Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

Wehnert, Gabi U.; Abratt, Valerie R.; Woods, David R.

doi:10.1016/0147-619x(92)90027-8

Cited by 8 publications

(3 citation statements)

References 17 publications

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“…No significant homology with any published sequences was found. In particular, the nim genes share no relationship with the gene isolated from another strain of B. fragilis which would encode a product involved in metronidazole resistance in E. coli (25).…”

Section: Materuils and Methodsmentioning

confidence: 99%

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes

Haggoud

Reysset

Azeddoug

et al. 1994

Antimicrob Agents Chemother

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DNA sequence analysis of regions from plasmid pIP417 and chromosome BF8 which encode 5-nitroimidazole resistance in Bacteroides strains allowed the identification of two open reading frames corresponding to new genes, nimA (528 bp) and nimB (492 bp). Either gene may confer 5-nitroimidazole resistance to susceptible strains of Bacteroides. The encoded polypeptides have deduced molecular masses of 20.1 and 18.6 kDa, respectively, and share about 73% identity and 85% similarity. A new insertion sequence (IS) element named IS1168 lies 14 bases upstream of the nimA gene. The complete sequence of IS1168 was determined. A similar IS exists 12 bp upstream of the nimB gene. About 60% of the BF8 IS element was also sequenced and shown to be almost identical to IS1168.

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Section: Materuils and Methodsmentioning

confidence: 99%

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes

Haggoud

Reysset

Azeddoug

et al. 1994

Antimicrob Agents Chemother

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“…Southern blot analysis was performed according to standard protocols 15 using genomic DNA (20 µg) of both B. thetaiotaomicron VPI-5482 and the B. thetaiotaomicron Tn Met R mutant, as well as 20 ng of the 13.1 kb transposon delivery vector, pYT646B, each digested to completion with restriction enzyme PvuII. 15 B. thetaiotaomicron total genomic DNA was prepared according to the method of Wehnert et al 19 and E. coli plasmid DNA was prepared by the alkali-hydrolysis method of Ish-Horowicz and Burke. 20 The DNA probe used for hybridization was a 0.9 kb internal fragment of the pYT646B tetQ gene generated by digesting it with SacI and EcoRI.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Patel

Paul

Casanueva

et al. 2009

Journal of Antimicrobial Chemotherapy

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ObjectivesThe aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance.MethodsThe genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays.ResultsA metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose.ConclusionsThese data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron

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“…Southern blot analysis was performed according to standard protocols 15 using genomic DNA (20 mg) of both B. thetaiotaomicron VPI-5482 and the B. thetaiotaomicron Tn Met R mutant, as well as 20 ng of the 13.1 kb transposon delivery vector, pYT646B, each digested to completion with restriction enzyme PvuII. 15 B. thetaiotaomicron total genomic DNA was prepared according to the method of Wehnert et al 19 and E. coli plasmid DNA was prepared by the alkali-hydrolysis method of Ish-Horowicz and Burke. 20 The DNA probe used for hybridization was a 0.9 kb internal fragment of the pYT646B tetQ gene generated by digesting it with SacI and EcoRI.…”

Section: Tn4400 0 Transposon Mutagenesis Southern Hybridization and mentioning

confidence: 99%

Bacteroides thetaiotaomicron

2008

Encyclopedia of Genetics, Genomics, Proteomics and Informatics

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The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance. Methods: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays. Results: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose. Conclusions: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron

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Molecular analysis of a gene fromBacteroides fragilis involved in metronidazole resistance inEscherichia coli

Cited by 8 publications

References 17 publications

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron

Bacteroides thetaiotaomicron

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