Characterization of Mutations Contributing to Sulfathiazole Resistance in Escherichia coli

ABSTRACT A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia colirepeatedly selected for sulfathiazole resistance was mapped tofolP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C→T transition resulted in a Pro→Ser substitution at amino acid position 64. Replacement of the mutantfolP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance,sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by thesepurU strains.

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Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

Biochemical Journal ◽

10.1042/bj3140063 ◽

1996 ◽

Vol 314 (1) ◽

pp. 63-71 ◽

Cited By ~ 10

Author(s):

Johanneke L. H. BUSCH ◽

Jacques L. J. BRETON ◽

Barry M. BARTLETT ◽

Richard JAMES ◽

E. Claude HATCHIKIAN ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Magnetic Circular Dichroism ◽

Wild Type ◽

E Coli ◽

Excess Iron ◽

Expression In Escherichia Coli ◽

Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

Journal of Bacteriology ◽

10.1128/jb.181.14.4318-4325.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4318-4325 ◽

Cited By ~ 42

Author(s):

Masaru Ohara ◽

Henry C. Wu ◽

Krishnan Sankaran ◽

Paul D. Rick

Keyword(s):

Escherichia Coli ◽

Direct Consequence ◽

Insertion Mutant ◽

Polynucleotide Phosphorylase ◽

Wild Type ◽

E Coli ◽

K 12 ◽

Identification And Characterization ◽

Lines Of Evidence

ABSTRACT We report here the identification of a new lipoprotein, NlpI, inEscherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp(polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.

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Differential effects of a molybdopterin synthase sulfurylase (moeB) mutation on Escherichia coli molybdoenzyme maturation

Biochemistry and Cell Biology ◽

10.1139/o02-131 ◽

2002 ◽

Vol 80 (4) ◽

pp. 435-443 ◽

Cited By ~ 10

Author(s):

Damaraju Sambasivarao ◽

Raymond J Turner ◽

Peter T Bilous ◽

Richard A Rothery ◽

Gillian Shaw ◽

...

Keyword(s):

Escherichia Coli ◽

Nitrate Reductase ◽

Mutant Strain ◽

Physiological Function ◽

Signal Sequence ◽

Molybdenum Cofactor ◽

Wild Type ◽

Dmso Reductase ◽

E Coli ◽

Membrane Bound

We have generated a chromosomal mutant of moeB (moeBA228T) that demonstrates limited molybdenum cofactor (molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD)) availability in Escherichia coli and have characterized its effect on the maturation and physiological function of two well-characterized respiratory molybdoenzymes: the membrane-bound dimethylsulfoxide (DMSO) reductase (DmsABC) and the membrane-bound nitrate reductase A (NarGHI). In the moeBA228T mutant strain, E. coli F36, anaerobic respiratory growth is possible on nitrate but not on DMSO, indicating that cofactor insertion occurs into NarGHI but not into DmsABC. Fluorescence analyses of cofactor availability indicate little detectable cofactor in the moeBA228T mutant compared with the wild-type, suggesting that NarGHI is able to scavenge limiting cofactor, whereas DmsABC is not. MoeB functions to sulfurylate MoaD, and in the structure of the MoeBMoaD complex, Ala-228 is located in the interface region between the two proteins. This suggests that the moeBA228T mutation disrupts the interaction between MoeB and MoaD. In the case of DmsABC, despite the absence of cofactor, the twin-arginine signal sequence of DmsA is cleaved in the moeBA228T mutant, indicating that maturation of the holoenzyme is not cofactor-insertion dependent.Key words: mdybdenum cofactor, DMSO reductase, nitrate reductase.

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ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000441640 ◽

2015 ◽

Vol 25 (6) ◽

pp. 394-402 ◽

Cited By ~ 1

Author(s):

Taylor L. Fischer ◽

Robert J. White ◽

Katherine F.K. Mares ◽

Devin E. Molnau ◽

Justin J. Donato

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Wild Type ◽

E Coli ◽

Enoyl Acp Reductase ◽

Selection For

Background/Aims: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. Methods: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. Results: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. Conclusion: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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Isolation and characterization of catabolite repression-resistant mutants of Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/m77-207 ◽

1977 ◽

Vol 23 (10) ◽

pp. 1384-1393 ◽

Cited By ~ 2

Author(s):

Glen D. Armstrong ◽

Hiroshi Yamazaki

Keyword(s):

Escherichia Coli ◽

Catabolite Repression ◽

Coli Strain ◽

Wild Type ◽

Phosphotransferase System ◽

E Coli ◽

Isolation And Characterization ◽

In The Wild ◽

Resistant Mutants

A method has been developed for the isolation of Escherichia coli mutants which are resistant to catabolite repression. The method is based on the fact that a mixture of glucose and gluconate inhibits the development of chemotactic motility in the wild type, but not in the mutants. A motile E. coli strain was mutagenized and grown in glucose and gluconate. Mutants which were able to swim into a tube containing a chemotactic attractant (aspartic acid) were isolated. Most of these mutants were able to produce β-galactosidase in the presence of glucose and gluconate and were normal in their ability to degrade adenosine 3′,5′-cyclic monophosphate. Some of these mutants were defective in the glucose phosphotransferase system.

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Characterization of Cefoxitin-Resistant Escherichia coli Isolates from Recreational Beaches and Private Drinking Water in Canada between 2004 and 2006

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01353-08 ◽

2009 ◽

Vol 53 (7) ◽

pp. 3126-3130 ◽

Cited By ~ 27

Author(s):

L. F. Mataseje ◽

N. Neumann ◽

B. Crago ◽

P. Baudry ◽

G. G. Zhanel ◽

...

Keyword(s):

Escherichia Coli ◽

Drinking Water ◽

Multidrug Resistance ◽

Promoter Region ◽

Water Sources ◽

Wild Type ◽

Phylogenetic Groups ◽

E Coli ◽

Recreational Beaches

ABSTRACT A total of 142 cefoxitin-resistant Escherichia coli isolates from water sources were collected across Canada. Multidrug resistance was observed in 65/142 (45.8%) isolates. The bla CMY-2 gene was identified in 110/142 (77.5%) isolates. Sequencing of the chromosomal ampC promoter region showed mutations from the wild type, previously shown to hyperproduce AmpC. CMY-2-producing plasmids predominantly belonged to replicon groups I1-Iγ, A/C, and K/B. The majority of the E. coli isolates belonged to the nonvirulent phylogenetic groups A and B1.

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Identification of rcnA (yohM), a Nickel and Cobalt Resistance Gene in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.8.2912-2916.2005 ◽

2005 ◽

Vol 187 (8) ◽

pp. 2912-2916 ◽

Cited By ~ 121

Author(s):

Agnès Rodrigue ◽

Géraldine Effantin ◽

Marie-Andrée Mandrand-Berthelot

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Nickel Content ◽

Efflux System ◽

E Coli ◽

Cobalt Resistance ◽

Membrane Bound ◽

Cadmium Zinc ◽

Cobalt And Nickel

ABSTRACT We report here on the isolation and primary characterization of the yohM gene of Escherichia coli. We show that yohM encodes a membrane-bound polypeptide conferring increased nickel and cobalt resistance in E. coli. yohM was specifically induced by nickel or cobalt but not by cadmium, zinc, or copper. Mutation of yohM increased the accumulation of nickel inside the cell, whereas cells harboring yohM in multicopy displayed reduced intracellular nickel content. Our data support the hypothesis that YohM is the first described efflux system for nickel and cobalt in E. coli. We propose rcnA (resistance to cobalt and nickel) as the new denomination of yohM.

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Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits

Biochemical Journal ◽

10.1042/bj3370379 ◽

1999 ◽

Vol 337 (3) ◽

pp. 379-385 ◽

Cited By ~ 11

Author(s):

Javier VELASCO ◽

Santiago GUTIERREZ ◽

Sonia CAMPOY ◽

Juan F. MARTIN

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Protein Band ◽

Immunoaffinity Chromatography ◽

Enzymic Activity ◽

Acremonium Chrysogenum ◽

Wild Type ◽

E Coli ◽

Protein Levels

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

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Pet, an Autotransporter Enterotoxin from Enteroaggregative Escherichia coli

Infection and Immunity ◽

10.1128/iai.66.7.3155-3163.1998 ◽

1998 ◽

Vol 66 (7) ◽

pp. 3155-3163 ◽

Cited By ~ 163

Author(s):

Carlos Eslava ◽

Fernando Navarro-García ◽

John R. Czeczulin ◽

Ian R. Henderson ◽

Alejandro Cravioto ◽

...

Keyword(s):

Escherichia Coli ◽

Short Circuit ◽

Ussing Chamber ◽

Short Circuit Current ◽

Nucleotide Sequence Analysis ◽

E Coli ◽

Eaec Strain ◽

Heat Labile ◽

Tissue Resistance

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging cause of diarrheal illness. Clinical data suggest that diarrhea caused by EAEC is predominantly secretory in nature, but the responsible enterotoxin has not been described. Work from our laboratories has implicated a ca. 108-kDa protein as a heat-labile enterotoxin and cytotoxin, as evidenced by rises in short-circuit current and falls in tissue resistance in rat jejunal tissue mounted in an Ussing chamber. Here we report the genetic cloning, sequencing, and characterization of this high-molecular-weight heat-labile toxin. The toxin (designated the plasmid-encoded toxin [Pet]) is encoded on the 65-MDa adherence-related plasmid of EAEC strain 042. Nucleotide sequence analysis suggests that the toxin is a member of the autotransporter class of proteins, characterized by the presence of a conserved C-terminal domain which forms a β-barrel pore in the bacterial outer membrane and through which the mature protein is transported. The Pet toxin is highly homologous to the EspP protease of enterohemorrhagic E. coli and to EspC of enteropathogenicE. coli, an as yet cryptic protein. In addition to its potential role in EAEC infection, Pet represents the first enterotoxin within the autotransporter class of secreted proteins. We hypothesize that other closely related members of this class may also produce enterotoxic effects.

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Characterization of YvcJ, a Conserved P-Loop-Containing Protein, and Its Implication in Competence in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.01493-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1556-1564 ◽

Cited By ~ 10

Author(s):

Jennifer Luciano ◽

Elodie Foulquier ◽

Jean-Raphael Fantino ◽

Anne Galinier ◽

Frédérique Pompeo

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Transformation Efficiency ◽

Cellular Function ◽

Protein Family ◽

Wild Type ◽

Uncharacterized Protein ◽

E Coli ◽

Phosphate Synthase

ABSTRACT The uncharacterized protein family UPF0042 of the Swiss-Prot database is predicted to be a member of the conserved group of bacterium-specific P-loop-containing proteins. Here we show that two of its members, YvcJ from Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression was quite similar in the two strains. However, in mutants defective in yvcJ, the transformation efficiency and the fraction of cells that expressed competence were reduced. Furthermore, our data show that YvcJ positively controls the expression of late competence genes. The overexpression of comK or comS compensates for the decrease in competence of the yvcJ mutant. Our results show that even if YvcJ and YhbJ belong to the same family of P-loop-containing proteins, the deletion of corresponding genes has different consequences in B. subtilis and in E. coli.

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