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

Abstract

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Results</p> <p>A nitroreductase- encoding gene was identified in the <it>N. gonorrhoeae </it>FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the <it>Escherichia coli </it>nitroreductase gene, <it>nfsB</it>. Cell extracts from <it>N. gonorrhoeae </it>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 ~3 × 10^-6- 8 × 10^-8among the various strains tested. The <it>nfsB </it>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.</p> <p>Conclusion</p> <p>We found that <it>nfsB </it>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.</p