Journal of General Microbiology (I 973), 76,2 1-29 21 Printed in Great Britain Nitrite Reductase-deficient Mutants of Escherichia coli ~12 By J. A. COLE AND F. B. W A R D Department of Biochemistry, University of Birmingham, Birmingham, B I 5 2 TT (Received 2 June I 972 ; revised I 7 October I 972) SUMMARY Mutants of Escherichia coli K12 have been isolated which reduce nitrite 3 to 30 yi as rapidly as the wild-type. Activities of reduced nicotinamide adenine dinucleotide (NADH)-nitrite oxidoreductase were lower in cell-free extracts of these nirA* mutants than in the wild-type. The mutants grew on minimal agar, and their sulphite reductase activity was the same as in the wild-type. Double mutants deficient in both nitrite and sulphite reductases were con- structed, as well as recombinants which had regained one or both activities follow- ing recombination with Escherichia coli Hfr Hayes. The inability to reduce sulphite was due to an altered cysBt gene. Suspensions of nirA cysBf and nirA cysB bacteria reduced nitrite at similar rates, showing that sulphite reductase (which is a gratuitous nitrite reductase) contributes little to the rate of nitrite reduction in vivo. Cytochome c552 was synthesized by nirA+ cysB+ double recom- binants but not by nirA cysB or nirA cysBf bacteria. This data suggests that cytochrome c552 is involved in nitrite reduction in E. coli either as a component of NADH-NO2- oxidoreductase, or as an electron carrier whose synthesis is affected by the iiir gene. INTRODUCTION This paper describes the isolation and biochemical characteristics of mutants of Escherichia coli K I 2 which lack reduced nicotinamide adenine dinucleotide (NADH)-nitrite oxido- reductase activity (EC. I .6.6.4). We have estimated the relative contributions of this enzyme, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent sulphite reductase, to the rate of nitrite reduction in vivo. The incentive to isolate nitrite reductase-deficient mutants arose from unsuccessful attempts in several laboratories to define the metabolic function of cytochrome c552 in Escherichia coli. This cytochrome is synthesized during anaerobic growth with nitrate or nitrite (Gray, Wimpenny, Hughes & Ranlett, 1963; Fujita, 1966; Cole, 1968; Cole & Wimpenny, I 968) and maximum concentrations occur in bacterial extracts which reduce nitrite rapidly (Fujita & Sato, 1966; Cole, 1968). Although the cytochrome and nitrite reductase were separated by gel filtration, the cytochrome rich fractions did not stimulate nitrite reduction by the partially purified enzyme (Cole, 1968). The possibility arose, there- fore, that these bacteria synthesize other enzymes capable of reducing nitrite. The properties of an alternative cytochrome c,,,-dependent enzyme could be studied more easily in mutants deficient in the very active NADH-nitrite oxidoreductase. Conversely, cytochrome c5,,-deficient mutants should lack the alternative nitrite reductase, but retain the NADH- dependent activity. * nirA denotes nitrite reductase-deficient mutants, which show low activities of NADH-nitrite oxido- reductase when grown anaerobically in nutrient broth supplemented with 10 mwsodium nitrite. t cysB pleiotropic negative mutants of cysteine biosynthesis, lacking reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent sulphite reductase activity.