Analysis of partially purified crude extract of Escherichia coli K12 by chromatography and gel electrophoresis has resulted in the separation of three distinct activities which catalyse the reduction of nitrofurazone (semicarbazone of 5-nitro-2-furaldehyde) in the presence of oxygen (type I nitroreductases). The major enzymatic activity (type IA), which was dependent solely on NADPH as a cofactor, was absent from nitrofurazone-resistant strains NFR 402 and NFR 502, but present in SIL 41, a strain which is only marginally resistant to the nitrofuran. The remaining nitroreductase activities (IB1 and IB2) utilize either NADH or NADPH as a cofactor. These activities coelute from DEAE-cellulose at pH 7.2, but may be differentiated by their behaviour on CM-cellulose at pH 5.8. The reductase activity missing in SIL 41 was observed in extracts of strain NFR 402 but not NFR 502. This enzyme (IB1) though retained by DEAE-cellulose had no affinity for CM-cellulose. The only reductase present in extracts of NFR 502 (a nitrofuran-resistant strain selected after two mutational events) was type IB2. This activity, also detectable in SIL 41 and NFR 402, has not been mapped genetically. An interesting feature of the type IB2 enzyme is its apparent inactivation by MnCl2 which has been routinely used as a partial purification step in the past.

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Wild-type Escherichia coli cells are sensitive to nitrofurazone (NF) and many other nitrofuran derivatives. A variety of evidence indicated that these compounds are converted to toxic "active" metabolites by reductases present in the bacteria. Sensitive E. coli K-12 acquired threefold-greater resistance to NF in one mutational step. These partially resistant mutants could undergo a second mutation that made them 10 times as resistant as the wild type. Mutation of wild-type strain K-12 to the higher level of resistance in a single step was not observed. The first mutational step was associated with partial loss of reduced nicotinamide adenine dinucleotide phosphate-linked, 02-insensitive NF reductase activity, and the second step was associated with loss of the remaining activity. The two-step mutants did, however, contain other NF reductases that were inhibited by 02 and reduced NF only under anaerobic conditions. We designated the genes that control reductase activity "nitrofuran sensitivity genes" (nfsA and nfsB). Thus, wild-type strains are nfsA+ nfsB+, and the resistant double mutants are nfsA nfsB. A variety of crosses established that these genes are both located close to gal, that the most probable sequence is lac nfsB gal nfsA, and that the singlestep mutants with an intermediate level of resistance are nfsA nfsB+. The nfsA+ nfsB strains contained about 70 to 80% of the wild-type reductase I activity-apparently enough to confer wild-type sensitivity. This reductase activity was resistant to 2 M urea. The nfsA nfsB+ strains had only 20 to 30% of the wild-type activity, and this residual activity was sensitive to 2 M urea. Bacbmann, Yale University, New Haven, Conn. The isolation and properties of resistant mutants are described in Table 1 and in Results. Media and culture condition. Bacteria were routinely cultured on nutrient agar (Oxoid, London, England) plates or in nutrient broth no. 2 (Oxoid) or Penassay broth (Difco Laboratories, Detroit, Mich.). Defined medium consisted of Davis-Mingioli medium

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When Escherichia coli strain B/r is exposed to 10 to 20 ug of nitrofurazone per ml, mutants with roughly threefold resistance are obtained. Treatment of these mutants with higher concentrations of nitrofurazone yields strains with six-to sevenfold resistance over strain B/r. Each of these steps toward nitrofurazone resistance is accompanied by loss of soluble nitrofurazone reductase activity. When sensitive bacteria are exposed to labeled nitrofurazone or labeled 2-nitrofuran, a considerable amount of radioactivity becomes bound to the cold trichloroacetic acid-insoluble fraction. Very little activity becomes bound in the mutants with sixto seven-fold resistance; mutants with intermediate resistance show intermediate levels of binding. Partially purified nitrofurazone reductase preparations catalyze the conversion of nitrofurazone to compounds which bind to protein and are not removed by prolonged dialysis against 8 M urea or by cold acid. Nitrofurazone reduced by xanthine oxidase or electrolytically reduced also yields compounds which react with protein to form stable derivatives.