Arch Microbiol (1990) 154:349 - 354 Arch wes of Microbiology 9 Springer-Verlag1990 Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria Lisa Page, Lesley Griffiths, and Jeff A. Cole School of Biochemistry,Universityof Birmingham, Birmingham, B15 2TT, UK ReceivedDecember4, 1989/AcceptedApril 2, 1990 Abstract. Operon fusion strains and mutants of Escherichia coli K-12 lacking the NADH-dependent ni- trite reductase have been used to determine the regulation and physiological roles of two independent pathways for nitrite reduction to ammonia. Both the formate- and NADH-dependent pathways (Nrf and Nir, respectively) were totally repressed during aerobic growth, partially active during anaerobic growth in the absence of nitrite and further induced anaerobically by nitrite. Both were dependent upon a functional Fnr protein (a transcription activator of genes for anaerobic respiration). During an- aerobic growth in the presence of nitrate, the Nir pathway was fully induced but Nrf was strongly repressed. Mu- tants defective in the NarL protein, which induces tran- scription of nitrate reductase genes but represses fumarate reductase genes in the presence of nitrate, were derepressed for Nrf activity during growth with nitrate, but the Nir enzyme was less active. The synthesis of Nrf components was also sensitive to glucose repression and weak activation by NarL during growth in the absence of nitrate. These data indicate that the Nir pathway pro- vides a mechanism for detoxifying nitrite formed in the cytoplasm as a product of nitrate reduction. In contrast, the electrogenic reduction of nitrite by the Nrf pathway provides a secondary source of energy during anaerobic growth and is consequently repressed by the NarL protein when the thermodynamically more favourable electron acceptor, nitrate, is available. Two short DNA sequences, 5'-TACCAT-3' and 5'-CTCCTT-Y, were found in the promoters of operons known to be activated or repressed by the NarL protein. It is proposed that NarL activates nirB transcription by binding to one or both of these sequences located 5' to the RNA polymerase binding site, but represses other operons, including nrf, by binding close to the transcription start. Offprint requests to: J. A. Cole Abbreviations: Nit, nitrite reduction by NADH; Nrf, nitrite re- duction by formate Key words: Nitrite reduction - Anaerobic regulation - NarL protein - Two-component regulatory systems - Anaerobic respiration - Nitrite detoxification - Escherichia coli Anaerobic cultures of many enteric bacteria, including Escherichia coli K-12 strains, can use either NADH or formate as an electron donor to reduce nitrite rapidly to ammonia during anaerobic growth (Cole 1988). The two pathways are biochemically and genetically independent (Abou-Jaoud~ et al. 1979a, b; Jackson et al. 1981; Pope and Cole 1982; Cole 1988). The NADH-dependent en- zyme (the Nir pathway) is a soluble, cytoplasmic protein: consequently, no energy is conserved during nitrite re- duction by NADH (Cole 1978; Cole and Brown 1980; Jackson et al. 1981). In contrast, a membrane potential is generated during nitrite reduction by formate (the Nrf pathway) as electrons are transferred from formate in the cytoplasm via membrane-bound components to the hexa- haem cytochrome c5s2 in the periplasm (Motteram et al. 1981 ; Pope and Cole 1982; Abou-Jaoud6 et al. 1979a, b; Fujita and Sato 1966a, b). Mutants defective in NADH- dependent nitrite reduction reduce nitrite to ammonia at 5 to 20% of the rate of their Nir § parents: the Nir pathway therefore catalyses the majority of the nitrite reduction during anaerobic growth. The physiological role of a biochemical pathway can usually be deduced from its pattern of genetic regulation. Despite the biochemical differences between the two pathways for nitrite reduction, both are active only in bacteria which have been grown anaerobically; both are repressed by oxygen, induced by nitrite during anaerobic growth and are apparently dependent on the Fnr transcription activator protein (Cole and Ward 1973) Newman and Cole 1978; Chippaux et al. 1978; Jayaraman et al. 1987). This raises intriguing questions about why there are two similarly-regulated but indepen- dent pathways for catalysing the same biochemical reac-