Modified peptidoglycan transpeptidase activity in a carbenicillin-resistant mutant of Pseudomonas aeruginosa 18s.
A carbenicillin-resistant mutant of Pseudomonas aeruginosa 18s was found to possess peptidoglycan transpeptidase activity significantly more resistant to inhibition by benzyl penicillin, ampicillin, carbenicillin, and cephaloridine than that of the parent strain. The mutant was more resistant than the parent strain to all of the beta-lactam antibiotics tested, and 50% inhibition values for these compounds against membrane-bound model transpeptidase activity paralleled this increase. The resistance of the mutant to kanamycin, streptomycin, and chloramphenicol was unchanged. (+info)
Polyamine profiles within genera of the class Actinobacteria with LL-diaminopimelic acid in the peptidoglycan.
Polyamine patterns of coryne- and nocardioform representatives of the class Actinobacteria with LL-diaminopimelic acid in the peptidoglycan, comprising strains of the genera Aeromicrobium, Nocardioides, Intrasporangium, Terrabacter, Terracoccus, Propioniferax, Friedmanniella, Microlunatus, Luteococcus and Sporichthya, were analysed. The different polyamine patterns were in good agreement with the phylogenetic heterogeneity within this group of actinomycetes. Strains of the closely related genera Nocardioides and Aeromicrobium were characterized by the presence of cadaverine. The second cluster, consisting of the type strains of the species Friedmanniella antarctica, Propioniferax innocua, Microlunatus phosphovorus and Luteococcus japonicus, displayed as a common feature the presence of the two predominant compounds spermidine and spermine. The presence of putrescine was common to the type strains of the species Intrasporangium calvum, Terrabacter tumescens and Terracoccus luteus. Sporichthya polymorpha, which is a representative of a separate line of descent, displayed spermidine as the predominant polyamine. These data indicate that polyamine patterns are suitable for the classification of actinomycetes with LL-diaminopimelic acid in the peptidoglycan. (+info)
Selective inhibition of the bacterial peptidoglycan biosynthesis by the new types of liposidomycins.
We examined the inhibitory activity against bacterial peptidoglycan biosynthesis, mammalian glycoprotein biosynthesis and growth of BALB/3T3 cells of four different types of liposidomycins which have the structure with or without sulfate and/or 3-methylglutaric acid moieties. Liposidomycins inhibited peptidoglycan biosynthesis about 30 to 500 times more effectively than tunicamycin, whereas liposidomycins inhibited mammalian glycoprotein biosynthesis about 30 to 300 times less effectively than tunicamycin. When the cytotoxic effect of liposidomycins and tunicamycin on the growth of mammalian cells were compared, liposidomycins did not show toxicity against BALB/3T3 cell at 25 microg/ml, though tunicamycin inhibited cell growth by 50% at 0.05 microg/ml. On the basis of these results, it is concluded that liposidomycins are selective antibiotics showing highly specific inhibition toward bacterial peptidoglycan biosynthesis. (+info)
Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium.
Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space. (+info)
Electron microscopy studies of cell-wall-anchored cellulose (Avicel)-binding protein (AbpS) from Streptomyces reticuli.
Streptomyces reticuli produces a 35-kDa cellulose (Avicel)-binding protein (AbpS) which interacts strongly with crystalline cellulose but not with soluble types of cellulose. Antibodies that were highly specific for the NH2-terminal part of AbpS were isolated by using truncated AbpS proteins that differed in the length of the NH2 terminus. Using these antibodies for immunolabelling and investigations in which fluorescence, transmission electron, or immunofield scanning electron microscopy was used showed that the NH2 terminus of AbpS protrudes from the murein layer of S. reticuli. Additionally, inspection of ultrathin sections of the cell wall, as well as biochemical experiments performed with isolated murein, revealed that AbpS is tightly and very likely covalently linked to the polyglucane layer. As AbpS has also been found to be associated with protoplasts, we predicted that a COOH-terminal stretch consisting of 17 hydrophobic amino acids anchors the protein to the membrane. Different amounts of AbpS homologues of several Streptomyces strains were synthesized. (+info)
Surface proteins of gram-positive bacteria and mechanisms of their targeting to the cell wall envelope.
The cell wall envelope of gram-positive bacteria is a macromolecular, exoskeletal organelle that is assembled and turned over at designated sites. The cell wall also functions as a surface organelle that allows gram-positive pathogens to interact with their environment, in particular the tissues of the infected host. All of these functions require that surface proteins and enzymes be properly targeted to the cell wall envelope. Two basic mechanisms, cell wall sorting and targeting, have been identified. Cell well sorting is the covalent attachment of surface proteins to the peptidoglycan via a C-terminal sorting signal that contains a consensus LPXTG sequence. More than 100 proteins that possess cell wall-sorting signals, including the M proteins of Streptococcus pyogenes, protein A of Staphylococcus aureus, and several internalins of Listeria monocytogenes, have been identified. Cell wall targeting involves the noncovalent attachment of proteins to the cell surface via specialized binding domains. Several of these wall-binding domains appear to interact with secondary wall polymers that are associated with the peptidoglycan, for example teichoic acids and polysaccharides. Proteins that are targeted to the cell surface include muralytic enzymes such as autolysins, lysostaphin, and phage lytic enzymes. Other examples for targeted proteins are the surface S-layer proteins of bacilli and clostridia, as well as virulence factors required for the pathogenesis of L. monocytogenes (internalin B) and Streptococcus pneumoniae (PspA) infections. In this review we describe the mechanisms for both sorting and targeting of proteins to the envelope of gram-positive bacteria and review the functions of known surface proteins. (+info)
Effects of antibiotics on metabolism of peptidoglycan, protein, and lipids in Bifidobacterium bifidum subsp. pennsylvanicus.
The formation of cell envelope components of Bifidobacterium bifidum subsp. pennsylvanicus was studied by measuring the incorporation of [(3)H]glycine, (14)C-labeled fatty acids, and N-benzoyl-[(14)C]glucosamine into the membrane protein, membrane lipids, and cell wall peptidoglycan, respectively. Inhibition of peptidoglycan synthesis by antibiotics (penicillin G, vancomycin, d-cycloserine, and bacitracin) and by the omission of glucosamine-containing growth factors caused a marked decrease in glycine incorporation into cellular as well as membrane protein, which was accompanied by a considerable enhancement of fatty acid incorporation. The uncoupling of protein and lipid synthesis led to the release of marked amounts of lipids from the cell under these conditions. Arrestment of protein synthesis by antibiotics (chloramphenicol, tetracycline, and actinomycin D) decreased peptidoglycan and lipid synthesis only partially, but did not lead to lipid release. Mg(2+) deficiency of the medium caused about 60% inhibition of growth and lipid synthesis, but protein synthesis and especially peptidoglycan synthesis were much less inhibited. Staphylococcin 1580 arrested the growth and also the synthesis of protein and peptidoglycan. However, the synthesis and turnover of lipids were considerably increased and a release of large amounts of lipids was observed. Peptidoglycan and cellular protein did not show any turnover either during normal growth or after the inhibition of cell wall and protein synthesis. (+info)
Quantitative determination of N-acetylglucosamine residues at the non-reducing ends of peptidoglycan chains by enzymic attachment of [14C]-D-galactose.
The ability of human milk galactosyltransferase to attach D-galactose residues quantitatively to the C-4 of N-acetylglucosamine moieties at the ends of oligosaccharides has been utilized for the specific labeling and quantitative determination of the chain length of the glycan moiety of the bacterial cell wall. The average polysaccharide chain length of the soluble, uncrosslinked peptidoglycan secreted by Micrococcus luteus cells on incubation with penicillin G was studied with this technique and found to be approximately 70 hexosamines long. Furthermore, the peptidoglycan chain length of Escherichia coli sacculi of different cell shapes and dimensions was determined both in rod-shaped cells and in filaments induced by temperature shift of a division mutant or by addition of cephalexin or nalidixic acid. The average chain length found in most of these sacculi was between 70 and 100 hexosamines long. Small spherical 'mini' cells had chain lengths similar to those of the isogenic rod-like cells. (+info)