Peptidoglycan

Bacterial surface proteins are important molecules in the infectivity and survival of pathogens. Surface proteins on gram-positive bacteria have been shown to attach via a transpeptidase, termed sortase, that cleaves an LPXTG sequence found close to the C termini of nearly all surface proteins on these bacteria. We previously identified a unique enzyme (LPXTGase) from Streptococcus pyogenes that also cleaves the LPXTG motif with a catalytic activity higher than that of sortase, suggesting that it plays an important role in the attachment process. We have now purified and characterized an LPXTGase from Staphylococcus aureus and found that it has both similar and unique features compared to the S. pyogenes enzyme. The S. aureus enzyme is glycosylated and contains unusual amino acids, like its streptococcal counterpart. Like the streptococcal enzyme, staphylococcal LPXTGase has an overrepresentation of amino acids found in the peptidoglycan, i.e., glutamine/glutamic acid, glycine, alan...

The ability of stationary-phase cells of Escherichia coli W7 to incorporate radioactive precursorsinto macromolecular murein has been studied. During the initial 6 h of the stationary phase, resting cells incorporated meso-[3H]diaminopimelic acid at a rate corresponding to theinsertion ...

Our earlier studies demonstrated that representative microbial substances--lipopolysaccharide, peptidoglycan, and poly-inosine: poly-cytosine (poly(I):(C))--increased the spontaneous discharge rates and sensory-evoked responses of isolated locus coeruleus (LC) neurons in a dose- and time-related manner after i.p. injection into rats. We then turned our attention to the mechanism by which microbial substances administered into the peritoneal cavity affect the LC neurons. The involvement of the subdiaphragmatic vagus nerves was examined in this regard since several brain responses to peripherally administered lipopolysaccharide have been found to depend upon the integrity of these nerves. The experiments reported here show that lipopolysaccharide, peptidoglycan, and poly(I):(C) all failed to excite LC neurons after i.p. injection into rats that had previously been subjected to complete transection of the subdiaphragmatic vagus nerves. Furthermore, selective transection of the subdiaph...

Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in s...

A yellow Gram-stain-positive, non-motile, non-endospore -forming, spherical endophytic actinobacterium, designated strain AE-6(T), was isolated from the inner fleshy leaf tissues of Aloe barbadensis (Aloe vera) collected from Pune, Maharashtra, India. Strain AE-6(T) grew at high salt concentrations [10% (w/v) NaCl], temperatures of 15-41 °C and a pH range of 5-12. It showed highest (99.7%) 16S rRNA gene sequence similarity with Micrococcus yunnanensis YIM 65004(T) followed by Micrococcus luteus NCTC 2665(T) (99.6%) and Micrococcus endophyticus YIM 56238(T) (99.0%). Ribosomal protein profiling by MALDI-TOF/MS also showed it was most closely related to M. yunnanensis YIM 65004(T) and M. luteus NCTC 2665(T). Like other members of the genus Micrococcus, strain AE-6(T) had a high content of branched chain fatty acids (iso-C15:0 and anteiso-C15:0). MK-8(H2) and MK-8 were the predominant isoprenoid quinones. Cell wall analysis showed an 'A2 L-Lys-peptide subunit' type of peptidogly...

In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by...

A method for the preparation of heparan sulfate from peptidoglycan heparin is described. The objective of this research was to provide a basis for the development and validation of an industrial process to support the preclinical development of heparan sulfate and/or heparan sulfate derivatives. In the preparation of heparan sulfate, heparin was recovered by alcohol fractionation and dermatan sulfate was

P128 is an anti-staphylococcal protein consisting of the Staphylococcus aureus phage-K-derived tail-associated muralytic enzyme (TAME) catalytic domain (Lys16) fused with the cell-wall-binding SH3b domain of lysostaphin. In order to understand the mechanism of action and emergence of resistance to P128, we isolated mutants of Staphylococcus spp., including meticillin-resistant Staphylococcus aureus (MRSA), resistant to P128. In addition to P128, the mutants also showed resistance to Lys16, the catalytic domain of P128. The mutants showed loss of fitness as shown by reduced rate of growth in vitro. One of the mutants tested was found to show reduced virulence in animal models of S. aureus septicaemia suggesting loss of fitness in vivo as well. Analysis of the antibiotic sensitivity pattern showed that the mutants derived from MRSA strains had become sensitive to meticillin and other β-lactams. Interestingly, the mutant cells were resistant to the lytic action of phage K, although the...

The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure–function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand...

A novel actinobacterial strain, designated NCCP-696T, was isolated from the Thal desert in Punjab, Pakistan, and characterized by using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain NCCP-696T belongs to the genus Nocardioides and showed the highest level of sequence similarity with respect to Nocardioides panacisoliGsoil 346T (98.2 %) and less than 96.4 % to the strains of other species of the genus Nocardioides. Cells of strain NCCP-696T were Gram-positive, aerobic, non-motile rods and formed cream-coloured colonies. The strain was positive for oxidase and catalase. Growth occurred at 20-42 °C (optimum 30-37 °C) at pH 5.5-9.0 (optimum pH 7.0) and with 0-4 % NaCl (optimum 0-2 %, w/v). Strain NCCP-696T contained Iso-C16 : 0, C18 : 1ω9c, C17 : 1ω8c and C17 : 0 as the predominant fatty acids and was found to have LL-2,6-diaminopimelic acid in the cell-wall peptidoglycan. The polar lipid profile consisted of phosphatidylinosito...

Three strains of gram-positive, motile, rod-shaped and boron (B)-tolerant bacterium were isolated from naturally B containing soil of Hisarcik area in the Kutahya Province, Turkey. The strains, designated as T-14A, T-15ZT and T-17s, produced spherical or ellipsoidal endospores in a terminal bulging sporangium. The strains required B for the growth and can tolerate more than 450 mM B. These also tolerated up to 7.0% (w/v) NaCl in the presence of 50 mM B in agar medium but grew optimally without NaCl. The temperature range for growth was 16–37°C (optimal of 30°C), whereas the pH range was 6.5–9.0 (optimal of 7.5–8.5). The DNA G + C content was 41.1–42.2 mol% and the predominant cellular fatty acid was iso-C15:0. The major respiratory quinone system was detected as MK-7 and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis of 16S rRNA gene sequences data and DNA–DNA re-association values, we concluded that the three strains belong to a novel species of the genus Bacillus, the type strain of which is T-15ZT and for which we proposed the name, B. boroniphilus sp. nov. (DSM 17376T = IAM 15287T = ATCC BAA-1204T).