scholarly journals Topological Analysis of the Aerobic Membrane-Bound Formate Dehydrogenase of Escherichia coli

1998 ◽  
Vol 180 (24) ◽  
pp. 6625-6634 ◽  
Author(s):  
Stéphane Benoit ◽  
Hafid Abaibou ◽  
Marie-Andrée Mandrand-Berthelot
Keyword(s):  
Escherichia Coli ◽  
Formate Dehydrogenase ◽  
Sequence Similarity ◽  
Topological Analysis ◽  
Physiological Role ◽  
Respiratory Pathway ◽  
High Sequence Similarity ◽  
Entire Sequence ◽  
Membrane Bound ◽  
Lactamase Gene

ABSTRACT Besides formate dehydrogenase N (FDH-N), which is involved in the major anaerobic respiratory pathway in the presence of nitrate,Escherichia coli synthesizes a second isoenzyme, called FDH-O, whose physiological role is to ensure rapid adaptation during a shift from aerobiosis to anaerobiosis. FDH-O is a membrane-bound enzyme complex composed of three subunits, α (FdoG), β (FdoH), and γ (FdoI), which exhibit high sequence similarity to the equivalent polypeptides of FDH-N. The topology of these three subunits has been studied by using blaM (β-lactamase) gene fusions. A collection of 47 different randomly generated Fdo-BlaM fusions, 4 site-specific fusions, and 3 sandwich fusions were isolated along the entire sequence of the three subunits. In contrast to previously reported predictions from sequence analysis, our data suggested that the αβ catalytic dimer is located in the cytoplasm, with a C-terminal anchor for β protruding into the periplasm. As expected, the γ subunit, which specifies cytochrome b, was shown to cross the cytoplasmic membrane four times, with the N and C termini exposed to the cytoplasm. Protease digestion studies of the35S-labelled FDH-O heterotrimer in spheroplasts add further support to this model. Consistently, prior studies regarding the bioenergetic function of formate dehydrogenase provided evidence for a mechanism in which formate is oxidized in the cytoplasm.

scholarly journals S-Methylmethionine Metabolism in Escherichia coli

1999 ◽  
Vol 181 (2) ◽  
pp. 662-665 ◽  
Author(s):  
Martin Thanbichler ◽  
Bernhard Neuhierl ◽  
August Böck
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Similarity ◽  
Physiological Role ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Amino Acid Permeases ◽  
Sequence Similarities ◽  
Homocysteine Methyltransferase

ABSTRACT Selenium-accumulating Astragalus spp. contain an enzyme which specifically transfers a methyl group fromS-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed thatEscherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is anS-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants inyagD which also possess defects in metE andmetH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine. Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in ametE metH genetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine. Methionine but notS-methylmethionine in the medium reduces the amount ofyagD protein. This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon. The physiological roles of the ykfDand yagD products appear to reside in the acquisition ofS-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Complete Nucleotide Sequence of an IncI2 Plasmid CoharboringblaCTX-M-55andmcr-1

2016 ◽  
Vol 60 (8) ◽  
pp. 5014-5017 ◽  
Author(s):  
Jian Sun ◽  
Xing-Ping Li ◽  
Run-Shi Yang ◽  
Liang-Xing Fang ◽  
Wei Huo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Complete Nucleotide Sequence ◽  
Sequence Similarity ◽  
Conjugative Plasmid ◽  
Insertion Sequences ◽  
Content Type ◽  
Extensive Sequence ◽  
Extended Spectrum ◽  
Lactamase Gene

ABSTRACTWe report the complete nucleotide sequence of a plasmid, pA31-12, carryingblaCTX-M-55andmcr-1from a chickenEscherichia coliisolate. pA31-12 has an IncI2 replicon that displays extensive sequence similarity with pHN1122-1-borneblaCTX-M-55and pHNSHP45-bornemcr-1. Insertion sequences ISEcp1and ISApl1are responsible for the mobilization ofblaCTX-M-55andmcr-1, respectively. The colocalization ofmcr-1with an extended-spectrum β-lactamase gene on a conjugative plasmid may accelerate the dissemination of both genes by coselection.

scholarly journals Evolution of Chi motifs in Proteobacteria

2020 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif

AbstractHomologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Wataru Hayashi ◽  
Hayato Tanaka ◽  
Yui Taniguchi ◽  
Masaki Iimura ◽  
Eiji Soga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Sequence Similarity ◽  
Multidrug Resistant ◽  
Common Source ◽  
High Sequence Similarity ◽  
Content Type ◽  
E Coli ◽  
Query Coverage ◽  
Food Animals

ABSTRACT This study focused on the detection of the plasmid-mediated mcr colistin resistance gene in Escherichia coli isolates from wastewater treatment plants (WWTPs). Seven influent samples were collected from three WWTPs in Nagano Prefecture, Japan, during August and December 2018. Colistin-resistant E. coli isolates were selected on colistin-supplemented CHROMagar ECC plates. mcr-1-positive isolates were subjected to whole-genome sequencing (WGS) analysis. From six influent samples, seven mcr-1-positive but extended-spectrum β-lactamase (ESBL)-negative isolates belonging to different genetic lineages, namely, B2-O25:H4-ST131-fimH22, B2-O2:H1-ST135-fimH2, B1-O8:H9-ST764-fimH32, B1-O23:H16-ST453-fimH31, A-O81:H27-ST10-fimH54, A-O16:H5-ST871-fimH25, and F-O11:H6-ST457-fimH145, were detected. The MICs of colistin for these isolates ranged from 4 to 16 mg/liter. The mcr-1 genes were located on plasmids belonging to IncX4 and IncI2 in five and two isolates, respectively. Four IncX4 plasmids with the same size (33,309 bp) showed high sequence similarity (4 single-nucleotide variations). The remaining one IncX4 plasmid, with a size of 33,858 bp, carried the mcr-1 gene with the single synonymous nucleic substitution T27C. Two IncI2 plasmids with sizes of 60,710 bp and 60,733 bp had high sequence similarity (99.9% identity; 100% query coverage). Two of five isolates carrying IncX4 plasmids and both of the isolates carrying IncI2 plasmids harbored ColV plasmids carrying virulence-associated genes of avian pathogenic E. coli (APEC). In addition, another isolate of the B2-O25:H4-ST131-fimH22 lineage had those APEC-associated virulence genes on its chromosome. In conclusion, mcr-1-positive E. coli environmental isolates were mostly characterized as positive for APEC-associated virulence genes. The copresence of those genes may suggest the existence of a common source in animals and/or their associated environments. IMPORTANCE Colistin is considered a last-line therapeutic option in severe infections due to multidrug-resistant Gram-negative bacteria, in particular carbapenemase-producing Enterobacteriaceae and multidrug-resistant Acinetobacter baumannii. An increasing prevalence of mcr genes in diverse Enterobacteriaceae species, mainly Escherichia coli and Klebsiella pneumoniae from humans and food animals, has become a significant concern to public health all over the world. In Japan, mcr genes have so far been detected in food animals, raw meat, wastewater, and human clinical samples. This study reports the copresence of mcr-1 and avian pathogenic E. coli (APEC)-associated virulence genes in five of seven E. coli isolates recovered from aquatic environments in Japan. Our study highlights the importance and urgency of action to reduce environmental contamination by mcr genes that may likely occur due to exposure to untreated wastewater through combined sewer overflow by recent unusual weather.

scholarly journals Identification of GutQ from Escherichia coli as a d-Arabinose 5-Phosphate Isomerase

2005 ◽  
Vol 187 (20) ◽  
pp. 6936-6942 ◽  
Author(s):  
Timothy C. Meredith ◽  
Ronald W. Woodard
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Biochemical Properties ◽  
Phosphotransferase System ◽  
High Sequence Similarity ◽  
E Coli ◽  
Regulatory Molecule ◽  
Conditional Mutant ◽  
K 12 ◽  
Functional Copy

ABSTRACT The glucitol operon (gutAEBDMRQ) of Escherichia coli encodes a phosphoenolpyruvate:sugar phosphotransferase system that metabolizes the hexitol d-glucitol (sorbitol). The functions for all but the last gene, gutQ, have been previously assigned. The high sequence similarity between GutQ and KdsD, a d-arabinose 5-phosphate isomerase (API) from the 3-deoxy-d-manno-octulosonate (KDO)-lipopolysaccharide (LPS) biosynthetic pathway, suggested a putative activity, but its role within the context of the gut operon remained unclear. Accordingly, the enzyme was cloned, overexpressed, and characterized. Recombinant GutQ was shown to indeed be a second copy of API from the E. coli K-12 genome with biochemical properties similar to those of KdsD, catalyzing the reversible aldol-ketol isomerization between d-ribulose 5-phosphate (Ru5P) and d-arabinose 5-phosphate (A5P). Genomic disruptions of each API gene were constructed in E. coli K-12. TCM11[(ΔkdsD)] was capable of sustaining essential LPS synthesis at wild-type levels, indicating that GutQ functions as an API inside the cell. The gut operon remained inducible in TCM7[(ΔgutQ)], suggesting that GutQ is not directly involved in d-glucitol catabolism. The conditional mutant TCM15[(ΔgutQΔkdsD)] was dependent on exogenous A5P both for LPS synthesis/growth and for upregulation of the gut operon. The phenotype was suppressed by complementation in trans with a plasmid encoding a functional copy of GutQ or by increasing the amount of A5P in the medium. As there is no obvious obligatory role for GutQ in the metabolism of d-glucitol and there is no readily apparent link between d-glucitol metabolism and LPS biosynthesis, it is suggested that A5P is not only a building block for KDO biosynthesis but also may be a regulatory molecule involved in expression of the gut operon.

scholarly journals A Novel Highly Thermostable Multifunctional Beta-Glycosidase from CrenarchaeonAcidilobus saccharovorans

Archaea ◽  
2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Vadim M. Gumerov ◽  
Andrey L. Rakitin ◽  
Andrey V. Mardanov ◽  
Nikolai V. Ravin
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Half Life ◽  
Sequence Similarity ◽  
Hydrolytic Activity ◽  
Maximum Activity ◽  
Glycoside Hydrolases ◽  
Amino Acid Residues ◽  
High Tolerance ◽  
High Sequence Similarity

We expressed a putativeβ-galactosidase Asac_1390 from hyperthermophilic crenarchaeonAcidilobus saccharovoransinEscherichia coliand purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity withp-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg−1), pNP-β-D-glucopyranoside (246 U mg−1), pNP-β-D-xylopyranoside (72 U mg−1), and pNP-β-D-mannopyranoside (28 U mg−1). Thus the enzyme was actually a multifunctionalβ-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

scholarly journals Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani

2017 ◽  
Vol 199 (17) ◽  
Author(s):  
David L. Cech ◽  
Katherine Markin ◽  
Ronald W. Woodard
Keyword(s):  
Sequence Similarity ◽  
Physiological Role ◽  
Gram Negative Bacteria ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Clostridium Tetani ◽  
High Sequence Similarity ◽  
Gram Negative ◽  
Content Type ◽  
Sugar Phosphates

ABSTRACT d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium. IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes

1975 ◽  
Vol 148 (2) ◽  
pp. 329-333 ◽  
Author(s):  
M B Kemp ◽  
B A Haddock ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytochrome B ◽  
Cytoplasmic Membrane ◽  
Coli Strain ◽  
Respiratory Pathway ◽  
Aminolaevulinic Acid ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

The synthesis of nitrate reductase and its incorporation into the cytoplasmic membrane of Escherichia coli strain A1004a (5-aminolaevulinic acid auxotroph) does not require synthesis of cytochrome b. The synthesis of the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to nitrate appears to be inhibited by the absence of haem. No member of the respiratory pathway from NADH to oxygen is capable of reducing nitrate reductase directly. The site on nitrate reductase that oxidizes FMNH2 is located on the cytoplasmic aspect of the cytoplasmic membrane.

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