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 Mutagenesis of the Rns regulator of enterotoxigenic Escherichia coli reveals roles for a linker sequence and two helix–turn–helix motifs

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2796-2806 ◽  
Author(s):  
Vivienne Mahon ◽  
Cyril J. Smyth ◽  
Stephen G. J. Smith
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein A ◽  
Diarrhoeal Disease ◽  
Enterotoxigenic Escherichia Coli ◽  
Insertion Mutagenesis

The pathogenesis of diarrhoeal disease due to human enterotoxigenic Escherichia coli absolutely requires the expression of fimbriae. The expression of CS1 fimbriae is positively regulated by the AraC-like protein Rns. AraC-like proteins are DNA-binding proteins that typically contain two helix–turn–helix (HTH) motifs. A program of pentapeptide insertion mutagenesis of the Rns protein was performed, and this revealed that both HTH motifs are required by Rns to positively regulate CS1 fimbrial gene expression. Intriguingly, a pentapeptide insertion after amino acid C102 reduced the ability of Rns to transactivate CS1 fimbrial expression. The structure of Rns in this vicinity (NACRS) was predicted to be disordered and thus might act as a flexible linker. This hypothesis was confirmed by deletion of this amino acid sequence from the Rns protein; a truncated protein that lacked this sequence was no longer functional. Strikingly, this sequence could be functionally substituted in vivo and in vitro by a flexible seven amino acid sequence from another E. coli AraC-like protein RhaS. Our data indicate that HTH motifs and a flexible sequence are required by Rns for maximal activation of fimbrial gene expression.

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 Ribosomal stalling landscapes revealed by high-throughput inverse toeprinting of mRNA libraries

2018 ◽  
Vol 1 (5) ◽  
pp. e201800148 ◽  
Author(s):  
Britta Seip ◽  
Guénaël Sacheau ◽  
Denis Dupuy ◽  
C Axel Innis
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Throughput ◽  
Quantitative Measure ◽  
Ribosome Profiling ◽  
Coding Region ◽  
Versatile Tool

Although it is known that the amino acid sequence of a nascent polypeptide can impact its rate of translation, dedicated tools to systematically investigate this process are lacking. Here, we present high-throughput inverse toeprinting, a method to identify peptide-encoding transcripts that induce ribosomal stalling in vitro. Unlike ribosome profiling, inverse toeprinting protects the entire coding region upstream of a stalled ribosome, making it possible to work with random or focused transcript libraries that efficiently sample the sequence space. We used inverse toeprinting to characterize the stalling landscapes of free and drug-boundEscherichia coliribosomes, obtaining a comprehensive list of arrest motifs that were validated in vivo, along with a quantitative measure of their pause strength. Thanks to the modest sequencing depth and small amounts of material required, inverse toeprinting provides a highly scalable and versatile tool to study sequence-dependent translational processes.

scholarly journals tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3467-3476 ◽  
Author(s):  
Zhijun Wang ◽  
Zhenghong Yuan ◽  
Li Xiang ◽  
Junjie Shao ◽  
Grzegorz Węgrzyn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Plasmid Dna ◽  
Copy Number ◽  
E Coli ◽  
Cole1 Plasmid ◽  
Specific Degradation ◽  
Rna I

Effects of tRNAAla(UGC) and its derivative devoid of the 3′-ACCA motif [tRNAAla(UGC)ΔACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysed in vivo and in vitro. In an amino-acid-starved relA mutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenic relA + host. Overexpression of tRNAAla(UGC), which under such conditions occurs in Escherichia coli mostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3′-ACCA-truncated tRNAAla(UGC). Moreover, tRNAAla(UGC), but not tRNAAla(UGC)ΔACCA, caused RNA I cleavage in vitro in the presence of MgCl2. These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearing E. coli, and demonstrate that tRNAAla(UGC) participates in specific degradation of RNA I in vivo and in vitro. This reaction is dependent on the presence of the 3′-ACCA motif of tRNAAla(UGC).

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

scholarly journals BglF, the Escherichia coli β-Glucoside Permease and Sensor of the bgl System: Domain Requirements of the Different Catalytic Activities

1999 ◽  
Vol 181 (2) ◽  
pp. 462-468 ◽  
Author(s):  
Qing Chen ◽  
Orna Amster-Choder
Keyword(s):  
Escherichia Coli ◽  
Specific Interactions ◽  
Phosphotransferase System ◽  
Structural Requirements ◽  
Catalytic Activities ◽  
Membrane Bound ◽  
Covalently Linked ◽  
The Individual

ABSTRACT The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of β-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG. In the presence of β-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports β-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIAbgland IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl. Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i.e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgland IICbgl which occur upon the interaction of BglF with the sugar.

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.

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