Muramyl Residues Research Articles

Hoyosella lacisalsi sp. nov., a halotolerant actinobacterium isolated from the Lake Gudzhirganskoe.

A Gram-stain-positive, aerobic, non-motile, non-spore-forming and coccus-shaped strain, designated strain G463T, was isolated from the rhizosphere soil of Salicornia europaea L. collected from Lake Gudzhirganskoe in Siberia. Based on 16S rRNA gene phylogeny, strain G463T belonged to the genus Hoyosella, with the highest 16S rRNA gene sequence similarity to Hoyosella altamirensis DSM 45258T (96.1%). The major fatty acids were C17:1 ω8c, C16:0, C15 : 0 and C17:0. The strain contained meso-diaminopimelic acid as the cell-wall diagnostic diamino acid and arabinose, galactose and ribose as the whole-cell sugars. MK-8 and MK-7 were the predominant menaquinones. The polar lipid profile comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, one unidentified phosphoglycolipid, two unidentified glycolipids and several unidentified lipids. Acetyl was the muramyl residue. Mycolic acids (C28-C34) were present. The G+C content of the genomic DNA was 68.3 mol%. Based on its phylogenetic, phenotypic and chemotaxonomic features, strain G463T was considered to represent a novel species of the genus Hoyosella, for which the name Hoyosella lacisalsi sp. nov. is proposed. The type strain is G463T (=JCM 33650T=CGMCC 1.17230T).

Planctomonas psychrotolerans sp. nov., isolated from rhizosphere soil of Suaeda salsa

A psychrotolerant actinobacterium, designated strain J5903T, was isolated from an alkaline soil sample from the rhizosphere of Suaeda salsa collected in desertification land surrounding Jiuliancheng Nur in Hebei Province, PR China. Cells of the isolate were Gram-stain-positive, aerobic, non-motile and non-spore-forming cocci. Strain J5903T grew optimally at 20‒25 °C, at pH 7.0‒7.5 and with <1 % (w/v) NaCl. The cell-wall peptidoglycan type was B2γ with d-2,4-diaminobutyric acid and l-2,4-diaminobutyric acid as diagnostic amino acids. The muramyl residue was acetyl type. The menaquinones were MK-11, MK-12, MK-10 and MK-13. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid. The major whole-cell fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The genomic DNA G+C content was 69.1 mol%. It shared the highest average nucleotide identity and digital DNA-DNA hybridization values with Planctomonas deserti 13S1-3T. Phylogenies based on genome sequence showed that strain J5903T and P. deserti 13S1-3T formed a robust cluster with high bootstrap support. Strain J5903T shared typical chemotaxonomic characteristics with P. deserti 13S1-3T. Combining the polyphasic taxonomic evidence, strain J5903T represents a novel species of the genus Planctomonas, for which the name Planctomonas psychrotolerans sp. nov. is proposed. The type strain is J5903T (=DSM 101894T=CGMCC 1.15523T).

Hoyosella rhizosphaerae sp. nov., a halotolerant actinobacterium isolated from rhizosphere soil of Suaeda salsa, and emended description of the genus Hoyosella.

A halotolerant actinobacterium, designated J12GA03T, was isolated from a rhizosphere soil sample of Suaeda salsa collected from a dried saline lake in Hebei Province, China. Cells were Gram-staining-positive, non-motile and non-spore-forming cocci. Strain J12GA03T grew optimally at 28‒37 °C, 0‒3 % NaCl (w/v) and pH 6.5‒7.5. It contained meso-diaminopimelic acid as the diagnostic diamino acid and arabinose, galactose and ribose as the diagnostic whole-cell sugars. MK-8 and MK-7 were detected as predominant menaquinones. Major fatty acids were C17 : 1ω8c, C16 : 0 and C17 : 0. Polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol, phosphatidylglycerol, phosphoglycolipids, glycolipids, unidentified phospholipids and additional lipids. The muramyl residue was acetyl. Mycolic acids (34-38 carbon atoms) were present. The G+C content of the genomic DNA was 55.8 mol%. It shared the highest 16S rRNA gene sequence similarities with Amycolicicoccus subflavus DQS3-9A1T (98.18 %) and Hoyosella altamirensis OFN S31T (97.75 %). Phylogenetic trees showed that strain J12GA03T firmly formed a distinct monophyletic branch in the clade with A.subflavus DQS3-9A1T and H.altamirensis DSM 45258T. The levels of DNA-DNA relatedness with A.subflavus DSM 45089T and H.altamirensis DSM 45258T were 39.7±3.9 % and 35.7±3.0 %, respectively. Combining the evidence from the polyphasic taxonomic study, strain J12GA03T represents a novel species of the genus Hoyosella, for which the name Hoyosella rhizosphaerae sp. nov. is proposed. The type strain is J12GA03T (=DSM 101985T=CGMCC 1.15478T).

Substrate Specificity and Kinetic Characterization of Peptidoglycan O-Acetyltransferase B from Neisseria gonorrhoeae

The O-acetylation of the essential cell wall polymer peptidoglycan is a major virulence factor identified in many bacteria, both Gram-positive and Gram-negative, including Staphylococcus aureus, Bacillus anthracis, Neisseria gonorrhoeae, and Neisseria meningitidis. With Gram-negative bacteria, the translocation of acetyl groups from the cytoplasm is performed by an integral membrane protein, PatA, for its transfer to peptidoglycan by O-acetyltransferase PatB, whereas a single bimodal membrane protein, OatA, appears to catalyze both reactions of the process in Gram-positive bacteria. Only phenotypic evidence existed in support of these pathways because no in vitro biochemical assay was available for their analysis, which reflected the complexities of investigating integral membrane proteins that act on a totally insoluble and heterogeneous substrate, such as peptidoglycan. In this study, we present the first biochemical and kinetic analysis of a peptidoglycan O-acetyltransferase using PatB from N. gonorrhoeae as the model system. The enzyme has specificity for muropeptides that possess tri- and tetrapeptide stems on muramyl residues. With chitooligosaccharides as substrates, rates of reaction increase with increasing degrees of polymerization to 5/6. This information will be valuable for the identification and development of peptidoglycan O-acetyltransferase inhibitors that could represent potential leads to novel classes of antibiotics.

Characterization of oligoglucan-containing products derived fromMesorhizobium lotiHAMBI 1148 murein after lysozyme digestion and β-elimination

Summary Short glucan oligomers were found to be covalently linked to the peptidoglycan fragments liberated by partial lysozyme digestion of Mesorhizobium loti HAMBI 1148 murein and among some muropeptide mono and oligomers subsequently subjected to alkaline β-elimination. The mass spectra of the preparation before and after treatment with alkali contained deprotonated ions [M-H]− recorded at m/z 3107 and 2856 which represented muropeptide fragments with 2 or 3 glucose residues, respectively, at the reducing end. Because of the lack of a free reducing group, muramyl residues remained untouched during the β-elimination reaction in a manner comparable with muropeptide fragments protected by 1,6-anhydromuramic acid represented in the spectra by [M-H]− ions with masses of 2451 and 2762 m/z. The ion [M-H]− at m/z 1615 which was proposed to represent a disaccharide fragment with one Glc residue linked had no counterpart in the spectra of lysozyme-liberated muropeptides and was supposed to have arisen from a dime...

Total Synthesis of N-Acetylglucosamine-1,6-anhydro-N-acetylmuramylpentapeptide and Evaluation of Its Turnover by AmpD from Escherichia coli

The bacterial cell wall is recycled extensively during the course of cell growth. The first recycling event involves the catalytic action of the lytic transglycosylase enzymes, which produce an uncommon 1,6-anhydropyranose moiety during separation of the muramyl residues from the peptidoglycan, the major constituent of the cell wall. This product, an N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramylpeptide, is either internalized to initiate the recycling process or diffuses into the milieu to cause stimulation of the pro-inflammatory responses by the host. We report the total syntheses of N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-meso-DAP-D-Ala-D-Ala (compound 1, the product of lytic transglycosylase action on the cell wall of gram-negative bacteria) and N-acetyl-beta-D-glucosamine-(1-->4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (compound 2, from lytic transglycosylase action on the cell wall of gram-positive bacteria). The syntheses were accomplished in 15 linear steps. Compound 1 is shown to be a substrate of the AmpD enzyme of the gram-negative bacterium Escherichia coli, an enzyme that removes the peptide from the disaccharide scaffold in the early cytoplasmic phase of cell wall turnover.

Localization of the attachment site of oligoglucans to Mesorhizobium loti HAMBI 1148 murein.

The location and nature of the linkage between peptidoglycan and oligoglucans in the cell wall of Mesorhizobium loti HAMBI 1148 have been defined by the analysis of nitrous acid deamination of peptidoglycan glucosaminyl residues. The MurNH(2)-Glc(n) fraction was obtained after converting deaminoacylated and N-deacetylated muramyl residues in the cell wall preparation to lactam forms which were stable during subsequent deamination, followed by reduction and opening of the lactams. GC/MS analysis of this material, subjected to partial hydrolysis and reduction or to methanolysis followed by peracetylation, confirmed the presence of glucosyl residues glycosidically attached to muramic acid. The MALDI-TOF spectroscopic analysis of the deaminated material also revealed the presence of [M-H](-) or [M+Na-2H](-) ions representing fragments containing muramic acid with one to three linked glucose residues. The analysis of fully methylated neutral oligosaccharides released from the peptidoglycan with lysozyme followed by borohydride reduction showed the presence of di- and trisaccharides lacking the reducing end.

Mycobacterial Lipid II Is Composed of a Complex Mixture of Modified Muramyl and Peptide Moieties Linked to Decaprenyl Phosphate

Structural analysis of compounds identified as lipid I and II from Mycobacterium smegmatis demonstrated that the lipid moiety is decaprenyl phosphate; thus, M. smegmatis is the first bacterium reported to utilize a prenyl phosphate other than undecaprenyl phosphate as the lipid carrier involved in peptidoglycan synthesis. In addition, mass spectrometry showed that the muropeptides from lipid I are predominantly N-acetylmuramyl-L-alanine-D-glutamate-meso-diaminopimelic acid-D-alanyl-D-alanine, whereas those isolated from lipid II form an unexpectedly complex mixture in which the muramyl residue and the pentapeptide are modified singly and in combination. The muramyl residue is present as N-acetylmuramic acid, N-glycolylmuramic acid, and muramic acid. The carboxylic functions of the peptide side-chains of lipid II showed three types of modification, with the dominant one being amidation. The preferred site for amidation is the free carboxyl group of the meso-diaminopimelic acid residue. Diamidated species were also observed. The carboxylic function of the terminal D-alanine of some molecules is methylated, as are all three carboxylic acid functions of other molecules. This study represents the first structural analysis of mycobacterial lipid I and II and the first report of extensive modifications of these molecules. The observation that lipid I was unmodified strongly suggests that the lipid II intermediates of M. smegmatis are substrates for a variety of enzymes that introduce modifications to the sugar and amino acid residues prior to the synthesis of peptidoglycan.

N Glycolylation of the Nucleotide Precursors of Peptidoglycan Biosynthesis of Mycobacterium spp. Is Altered by Drug Treatment

The peptidoglycan of Mycobacterium spp. reportedly has some unique features, including the occurrence of N-glycolylmuramic rather than N-acetylmuramic acid. However, very little is known of the actual biosynthesis of mycobacterial peptidoglycan, including the extent and origin of N glycolylation. In the present work, we have isolated and analyzed muramic acid residues located in peptidoglycan and UDP-linked precursors of peptidoglycan from Mycobacterium tuberculosis and Mycobacterium smegmatis. The muramic acid residues isolated from the mature peptidoglycan of both species were shown to be a mixture of the N-acetyl and N-glycolyl derivatives, not solely the N-glycolylated product as generally reported. The isolated UDP-linked N-acylmuramyl-pentapeptide precursor molecules also contain a mixture of N-acetyl and N-glycolyl muramyl residues in apparent contrast to previous observations in which the precursors isolated after treatment with d-cycloserine consisted entirely of N-glycolyl muropeptides. However, nucleotide-linked peptidoglycan precursors isolated from M. tuberculosis treated with d-cycloserine contained only N-glycolylmuramyl-tripeptide precursors, whereas those from similarly treated M. smegmatis consisted of a mixture of N-glycolylated and N-acetylated residues. The full pentapeptide intermediate, isolated following vancomycin treatment of M. smegmatis, consisted of the N-glycolyl derivative only, whereas the corresponding M. tuberculosis intermediate was a mixture of both the N-glycolyl and N-acetyl products. Thus, treatment with vancomycin and d-cylcoserine not only caused an accumulation of nucleotide-linked intermediate compounds but also altered their glycolylation status, possibly by altering the normal equilibrium maintained by de novo biosynthesis and peptidoglycan recycling.

Assay for Lytic Transglycosylases: A Family of Peptidoglycan Lyases

An assay has been developed to monitor the activity of the lytic transglycosylases which does not involve the use of radiolabel. Samples of lytic transglycosylase were incubated with isolated and purified insoluble peptidoglycan as substrate for varying lengths of time. Residual insoluble material was removed by ultracentrifugation in a microfuge and the solubilized components were treated with sodium borohydride prior to acid hydrolysis. The optimal conditions for this acid hydrolysis were established to be incubation at 96°C for 1 h in 6 M HCl, in vacuo. The hydrolyzed samples were subjected to amino acid/sugar analysis by cation-exchange chromatography on a Beckman System Gold amino acid analyzer. To effect a clear resolution of muramic acid from serine and glutamic acid, the equilibration buffer was modified to be composed of 33 mM sodium citrate, pH 3.12. The product of the lyase reaction of the lytic transglycosylases are 1,6-anhydromuramyl residues, which are not reduced by the sodium borohydride treatment. On the other hand, the muramyl residues arising at the reducing ends of peptidoglycan after treatment with muramidases (hydrolyases) are reduced to muramitol residues, which elute from the amino acid analyzer prior to aspartic acid. This assay thus distinguishes the activity of the two enzymes and was applied to determine the initial activities of increasing concentrations of a soluble derivative of lytic transglycosylase B from the opportunistic pathogen Pseudomonas aeruginosa.

A new rapid method of glycolate test by diethyl ether extraction, which is applicable to a small amount of bacterial cells of less than one milligram.

The glycolate test is a method to discriminate N-acyl groups of muramyl residue in peptidoglycan of bacterial cell walls by color reaction without purification of the cell walls. The glycolyl residue presents red purple color by heating with 0.02% 2,7-dihydroxynaphthalene (DON) dissolved in concentrated H(2)SO(4). Instead of the previous column methods for quantitative analysis, a qualitative method by solvent works was developed to simplify and to miniaturize the analysis. In this method, solvents played two roles, removal of interfering materials and extraction of glycolic acid from the cell hydrolysates. Of several solvent systems tested, diethyl ether was studied in detail on such properties as the efficiency of glycolic acid extraction under several conditions, the ability of removing various interfering compounds, and the advantage on evaporation procedure of the solvent from extracts. DON reaction of the second diethyl ether extract from cell hydrolysate of "Micromonospora nigra" JCM 3328 showed a clear red purple color of a strong absorbance at 530 nm, which is the same as that of authentic glycolic acid. The solvent method was applied to 20 strains of typical actinomycete species whose acyl types have already been known (Uchida and Seino, 1997). All glycolate test positive strains showed the clear red purple color mentioned above, whereas acetyl type strains revealed no apparent color by the same procedures. Additional experiments indicated that the glycolate test could be determined with less than 1 mg of actinomycete cells by using a smaller amount of DON reagent and ordinary polypropylene tubes. The new method was discussed for advantages in the identification of actinomycetes and for possible applications to other fields.

Intra- and Intergeneric Relationships of Various Actinomycete Strains Based on the Acyl Types of the Muramyl Residue in Cell Wall Peptidoglycans Examined in a Glycolate Test

A total of 106 actinomycete strains representing 40 genera were studied to gain a better understanding of their intra- and intergeneric relationships by examining the types of acyl groups on the muramyl residues of the peptidoglycans in a glycolate test. The glycolyl type was found in about 43% of the actinomycete strains examined. These strains were distributed in the following two characteristic taxa: the actinoplanetes and their relatives, including species of the genera Actinoplanes, Catellatospora, Couchioplanes, Dactylosporangium, Glycomyces, Micromonospora, and Pilimelia; and the nocardioforms and species of the related genera Gordona, Nocardia, Rhodococcus, Tsukamurella, and Mycobacterium. Only glycolyl type bacteria were found to be present among the strains of these genera tested, except for a few doubtful strains. In contrast, the acetyl type was found extensively in bacteria belonging to the families Pseudonocardiaceae, Streptomycetaceae, Streptosporangiaceae, and Thermomonosporaceae and other taxa. The pattern of acyl type distribution among the actinomycetes correlates very well with the cell wall chemotype sensu Lechevalier and Lechevalier; that is, the chemotype I and III genera were all acetyl type taxa, the chemotype II genera were glycolyl type taxa, and the chemotype IV genera were split into a glycolyl, mycolate-containing group and an acetyl, non-mycolate group. We discuss the idea that combination of both the acyl type system and the cell wall chemotype system should provide a more useful tool for taxonomy of members of the order Actinomycetales.

Evidence for the nature of the link between the arabinogalactan and peptidoglycan of mycobacterial cell walls.

The long-posed question of the nature of the link between the mycolylarabinogalactan and the underlying peptidoglycan of the cell walls of Mycobacterium sp. has been addressed. The insoluble cell wall matrix of Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis was partially hydrolyzed with acid either before or after per-O-methylation and the resulting oligosaccharides further derivatized and analyzed by gas chromatography/mass spectrometry. The structures of fragments arising from the reducing end of arabinogalactan demonstrated the existence of the terminal sequence----5)-D-Galf-(1----4)-L-Rhap-(1---3)-D-GlcNAc. Other analyses confirmed the presence of muramyl-6-P within the peptidoglycan of these mycobacteria. Based on the acid lability of the 3-linked GlcNAc unit, the presence of about equimolar amounts of Rhap-(1----3)-D-GlcNAc and muramyl-6-P in an isolated cell wall fragment, and 31P NMR analysis, it was concluded that the GlcNAc residue of the terminal triglycosyl unit of arabinogalactan is joined by 1-O-phosphoryl linkage to the 6-position of some muramyl residues within the peptidoglycan. Thus, it is reasoned that the massive mycolylarabinogalactan of mycobacteria, responsible for aspects of disease pathogenesis and much of the antibody response in infections, is attached to the peptidoglycan framework by the actinomycete-specific diglycosylphosphoryl bridge, L-Rhap-(1----3)-D-GlcNAc-(1----P, perhaps thereby providing a unique target for site-directed chemotherapy of mycobacterial infections.

Blood-platelets-damaging activity of some synthetic streptococcus peptidoglycan subunits and analogues

A series of 25 synthetic subunits or analogues of streptococcal peptidoglycan was tested for their ability to damage rabbit blood platelets. The morphological changes of the platelets were studied on an ultrastructural level. Minimal subunit structure able to produce a complete lysis of the platelets was found to be muramyldipeptide (MDP). Comparable lysis of platelets was also caused by muramyltetrapeptide and MDP containing D-Ala instead of L-Ala. The lytic effect was dose-dependent and was exhibited rather after high doses of the substances used (up to 500 μg/ml). For the lytic reaction, the configuration of C3 and C4 in the muramyl residue of MDP was essential. Many substances produced only degranulation without lysis of the platelets and some of them did not influence the platelet ultrastructure at all. The paper presents some structure-to-function relationships of the compounds and shows that the platelet-damaging activity of peptidoglycan may be related to certain portions of the peptidoglycan molecule. This activity should be tested when the immunomodulatory substances derived from the bacterial peptidoglycan are searched for.

Biological Activity of Synthetic Subunits of Streptococcus Peptidoglycan

A series of synthetic subunits and analogues of streptococcal peptidoglycan was prepared and used in fever and tolerance experiments on rabbits. The lengthening of the chain of the peptide moiety of peptidoglycan did not result in pyrogenic activity, except for hexa-peptide. Attachment of the muramyl residue rendered the peptides pyrogenic. The activity of such materials varied in degree and was rather in an indirect relation to peptide chain length. A change in the configuration of C4-OH or C3-OR in the muramyl residue resulted in a profound decrease in pyrogenicity. No inhibitory effect of N-acetylmuramyl-.&lt;i&gt;D&lt;/i&gt;-alanyl- &lt;i&gt;D&lt;/i&gt;-iso-glutamine on muramyldipeptide (MDP) pyrogenicity could be demonstrated. Repeated administration of MDP resulted in the induction of tolerance to the pyrogenicity of this substance in rabbits. These animals were not tolerant to the pyrogenicity of peptidoglycan. Nontolerance was also observed in reciprocal experiments with these materials as well as in trials with hexapeptide and peptidoglycan given in either order. The data are consistent with the assumption that peptidoglycan contains more than one biologically active subunit. There is a structure-to-function relationship. The knowledge of the biological effects of the synthetic analogues is essential for the prospect of their use under model or human conditions.

Analysis of the antigenic relationship of various derivatives of [formula omitted] (MDP), using anti-MDP antibodies

Antibodies to N- acetyl-muramyl- l-alanyl- d-isoglutamine (MDP) were produced in rabbits by injection of MDP conjugated to various carriers [bovine gamma globulin (BGG), methylated BSA or sheep erythrocytes]. The anti-MDP was assayed by a direct enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase linked to MDP-Lys. Various derivatives of MDP were employed in an inhibition of ELISA for analysis of specificity of antibodies and study of the relationship of configuration to biological activity. The results confirmed previous findings that MDP alone is not immunogenic but can act as a hapten when conjugated to carriers. The antibodies were shown to be primarily directed against the muramyl residue. Modifications of this region of MDP yielded derivatives with weak reactivity against anti-MDP, while some changes of other regions had no effect on its antigenicity. Optical isomers of MDP had reduced activity as compared to MDP and polymeric MDP was a strong inhibitor. The structure and function relationship is discussed for some derivatives.

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer : II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNac- L-Ala- D-isoglutamine- meso-diaminopimelic acid- D-Ala- D-Ala), labeled with 14C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood cells, plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10–50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [ 14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an N- acetylmuramoyl- L-alanine amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.