OXYGEN-INSENSITIVE NITROREDUCTASES - ANALYSIS OF THE ROLES OF NFSA AND NFSB IN DEVELOPMENT OF RESISTANCE TO 5-NITROFURAN DERIVATIVES IN ESCHERICHIA-COLI

Nitroheterocyclic and nitroaromatic compounds constitute an enormous r ange of chemicals whose potent biological activity has significant hum an health and environmental implications. The biological activity of n itro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase acti vities. Resistance of bacteria to nitro-substituted compounds is belie ved to result primarily from mutations in genes encoding oxygen-insens itive nitroreductases. We have characterized the nfsA and nfsB genes o f a large number of nitrofuran-resistant mutants of Escherichia coli a nd have correlated mutation with cell extract nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an nfsA mutation, while the increased resi stance associated with second-step mutants is a consequence of an nfsB mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, especially with regard t o the identification of flavin mononucleotide binding sites. We show t hat expression of plasmid-carried nfsA and nfsB genes in resistant mut ants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and 49%, respectivel y, contained insertion sequence elements in nfsA and nfsB. ISI integra ted in both genes, while IS30 and IS186 were found only in nfsA and IS 2 and IS5 were observed only in nfsB. Insertion hot spots for IS30 and IS186 are indicated in nfsA, and a hot spot for IS5 insertion is evid ent in nfsB. We discuss potential regional and sequence-specific deter minants for insertion sequence element integration in nfsA and nfsB.