Inhibitors of nitrofuran reduction in Escherichia coli: evidence for their existence, partial purification, binding of nitrofurantoin in vitro, and implications for nitrofuran resistance.
Nitrofurantoin (NF)-resistant mutants of Escherichia coli were isolated as described previously (18). One of the mutants (SSJ-2) was found to possess NF reductase activity equal to that of its parent (E. coli KL16). Two NF-resistant transductional derivatives, SSJ-2A and SSJ-2B, were isolated using SSJ-2 as the donor. SSJ-2 was found to be a double mutant carrying two mutations, nfnA and nfnB, while SSJ-2A (nfnA) and SSJ-2B (nfnB) carried these mutations individually. Heated extracts from SSJ-2A and SSJ-2B were found to inhibit the reduction of NF by unheated extracts of the NF-sensitive strain E. coli KL16 in vitro. Unheated extracts of these mutants reduced NF poorly relative to E. coli KL16. The poor reduction of NF by unheated extracts of SSJ-2A and SSJ-2B was greatly stimulated by heated extracts of SSJ-2B and SSJ-2A, respectively, and also by heated extracts of E. coli KL16. When heated extracts of SSJ-2A and SSJ-2B were mixed in a particular ratio and added to unheated extracts of E. coli KL16 they lost their inhibitory activity. Two proteins, designated inhibitor A and inhibitor B, have been partially purified from heated extracts of SSJ-2B and SSJ-2A, respectively. Their respective molecular weights, as determined by gel chromatography, were 37,000 and 20,500. The two inhibitors bound nitrofurantoin in vitro, and the NF-binding ability was lost when mixed in the molar ration of 3/1 (B/A). These observations were rationalized in terms of a hypothesis which explains (i) maximal NF reduction in wild-type cells, (ii) maximal NF reduction of nfnA-nfnB- double mutant, and (iii) poor NF reduction in nfnA- or nfnB- single mutants. The possible role of these inhibitors in nitrofurantoin resistance is also discussed.
D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D009154:Mutation; D009582:Nitrofurantoin / Q000378:metabolism* / Q000494:pharmacology; D009601:Nitroreductases; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D011485:Protein Binding
D009582:Nitrofurantoin; D010088:Oxidoreductases; C029492:aromatic NADH-dependent nitroreductase; D009601:Nitroreductases