Dataset Information

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae.

ABSTRACT: Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions).A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of approximatelty 3 x 10(-6) - 8 x 10(-8) among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene.We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.

SUBMITTER: Stein DC

PROVIDER: S-EPMC2788575 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae.

Stein Daniel C DC Carrizosa Esteban E Dunham Stephen S

BMC microbiology 20091123

<h4>Background</h4>Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions).<h4>Results</h ...[more]

PMID: 19930647

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Project description:BACKGROUND:New approaches are urgently required to address increasing rates of gonorrhoea and the emergence and global spread of antibiotic-resistant Neisseria gonorrhoeae. We used whole-genome sequencing to study transmission and track resistance in N gonorrhoeae isolates. METHODS:We did whole-genome sequencing of isolates obtained from samples collected from patients attending sexual health services in Brighton, UK, between Jan 1, 2011, and March 9, 2015. We also included isolates from other UK locations, historical isolates from Brighton, and previous data from a US study. Samples from symptomatic patients and asymptomatic sexual health screening underwent nucleic acid amplification testing; positive samples and all samples from symptomatic patients were cultured for N gonorrhoeae, and resulting isolates were whole-genome sequenced. Cefixime susceptibility testing was done in selected isolates by agar incorporation, and we used sequence data to determine multi-antigen sequence types and penA genotypes. We derived a transmission nomogram to determine the plausibility of direct or indirect transmission between any two cases depending on the time between samples: estimated mutation rates, plus diversity noted within patients across anatomical sites and probable transmission pairs, were used to fit a coalescent model to determine the number of single nucleotide polymorphisms expected. FINDINGS:1407 (98%) of 1437 Brighton isolates between Jan 1, 2011, and March 9, 2015 were successfully sequenced. We identified 1061 infections from 907 patients. 281 (26%) of these infections were indistinguishable (ie, differed by zero single nucleotide polymorphisms) from one or more previous cases, and 786 (74%) had evidence of a sampled direct or indirect Brighton source. We observed multiple related samples across geographical locations. Of 1273 infections in Brighton (including historical data), 225 (18%) were linked to another case elsewhere in the UK, and 115 (9%) to a case in the USA. Four lineages initially identified in Brighton could be linked to 70 USA sequences, including 61 from a lineage carrying the mosaic penA XXXIV allele, which is associated with reduced cefixime susceptibility. INTERPRETATION:We present a whole-genome-sequencing-based tool for genomic contact tracing of N gonorrhoeae and demonstrate local, national, and international transmission. Whole-genome sequencing can be applied across geographical boundaries to investigate gonorrhoea transmission and to track antimicrobial resistance. FUNDING:Oxford National Institute for Health Research Health Protection Research Unit and Biomedical Research Centre.

Dataset Information

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae.

Publications

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae.

Similar Datasets

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