Blautia argi sp. nov., a new anaerobic bacterium isolated from dog faeces.

Two isolates of a Gram-positive, non-motile, coccoid or oval-shaped anaerobic bacterium, designated strains N6H1-15T and YH1_16, were isolated from faecal samples obtained from a mature dog. Analysis of 16S rRNA gene sequences indicated that the isolates belonged to the Blautia coccoidesrRNA gene group (cluster XIVa) and were closely related to Blautia hansenii KCTC 5951T, Blautia stercoris KCTC 5981T, Blautia producta producta KCTC 3695T and B. coccoides DSM 15327T, with 96.7, 94.4, 94.2 and 93.9 % sequence similarity, respectively. The two isolates contained m-diaminopimelic acid within their peptidoglycans. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the major fatty acids were C16 : 0 (18.5 %), C16 : 0 (18.0 %) and C18 : 1cis 9 (16.2 %). The predominant metabolic end products of glucose fermentation were acetic and lactic acids, and the G+C content was 44.2 mol%. Thus, the polyphasic data suggest that the two new isolates represent a new species, proposed as Blautia argi sp. nov. The type strain is N6H1-15T (=KCTC 15426=JCM 31394).

Combination of site directed mutagenesis and secondary structure analysis predicts the amino acids essential for stability of M. leprae MurE.

The life-threatening infections caused by Mycobacterium leprae (Mle) remain a major challenge in developing countries as well as globe and there is a need to design potent anti-leprosy drugs. In our previous studies, ATP-dependent Mle-MurE ligase involved in biosynthesis of peptidoglycan was identified as one of the common drug targets, homology modeled and reported. In this work in silico site directed mutagenesis study was carried out on the homology modeled Mle-MurE ligase. This predicted the amino acids essential for stability. In addition, the distribution of these residues in different secondary structures and in active sites was analyzed. Finally, the role of the conserved residues in stability and function was analyzed. The availability of Mle-MurE ligase built model together with insights gained from stability studies and docking studies will promote the rational design of potent and selective Mle-MurE ligase inhibitors as anti-leprosy therapeutics.

Unique structural features of the peptidoglycan of Mycobacterium leprae.

The peptidoglycan structure of Mycobacterium spp. has been investigated primarily with the readily cultivable Mycobacterium smegmatis and Mycobacterium tuberculosis and has been shown to contain unusual features, including the occurrence of N-glycolylated, in addition to N-acetylated, muramic acid residues and direct cross-linkage between meso-diaminopimelic acid residues. Based on results from earlier studies, peptidoglycan from in vivo-derived noncultivable Mycobacterium leprae was assumed to possess the basic structural features of peptidoglycans from other mycobacteria, other than the reported replacement of l-alanine by glycine in the peptide side chains. In the present study, we have analyzed the structure of M. leprae peptidoglycan in detail by combined liquid chromatography and mass spectrometry. In contrast to earlier reports, and to the peptidoglycans in M. tuberculosis and M. smegmatis, the muramic acid residues of M. leprae peptidoglycan are exclusively N acetylated. The un-cross-linked peptide side chains of M. leprae consist of tetra- and tripeptides, some of which contain additional glycine residues. Based on these findings and genome comparisons, it can be concluded that the massive genome decay in M. leprae does not markedly affect the peptidoglycan biosynthesis pathway, with the exception of the nonfunctional namH gene responsible for N-glycolylmuramic acid biosynthesis.

An unusual mutation results in the replacement of diaminopimelate with lanthionine in the peptidoglycan of a mutant strain of Mycobacterium smegmatis.

Mycobacterial peptidoglycan contains L-alanyl-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the L-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate D-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to beta-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine beta-synthase, an enzyme that is a part of the cysteine biosynthetic pathway.

The envelope of mycobacteria.

Mycobacteria, members of which cause tuberculosis and leprosy, produce cell walls of unusually low permeability, which contribute to their resistance to therapeutic agents. Their cell walls contain large amounts of C60-C90 fatty acids, mycolic acids, that are covalently linked to arabinogalactan. Recent studies clarified the unusual structures of arabinogalactan as well as of extractable cell wall lipids, such as trehalose-based lipooligosaccharides, phenolic glycolipids, and glycopeptidolipids. Most of the hydrocarbon chains of these lipids assemble to produce an asymmetric bilayer of exceptional thickness. Structural considerations suggest that the fluidity is exceptionally low in the innermost part of bilayer, gradually increasing toward the outer surface. Differences in mycolic acid structure may affect the fluidity and permeability of the bilayer, and may explain the different sensitivity levels of various mycobacterial species to lipophilic inhibitors. Hydrophilic nutrients and inhibitors, in contrast, traverse the cell wall presumably through channels of recently discovered porins.

On structural aspects of peptidoglycan of bacterial cell wall with special attention on mycobacteria by computer modelling.

The cell wall components of mycobacteria are said to be vitally linked with their pathogenicity. Peptidoglycan, one of the major cell wall component in most of the bacteria are multilayered in gram positive bacteria and it is diverse in nature for the Gram positive strain rather than gram negative. The cell wall of bacteria are primary targets for many drugs and antibiotics and conformation of the major cell wall components provide invaluable information and understanding at molecular level to medicinal chemists and drug designers. Mycobacterial peptidoglycan has been studied critically by computer modelling on various aspects. A plausible structure and conformation has been identified and glycan chain is found to have a pseudo two fold symmetry taking disaccharide unit as monomer with Knox & Murthy H-bond scheme. This paper attempts to clarify the understanding of organisation and possible interaction mode of peptidoglycan of organisation in complex mycobacterial cell wall structure.

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.