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

BMC Microbiology
Daniel C SteinStephen Dunham

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 ...Continue Reading

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Citations

Sep 6, 2012·Antimicrobial Agents and Chemotherapy·Belinda S HallShane R Wilkinson

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Datasets Mentioned

BETA
GU112781
GU112782
GU112783
GU112770
GU112771
GU112772
GU112773
GU112774
GU112775
GU112776

Methods Mentioned

BETA
PCR
electrophoresis
protein assay

Software Mentioned

tblastn
ClustalW

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Antimicrobial resistance poses a significant threat to the continued successful use of antimicrobial agents for the treatment of bacterial infections.