A Novel in vivo Cell-Wall Labeling Approach Sheds New Light on Peptidoglycan Synthesis in Escherichia coli

ChemBioChem ◽  
2011 ◽  
Vol 12 (7) ◽  
pp. 1124-1133 ◽  
Author(s):  
Nick K. Olrichs ◽  
Mirjam E. G. Aarsman ◽  
Jolanda Verheul ◽  
Christopher J. Arnusch ◽  
Nathaniel I. Martin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Peptidoglycan Synthesis ◽  
Labeling Approach

Properties of cell wall peptidoglycan synthesized by amino acid deprived relA mutants of Escherichia coli

1984 ◽  
Vol 30 (10) ◽  
pp. 1239-1246 ◽  
Author(s):  
Désirée Vanderwel ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Amino Acid ◽  
Free Form ◽  
Amino Acid Deprivation ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Covalently Linked

Cell wall peptidoglycan synthesis in Escherichia coli is under stringent control. During amino acid deprivation, peptidoglycan synthesis is inhibited in re1A+ bacteria but not in re1A mutants. The relaxed synthesis of peptidoglycan by amino acid deprived re1A bacteria was inhibited by Several β-lactam antibiotics at concentrations which inhibited cell elongation in growing cultures suggesting that the transpeptidase activity of penicillin-binding protein (PBP-1B) was involved in this process. Structural studies on the peptidoglycan also indicated the involvement of transpeptidation in relaxed peptidoglycan synthesis. The peptidoglycan synthesized during amino acid deprivation was cross-linked to the existing cell wall peptidoglycan, and the degree of cross-linkage was the same as that of peptidoglycan synthesized by growing control cells. The relaxed synthesis of peptidoglycan was also inhibited by moenomycin, an inhibitor of the in vitro transglycosylase activities of PBPs, but the interpretation of this result depends on whether the transglycosylases are the sole targets of moenomycin in vivo. Most of the peptidoglycan lipoprotein synthesized by histidine-deprived re1A+ bacteria was in the free form as previously reported, possibly because of the restriction in peptidoglycan synthesis. In support of this proposal, most of the lipoprotein synthesized during histidine deprivation of re1A mutants was found to be covalently linked to peptidoglycan. Nevertheless, the peptidoglycan synthesized by amino acid deprived re1A bacteria was apparently deficient in bound lipoprotein as compared with peptidoglycan synthesized by normal growing control bacteria suggesting that the rate of lipoprotein synthesis during amino acid deprivation may be limiting.

scholarly journals Induction of the Galactose Enzymes in Escherichia coli Is Independent of the C-1-Hydroxyl Optical Configuration of the Inducer d-Galactose

2008 ◽  
Vol 190 (24) ◽  
pp. 7932-7938 ◽  
Author(s):  
Sang Jun Lee ◽  
Dale E. A. Lewis ◽  
Sankar Adhya
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Carbon Source ◽  
The Other ◽  
Biosynthetic Pathways ◽  
In Vitro Experiments ◽  
Metabolizing Enzymes ◽  
Optical Configuration

ABSTRACT The two optical forms of aldohexose galactose differing at the C-1 position, α-d-galactose and β-d-galactose, are widespread in nature. The two anomers also occur in di- and polysaccharides, as well as in glycoconjugates. The anomeric form of d-galactose, when present in complex carbohydrates, e.g., cell wall, glycoproteins, and glycolipids, is specific. Their interconversion occurs as monomers and is effected by the enzyme mutarotase (aldose-1-epimerase). Mutarotase and other d-galactose-metabolizing enzymes are coded by genes that constitute an operon in Escherichia coli. The operon is repressed by the repressor GalR and induced by d-galactose. Since, depending on the carbon source during growth, the cell can make only one of the two anomers of d-galactose, the cell must also convert one anomer to the other for use in specific biosynthetic pathways. Thus, it is imperative that induction of the gal operon, specifically the mutarotase, be achievable by either anomer of d-galactose. Here we report in vivo and in vitro experiments showing that both α-d-galactose and β-d-galactose are capable of inducing transcription of the gal operon with equal efficiency and kinetics. Whereas all substitutions at the C-1 position in the α configuration inactivate the induction capacity of the sugar, the effect of substitutions in the β configuration varies depending upon the nature of the substitution; methyl and phenyl derivatives induce weakly, but the glucosyl derivative does not.

scholarly journals An activation pathway governs cell wall polymerization by a bacterial morphogenic machine

2018 ◽  
Author(s):  
Patricia D. A. Rohs ◽  
Jackson Buss ◽  
Sue Sim ◽  
Georgia Squyres ◽  
Veerasak Srisuknimit ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Elongation ◽  
System Function ◽  
System Activity ◽  
Activation Pathway ◽  
System A ◽  
Rod System

ABSTRACTCell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall (CW) assembly. InEscherichia coli, alterations in a CW synthase component of the system called PBP2 were identified that overcome other inactivating defects. Rod system activity was stimulated in the suppressors in vivo, and purified synthase complexes with these changes showed more robust CW synthesis in vitro. Polymerization of the actin-like MreB component of the Rod system was also found to be enhanced in cells with the activated synthase. The results suggest an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both CW glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient CW assembly. An analogous activation pathway involving similar enzymatic components is likely responsible for controlling CW synthesis by the division machinery.

scholarly journals FtsW is a peptidoglycan polymerase that is activated by its cognate penicillin-binding protein

2018 ◽  
Author(s):  
Atsushi Taguchi ◽  
Michael A. Welsh ◽  
Lindsey S. Marmont ◽  
Wonsik Lee ◽  
Daniel Kahne ◽  
...  
Keyword(s):  
Cell Wall ◽  
Side Wall ◽  
Polymerase Activity ◽  
Cell Wall Assembly ◽  
Peptidoglycan Synthesis ◽  
Lipid Ii ◽  
Peptidoglycan Cell Wall ◽  
Class B ◽  
A Cell

AbstractThe peptidoglycan cell wall is essential for the survival and shape maintenance ofbacteria.1 For decades it was thought that only penicillin-binding proteins (PBPs) effected peptidoglycan synthesis. Recently, it was shown that RodA, a member of the Rod complex involved in side wall peptidoglycan synthesis, acts as a peptidoglycan polymerase.2–4 RodA is absent or dispensable in many bacteria that contain a cell wall; however, all of these bacteria have a RodA homologue, FtsW, which is a core member of the divisome complex that is essential for septal cell wall assembly.5,6 FtsW was previously proposed flip the peptidoglycan precursor Lipid II to the peripasm,7,8 but we report here that FtsW polymerizes Lipid II. We show that FtsW polymerase activity depends on the presence of the class B PBP (bPBP) that it recruits to the septum. We also demonstrate that the polymerase activity of FtsW is required for its function in vivo. Our findings establish FtsW as a peptidoglycan polymerase that works with its cognate bPBP to produce septal peptidoglycan during cell division.

scholarly journals Biochemical and structural insights into the activation of PBP1b by the essential domain of FtsN

2020 ◽  
Author(s):  
Adrien Boes ◽  
Frederic Kerff ◽  
Raphael Herman ◽  
Thierry Touze ◽  
Eefjan Breukink ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Binding Pocket ◽  
Nonpermissive Temperature ◽  
Multiprotein Complex ◽  
Division Site ◽  
Structural Insights

AbstractPeptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division PG synthesis localizes at mid-cell under the control of a multiprotein complex, the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. The region L75 to Q93 of FtsN (EFtsN) was shown to be essential and sufficient for its functioning in vivo but the specific target and the molecular mechanism remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its GTase activity. We also report the crystal structure of PBP1b in complex with EFtsN which provides structural insights into the mode of binding of EFtsN at the junction between the GTase and UB2H domains of PBP1b. Interestingly, the mutations R141A/R397A of PBP1b, within the EFtsN binding pocket, reduce the activation of PBP1b by FtsN. This mutant was unable to rescue ΔponB-ponAts strain at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that PBP1b is a target of EFtsN and suggest that binding of FtsN to PBP1b contributes to trigger septal PG synthesis and cell constriction.

Mucopeptidhydrolasen in Escherichia coli B

1963 ◽  
Vol 18 (11) ◽  
pp. 956-964 ◽  
Author(s):  
H. Pelzer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Enzymatic Degradation ◽  
Quantitative Method ◽  
Degradation Products ◽  
In Vivo Studies ◽  
Partial Purification ◽  
E Coli ◽  
Escherichia Coli B

A quantitative method for the estimation of cell wall mucopeptides and their enzymatic degradation products by paper chromatography is described. The procedure can be used for measuring the activities of mucopeptidehydrolases as well as for in vivo studies of the metabolism of cell wall mucopeptides.A partial purification of E. coli mucopeptidehydrolases was achieved by column chromatography on DEAE-Sephadex.

scholarly journals CELL WALL COMPOSITION AND VIRULENCE IN ESCHERICHIA COLI

1968 ◽  
Vol 128 (3) ◽  
pp. 399-414 ◽  
Author(s):  
Donald N. Medearis ◽  
Bruce M. Camitta ◽  
Edward C. Heath
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Uridine Diphosphate ◽  
E Coli ◽  
Endotoxin Activity ◽  
A Cell ◽  
The Difference ◽  
Definition Of

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N-acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence.

scholarly journals Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

1998 ◽  
Vol 180 (17) ◽  
pp. 4406-4412 ◽  
Author(s):  
Dominique Mengin-Lecreulx ◽  
Juan Ayala ◽  
Ahmed Bouhss ◽  
Jean van Heijenoort ◽  
Claudine Parquet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Essential Gene ◽  
Cell Envelope ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Expression Of Genes

ABSTRACT Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (P mra ) on the chromosome and its replacement by the lac promoter (P mra ::P lac ) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P mra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P mra ::P lac promoter expression was repressed or fully induced. It was confirmed that the P mra promoter is required for expression of the first nine genes of the mra cluster:mraZ (orfC), mraW(orfB), ftsL (mraR),ftsI, murE, murF, mraY,murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P mra ::P lac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when P mra expression was repressed. It was concluded that the transcription of the genes located downstream fromftsW in the mra cluster, from murGto ftsZ, is also mainly (but not exclusively) dependent on the P mra promoter.

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