scholarly journals The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin

1974 ◽  
Vol 141 (1) ◽  
pp. 227-241 ◽  
Author(s):  
J. Barrie Ward
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Membrane Preparation ◽  
Deficient Mutant ◽  
Peptidoglycan Synthesis ◽  
Free Membrane ◽  
Negative Mutant

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, β-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by β-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of β-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2–3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with β-lactamase or by washing.

scholarly journals The direction of glycan synthesis in a bacterial peptidoglycan

1973 ◽  
Vol 135 (4) ◽  
pp. 721-728 ◽  
Author(s):  
J. Barrie Ward ◽  
Harold R. Perkins
Keyword(s):  
Bacillus Licheniformis ◽  
The Other ◽  
Membrane Preparation ◽  
Pimelic Acid ◽  
Smith Degradation ◽  
Alkaline Conditions ◽  
A Cell ◽  
Free Membrane ◽  
Bacterial Peptidoglycan ◽  
Mild Acid

A cell-free membrane preparation from a poorly lytic mutant of Bacillus licheniformis was used to synthesize radioactive peptidoglycan. The product was apparently un-cross-linked. When UDP-N-acetyl[14C]glucosamine was used and the final peptidoglycan subjected to Smith degradation, no radioactive glycerol was found. On the other hand, when peptidoglycan labelled with meso-diamino[14C]pimelic acid was first hydrolysed in 0.1m-HCl at 60°C for 2h and then subjected to alkaline conditions, radioactive lactyl-peptides were eliminated. The proportion of radioactive lactyl-peptide decreased with increasing time of incorporation. It is concluded that the glycan chains grow by extension at their reducing ends while remaining attached by some linkage labile to mild acid, such as a glycosyl link to undecaprenol pyrophosphate.

scholarly journals Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall

1974 ◽  
Vol 139 (3) ◽  
pp. 781-784 ◽  
Author(s):  
J. Barrie Ward ◽  
Harold R. Perkins
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Free Amino ◽  
Membrane Preparation ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Amino Group ◽  
Peptidoglycan Biosynthesis ◽  
Acetyl Group ◽  
Degraded Product

A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by 14C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.

Microanatomy of the Periplasm of Bacillus Licheniformis

1971 ◽  
Vol 29 ◽  
pp. 262-263
Author(s):  
B.K. Ghosh
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Bacillus Licheniformis ◽  
External Environment ◽  
Permeability Barrier ◽  
Periplasmic Space ◽  
Modified Technique ◽  
Gram Positive ◽  
Gram Negative ◽  
Gram Positive Bacteria

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

scholarly journals Membrane-Associated Proteins of a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis Activate the Inflammatory Response through Toll-Like Receptor 2

2001 ◽  
Vol 69 (4) ◽  
pp. 2230-2236 ◽  
Author(s):  
Robin R. Ingalls ◽  
Egil Lien ◽  
Douglas T. Golenbock
Keyword(s):  
Cell Wall ◽  
Inflammatory Response ◽  
Neisseria Meningitidis ◽  
Parental Strain ◽  
Host Responses ◽  
Deficient Mutant ◽  
Toll Like Receptor ◽  
Gram Negative ◽  
Negative Cell ◽  
Toll Like Receptor 2

ABSTRACT The recent isolation of a lipopolysaccharide (LPS)-deficient mutant of Neisseria meningitidis has allowed us to explore the roles of other gram-negative cell wall components in the host response to infection. The experiments in this study were designed to examine the ability of this mutant strain to activate cells. Although it was clearly less potent than the parental strain, we found the LPS-deficient mutant to be a capable inducer of the inflammatory response in monocytic cells, inducing a response similar to that seen with Staphylococcus aureus. Cellular activation by the LPS mutant was related to expression of CD14, a high-affinity receptor for LPS and other microbial products, as well as Toll-like receptor 2, a member of the Toll family of receptors recently implicated in host responses to gram-positive bacteria. In contrast to the parental strain, the synthetic LPS antagonist E5564 did not inhibit the LPS-deficient mutant. We conclude that even in the absence of LPS, the gram-negative cell wall remains a potent inflammatory stimulant, utilizing signaling pathways independent of those involved in LPS signaling.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

Cell wall teichoic acids of Bacillus licheniformis VKM B-511T, Bacillus pumilus VKM B-508T, and other strains previously assigned to Bacillus pumilus

Microbiology ◽  
2012 ◽  
Vol 81 (4) ◽  
pp. 425-434 ◽  
Author(s):  
G. M. Streshinskaya ◽  
A. S. Shashkov ◽  
Yu. I. Kozlova ◽  
E. M. Tul’skaya ◽  
E. B. Kudryashova ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Licheniformis ◽  
Bacillus Pumilus ◽  
Teichoic Acids ◽  
Wall Teichoic Acids

Biochemical characterization of a cortexless mutant of a variant of Bacillus cereus

1976 ◽  
Vol 22 (7) ◽  
pp. 1007-1012 ◽  
Author(s):  
Susanne M. Pearce
Keyword(s):  
Cell Wall ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Bacillus Cereus ◽  
Biochemical Characterization ◽  
Diaminopimelic Acid ◽  
Peptidoglycan Synthesis ◽  
Biochemical Lesion ◽  
Do So

Previous studies on this cortexless mutant of Bacillus cereus var. alesti indicated that the forespore membrane was the site of the biochemical lesion. This hypothesis is supported by the results presented here: fatty acid composition of sporulating cells of the mutant is altered, while in vegetative cells it is comparable to the parent; soluble precursors of peptidoglycan synthesis are accumulated in the mutant, at the time of cortex formation; homogenates of the mutant prepared at the time of cortex formation are unable to incorporate tritiated diaminopimelic acid into peptidoglycan, while homogenates of cells forming germ cell wall do so to an extent comparable to that of the parent; lipid-linked intermediates are formed by the mutant as in the parent. Apparently the mutant is unable either to transfer disaccharide penta-peptide units from the carrier lipid to the growing peptidoglycan acceptor, or to transport lipid-linked intermediates across the forespore membrane.

Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity

2015 ◽  
Vol 87 (2) ◽  
pp. 190-199 ◽  
Author(s):  
Michael F. Mesleh ◽  
Premraj Rajaratnam ◽  
Mary Conrad ◽  
Vasu Chandrasekaran ◽  
Christopher M. Liu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Bacterial Cell Wall ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan
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