scholarly journals In vivo and in vitro complementation of the N-terminal domain of enzyme I of the Escherichia coli phosphotransferase system by the cloned C-terminal domain

1998 ◽  
Vol 95 (15) ◽  
pp. 8491-8495 ◽  
Author(s):  
A. Fomenkov ◽  
A. Valiakhmetov ◽  
L. Brand ◽  
S. Roseman
Keyword(s):  
Escherichia Coli ◽  
Phosphotransferase System ◽  
Terminal Domain ◽  
Enzyme I

scholarly journals The Ascorbate Transporter of Escherichia coli

2003 ◽  
Vol 185 (7) ◽  
pp. 2243-2250 ◽  
Author(s):  
Zhongge Zhang ◽  
Mohammad Aboulwafa ◽  
Meghan H. Smith ◽  
Milton H. Saier,
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Energy Coupling ◽  
Phosphoryl Transfer ◽  
Transcriptional Level ◽  
Phosphotransferase System ◽  
Helical Segment ◽  
Enzyme I

ABSTRACT The sgaTBA genes of Escherichia coli encode a putative 12-transmembrane α-helical segment (12 TMS) transporter, an enzyme IIB-like protein and an enzyme IIA-like protein of the phosphotransferase system (PTS), respectively. We show that all three proteins as well as the energy-coupling PTS proteins, enzyme I and HPr, are required for the anaerobic utilization and uptake of l-ascorbate in vivo and its phosphoenolpyruvate-dependent phosphorylation in vitro. The transporter exhibits an apparent Km for l-ascorbate of 9 μM and is highly specific. The sgaTBA genes are regulated at the transcriptional level by the yjfQ gene product, as well as by Crp and Fnr. The yjfR gene product is essential for l-ascorbate utilization and probably encodes a cytoplasmic l-ascorbate 6-phosphate lactonase. We conclude that SgaT represents a novel prototypical enzyme IIC that functions with SgaA and SgaB to allow phosphoryl transfer from HPr(his-P) to l-ascorbate via the phosphoryl transfer pathway: PEP → enzyme I-P → HPr-P → IIA- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\mathrm{SgaA}}{\mathrm{P}}\) \end{document} → IIB- \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\mathrm{SgaB}}{\mathrm{P}}\) \end{document} \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\stackrel{\textstyle{\mathrm{IIC^{SgaT}}}}{{\rightarrow}}\) \end{document} l-ascorbate-6-P.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals PafR, a Novel Transcription Regulator, Is Important for Pathogenesis in Uropathogenic Escherichia coli

2014 ◽  
Vol 82 (10) ◽  
pp. 4241-4252 ◽  
Author(s):  
Mordechai Baum ◽  
Mobarak Watad ◽  
Sara N. Smith ◽  
Christopher J. Alteri ◽  
Noa Gordon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Gene Knockout ◽  
Sensory System ◽  
Phosphotransferase System ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Content Type

ABSTRACTThemetVgenomic island in the chromosome of uropathogenicEscherichia coli(UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termedpafRandpafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes intransin the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that bothpafRandpafPare important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes thepafgenesin vitro, suggesting that they arein vivoinduced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that thepafgenes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

scholarly journals An Atypical KdpD Homologue from the Cyanobacterium Anabaena sp. Strain L-31: Cloning, In Vivo Expression, and Interaction with Escherichia coli KdpD-CTD

2005 ◽  
Vol 187 (14) ◽  
pp. 4921-4927 ◽  
Author(s):  
Anand Ballal ◽  
Marc Bramkamp ◽  
Hema Rajaram ◽  
Petra Zimmann ◽  
Shree Kumar Apte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Response Regulator ◽  
Nitrilotriacetic Acid ◽  
Soluble Form ◽  
Filamentous Cyanobacterium ◽  
E Coli ◽  
Transmembrane Segments ◽  
Terminal Domain

ABSTRACT The kdpFABC operon of Escherichia coli, coding for the high-affinity K+ transport system KdpFABC, is transcriptionally regulated by the products of the adjacently located kdpDE genes. The KdpD protein is a membrane-bound sensor kinase consisting of a large N-terminal domain and a C-terminal transmitter domain interconnected by four transmembrane segments (the transmembrane segments together with the C-terminal transmitter domain of KdpD are referred to as CTD), while KdpE is a cytosolic response regulator. We have cloned and sequenced the kdp operon from a nitrogen-fixing, filamentous cyanobacterium, Anabaena sp. strain L-31 (GenBank accession. number AF213466 ). The kdpABC genes are similar in size to those of E. coli, but the kdpD gene is short (coding only for 365 amino acids), showing homology only to the N-terminal domain of E. coli KdpD. A kdpE-like gene is absent in the vicinity of this operon. Anabaena KdpD with six C-terminal histidines was overproduced in E. coli and purified by Ni2+-nitrilotriacetic acid affinity chromatography. With antisera raised against the purified Anabaena KdpD, the protein was detected in Anabaena sp. strain L-31 membranes. The membrane-associated or soluble form of the Anabaena KdpD(6His) could be photoaffinity labeled with the ATP analog 8-azido-ATP, indicating the presence of an ATP binding site. The coproduction of Anabaena KdpD with E. coli KdpD-CTD decreased E. coli kdpFABC expression in response to K+ limitation in vivo relative to the wild-type KdpD-CTD protein. In vitro experiments revealed that the kinase activity of the E. coli KdpD-CTD was unaffected, but its phosphatase activity increased in the presence of Anabaena KdpD(6His). To our knowledge this is the first report where a heterologous N-terminal domain (Anabaena KdpD) is shown to affect in trans KdpD-CTD (E. coli) activity, which is just opposite to that observed for the KdpD-N-terminal domain of E. coli.

scholarly journals Chitin, Chitin Oligosaccharide, and Chitin Disaccharide Metabolism of Escherichia coli Revisited: Reassignment of the Roles of ChiA, ChbR, ChbF, and ChbG

2021 ◽  
pp. 1-17
Author(s):  
Axel Walter ◽  
Simon Friz ◽  
Christoph Mayer
Keyword(s):  
Escherichia Coli ◽  
Catalytic Mechanism ◽  
Phosphotransferase System ◽  
E Coli ◽  
The Family ◽  
Acetyl Group ◽  
Bona Fide ◽  
Modeling Data

<i>Escherichia coli</i> is unable to grow on polymeric and oligomeric chitin, but grows on chitin disaccharide (GlcNAc-GlcNAc; <i>N,N</i>′-diacetylchitobiose) and chitin trisaccharide (GlcNAc-GlcNAc-GlcNAc; <i>N,N</i>′<i>,N</i>′′-triacetylchitotriose) via expression of the <i>chb</i> operon (<i>chbBCARFG</i>). The phosphotransferase system (PTS) transporter ChbBCA facilitates transport of both saccharides across the inner membrane and their concomitant phosphorylation at the non-reducing end, intracellularly yielding GlcNAc 6-phosphate-GlcNAc (GlcNAc6P-GlcNAc) and GlcNAc6P-GlcNAc-GlcNAc, respectively. We revisited the intracellular catabolism of the PTS products, thereby correcting the reported functions of the 6-phospho-glycosidase ChbF, the monodeacetylase ChbG, and the transcriptional regulator ChbR. Intracellular accumulation of glucosamine 6P-GlcNAc (GlcN6P-GlcNAc) and GlcN6P-GlcNAc-GlcNAc in a <i>chbF</i> mutant unraveled a role for ChbG as a monodeacetylase that removes the <i>N-</i>acetyl group at the non-reducing end. Consequently, GlcN6P- but not GlcNAc6P-containing saccharides likely function as coactivators of ChbR. Furthermore, ChbF removed the GlcN6P from the non-reducing terminus of the former saccharides, thereby degrading the inducers of the <i>chb</i> operon and facilitating growth on the saccharides. Consequently, ChbF was unable to hydrolyze GlcNAc6P-residues from the non-reducing end, contrary to previous assumptions but in agreement with structural modeling data and with the unusual catalytic mechanism of the family 4 of glycosidases, to which ChbF belongs. We also refuted the assumption that ChiA is a bifunctional endochitinase/lysozyme ChiA, and show that it is unable to degrade peptidoglycans but acts as a bona fide chitinase in vitro and in vivo, enabling growth of <i>E. coli</i> on chitin oligosaccharides when ectopically expressed. Overall, this study revises our understanding of the chitin, chitin oligosaccharide, and chitin disaccharide metabolism of <i>E. coli</i>.

scholarly journals Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli

2010 ◽  
Vol 192 (22) ◽  
pp. 5934-5942 ◽  
Author(s):  
Aurélie Barnéoud-Arnoulet ◽  
Marthe Gavioli ◽  
Roland Lloubès ◽  
Eric Cascales
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Signal Sequence ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Outer Membrane Receptor ◽  
Terminal Domain

ABSTRACT Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. We first produced KT fused to a signal sequence for periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.

scholarly journals A Single Mutation in Enzyme I of the Sugar Phosphotransferase System Confers Penicillin Tolerance to Streptococcus gordonii

2009 ◽  
Vol 54 (1) ◽  
pp. 259-266 ◽  
Author(s):  
A. Bizzini ◽  
J. M. Entenza ◽  
O. Michielin ◽  
I. Arnold ◽  
B. Erni ◽  
...  
Keyword(s):  
Sugar Uptake ◽  
Complete Disappearance ◽  
Single Mutation ◽  
Streptococcus Gordonii ◽  
Phosphotransferase System ◽  
Drug Induced ◽  
Nucleotide Mutation ◽  
Enzyme I

ABSTRACT Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log10 CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log10 CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

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