to define the structural determinants of MP-mediated somnogenic activity. One of these, i.e. N-acetylglucosaminyl-N-acetyl-1,6-anhydro-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine, was structurally identical to the major naturally occuring MP previously detected in mammalian brain and urine. The somnogenic potency of this MP was similar to that of the corresponding disaccharide pentapeptide containing an additional alanine at the C-terminus and the analogous anhydro-muramic acid-containing monosaccharide tetrapeptide lacking the glucosamine moiety. Infusion of as little as 1 pmol of these highly active MPs increased significantly the percentage of slow-wave sleep in experimental animals. In fact, each of 5 anhydro-muramyl disaccharide peptides tested was somnogenic at a dose of 10 pmol or less and, as far as tested, the activity was affected only slightly by the length or composition of the peptide side chain. However, none of a matched set of analogous MPs, differing only in replacement of the anhydro-muramyl end by a hydrated muramic acid residue, was somnogenic at this dose. A modified form of the hydrated muramyl tripeptide containing a free amide on the diaminopimelic acid residue was completely inactive in amounts up to 1000 pmol. Together, the current data suggested: (1) that the anhydro-muramic acid end (but not the glucosamine moiety) is essential for maximal somnogenic potency; and (2) that amidation of car☐yl groups on the peptide-side chain may block MP-mediated somnogenic activity.

Glucosamine substitution and muramidase susceptibility in Bacillus anthracis

Cell walls of Bacillus anthracis were found to be resistant to lysozyme, and partially resistant to mutanolysin, a muramidase from Streptomyces globisporus. Following treatment with acetic anhydride, it was observed that the walls were highly susceptible to hydrolysis by lysozyme or mutanolysin. Analyses of cell walls, prior to and following derivatization with fluorodinitrobenzene, revealed that approximately 88% of the glucosamine residues and 34% of the muramic acid residues of the peptidoglycan contained unsubstituted amino groups, thereby providing an explanation for the resistance of the walls to lysozyme. The walls of B. anthracis were approximately 19% cross-linked, based on the findings that 81% of the diaminopimelic acid residues could be modified by fluorodinitrobenzene. Walls of B. thuringiensis 4040 and B. cereus ATCC 19637 also contained high percentages of unsubstituted amino sugars, and unless acetylated, were also relatively resistant to lysozyme and mutanolysin. When B. anthracis, B. cereus, or B. thuringiensis were grown in the presence of 100 micrograms/mL lysozyme, there was a decrease in the average number of cells per chain, but there was no decrease in growth rates, suggesting that the enzyme was acting at septa. It is unlikely that lysozyme and autolysins act synergistically in Bacillus, because azide anion, which activates autolysins, did not enhance the lytic action of lysozyme in B. anthracis, B. cereus, or B. thuringiensis.

Production of Bacteriolytic Enzymes and Degradation of Bacterial Cell Walls During Growth of Agaricus bisporus on Bacillus subtilis

Summary: The mechanism by which the mycelium of Agaricus bisporus can use Bacillus subtilis as sole carbon and nitrogen source was investigated. During fungal growth more than 80% of the muramic acid and diaminopimelic acid residues in the bacterial walls disappeared from the microbial biomass and appeared as soluble glycopeptides. Culture supernatants dissolved purified walls of B. subtilis, and bacteriolytic enzymes were obtained by ion-exchange chromatography of these supernatants. The main bacteriolytic enzyme activity was found to be a β-N-acetylmuramidase. Soluble bacterial wall fragments were partially purified by chromatography of the culture fluids. The chemical properties of these fragments were consistent with their having been produced by β-N-acetylmuramidase action on the peptidoglycan of the B. subtilis cell wall.

Incorporation of diaminopimelic acid into the old poles of Escherichia coli.

The surface stress theory of the ontogeny of the bacterial rod depends critically on whether the old poles continue to incorporate new material into the stress-bearing murein. If insertion of peptidoglycan continues, then seemingly the shape must become gradually rounder due to the surface stress resulting from the internal hydrostatic pressure. We have reanalysed our earlier experimental data by classifying grains with respect to distance from the nearest pole, and not from the cell centre as was done previously, and conclude that old poles do incorporate new diaminopimelic acid residues. This eliminates the model we have proposed for Gram-positive rods, which assumed diffuse growth on the cylindrical sides and that poles once formed would be rigid. The new results are consistent with another model (presented elsewhere) in which insertion of new murein occurs all over the surface, although not equally. This new model leads to elongation and division if the energetics of wall expansion is altered by the cell in a control region at a particular point of the cycle by the cell.

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: solubilization of Escherichia coli peptidoglycan

During penetration of Bdellovibrio bacteriovorus into Escherchia coli, two enzymatic activities, a glycanase and a peptidase, rapidly solubilized some 10 to 15% of the E. coli peptidoglycan. The glycanase activity, which solubilizes peptidoglycan amino sugars, came to a sharp halt with completion of the penetration process. Peptidase activity, which cleaves diaminopimelic acid residues from the peptidoglycan, continued, but at a decreasing rate. By 90 min after bdellovibrio attack, some 30% of the initial E. coli diaminopimelic acid residues were solubilized and present in the culture fluid as free diaminopimelic acid. During bdellovibrio penetration some 25% of the lipopolysaccharide glucosamine was also solubilized by an as yet undefined enzymatic activity that yielded products having molecular weights below 2,000. The solubilization of E. coli lipopolysaccharide glucosamine also terminated at completion of bdellovibrio penetration. At the end of bdellovibrio growth, a second period of rapid solubilization of bdelloplast peptidoglycan began which resulted in lysis of the bdelloplast and complete solubilization of the peptidoglycan amino sugars and diaminopimelic acid. The final lytic enzyme(s) was synthesized just before the time of lysis.

Adjuvant activity in delayed hypersensitivity of the peptidic part of bacterial peptidoglycans.

IT is generally accepted that the presence of killed mycobacteria in complete Freund's adjuvant is necessary to induce a typical delayed hypersensitivity to soluble antigens1 and attempts have been made to isolate the active component. The preparations obtained initially were of a peptidoglycolipidic nature2. Hydrosoluble adjuvants containing an arabinogalactan and the wall peptidoglycan have been isolated3–5. We have shown previously that the purified cell wall peptidoglycan of several Gram-negative bacteria exhibit the same adjuvant effect as mycobacteria for the induction of delayed hypersensitivity6,7 and that the disaccharide peptide monomer (β-1,4-N-acetylglucosaminyl-N-acetylmuramyl-L- diaminopimelic acid (L)-D-Ala) was still active8. Similar findings have been made independently with the peptidoglycan of mycobacteria9,10, the subunit structure of which is only slightly different: N-acetylmuramic acid is replaced by N-glycolylmuramic acid and both Glu and diaminopimelic acid residues have amide substituents. The present study was undertaken to determine to what chemical structure of the peptidoglycan adjuvant activity was related.

Inhibition of Growth of Mycobacterium smegmatis and of Cell Wall Synthesis by d -Serine

d-Serine inhibited the growth of Mycobacterium smegmatis and induced the morphological alteration of the bacilli. The growth inhibitory action of d-serine was partially reduced by an equimolecular concentration of d-alanine. The combination of glycine with d-alanine reversed the growth inhibition produced by d-serine more than did d-alanine alone. In cells cultured in the presence of d-serine, the amounts of alanine, diaminopimelic acid, and glycine inserted into the cell wall mucopeptide were reduced, and serine was increased. The intracellular accumulation of a precursor of cell wall mucopeptide was increased by d-serine, and this accumulation was reduced by d-alanine. d-Serine competed with glycine for incorporation into the cell wall mucopeptide. The incorporation of l-aspartic acid into diaminopimelic acid residues in the cell wall mucopeptide was markedly inhibited by d-serine. Three mutants resistant to d-serine were isolated by nitrosoguanidine treatment. In these mutants the effects of d-serine on the sites of cell wall mucopeptide synthesis were all reduced. Thus, d-serine inhibition of the growth is due to replacement of glycine residues of the cell wall mucopeptide with d-serine and inhibition of the cell wall synthesis by blocking the formation of d-alanine and diaminopimelic acid.

Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg Strain 168. Separation of the products and characterization of the mucopeptide fragments.

1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-beta-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.

Autolysis of cell walls of Bacillus stearothermophilus B65 and the chemical structure of the peptidoglycan.

1. The cell walls of Bacillus stearothermophilus B65 contain glucosamine, muramic acid, alanine, alphain-diaminopimelic acid (Dap), glutamic acid, aspartic acid, glycine, and serine in the molecular proportions 0.60:0.64:2.30:0.85:1.00:0.11:0.13:0.31. 2. Both d- and l-alanine are present, but glutamic acid and diaminopimelic acid are present only as the d- and meso-isomers respectively. 3. The peptide fragments Ala-Dap, Dap-Ala, and Dap-Ala-Dap have been isolated from a partial acid hydrolysate of the cell walls. 4. The major products of autolysis of the cell wall were d-alanine, a peptide mixture, peptidoglycan material and a peptidoglycan-teichoic acid complex. 5. Separation of the peptide mixture into ten major peptides was achieved by DEAE-Sephadex and paper chromatography, and paper electrophoresis. 6. The structures of these peptides have been determined and they fall into four groups, the individual members of each group differing only in number or position of carboxamide substituents. 7. The structures are I, a tripeptide l-Ala-d-Glu-meso-Dap; II, a pentapeptide made up by the tripeptide (I) linked through the in-amino group of its diaminopimelic acid residue to the carboxyterminal of the dipeptide meso-Dap-d-Ala; III, a heptapeptide made up by a similar linkage between the tripeptide (I) and the tetrapeptide l-Ala-d-Glu-meso-Dap-d-Ala; IV, a possible undecapeptide made up by a further tetrapeptide similarly linked to the heptapeptide (III) structure. 8. The structure of the peptidoglycan and the actions of the autolytic enzymes are discussed in terms of these peptide structures.

Peptide crosslinkage in cell wall murein of proteus mirabilis and its penicillin-induced unstable L-form

In mureins of normal rod-shaped P.mirabilis and its penicillin-induced unstable L-form approximately 33% of the diaminopimelic acid residues participate in peptide crosslinkage. No decrease of crosslinkage due to an effect of penicillin was observed.

The linkage of diaminopimelic acid in the peptidoglycan of Bacillus megaterium NCTC 7581

1. 1. It has been possible to demonstrate that diaminopimelic acid in the peptidoglycan of Bacillus megaterium NCTC 7581 is α-linked. One method utilized the photolability of the dinitrophenylamino group when such a group is linked directly to a carbon atom bearing a carboxyl group. Thus after irradiation of dinitrophenylated peptidoglycan and acid hydrolysis, only a trace of mono-(dinitrophenyl) diaminopimelic acid remained. When irradiated peptidoglycan was oxidized and hydrolyzed, the expected degradation product of diaminopimelic acid, α-aminoadipic acid was detected and estimated. 2. 2. The Lossen rearrangement was used to demonstrate that 75% of both glutamic acid and diaminopimelic acid residues in the peptidoglycan have one free carboxyl group. α-Aminoadipic acid was obtained from Lossen rearrangement of dinitrophenylated peptidoglycan after subsequent oxidation and hydrolysis, thus confirming that the diaminopimelic acid residues are α-linked.