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


RESULTS
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.


CONCLUSIONS
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.
Stein
Daniel C
DC
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA. dcstein@umd.edu
Carrizosa
Esteban
E
Dunham
Stephen
S
D000595:Amino Acid Sequence; D000892:Anti-Infective Agents, Urinary / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009344:Neisseria gonorrhoeae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D009582:Nitrofurantoin / Q000494:pharmacology; D009601:Nitroreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D000892:Anti-Infective Agents, Urinary; D009582:Nitrofurantoin; D009601:Nitroreductases