Parallel Pathways for Nitrite Reduction during Anaerobic Growth in Thermus thermophilus

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor). The expression and role of the nirSJM genes in nitrite respiration were analyzed. The three genes are expressed from two promoters, one (nirSp) producing a tricistronic mRNA under aerobic and anaerobic conditions and the other (nirJp) producing a bicistronic mRNA only under conditions of anoxia plus a nitrogen oxide. As for its nitrite reductase homologues, NirS is expressed in the periplasm, has a covalently bound heme c, and conserves the heme d₁ binding pocket. NirJ is a cytoplasmic protein likely required for heme d₁ synthesis and NirS maturation. NirM is a soluble periplasmic homologue of cytochrome c₅₅₂. Mutants defective in nirS show normal anaerobic growth with nitrite and nitrate, supporting the existence of an alternative Nir in the cells. Gene knockout analysis of different candidate genes did not allow us to identify this alternative Nir protein but revealed the requirement for Nar in NirS-dependent and NirS-independent nitrite reduction. As the likely role for Nar in the process is in electron transport through its additional cytochrome c periplasmic subunit (NarC), we concluded all the Nir activity takes place in the periplasm by parallel pathways.     

Expression of the narX, narL, narP, and narQ genes of Escherichia coli K-12: regulation of the regulators.

The products of four Escherichia coli genes (narX, narL, narQ, and narP) regulate anaerobic respiratory gene expression in response to nitrate and nitrite. We used lacZ gene and operon fusions to monitor the expression of these nar regulatory genes in response to different growth conditions. Maximal expression of the narXL operon required molybdate, nitrate, and integration host factor. Expression of the narP and narQ genes was weakly repressed by nitrate. The NarL and NarP proteins were required for full nitrate induction of narXL operon expression, whereas the nitrate repression of narP and narQ expression was mediated solely by the NarL protein. narXL operon expression was unaffected by anaerobiosis, whereas expression of narP and narQ was induced approximately fourfold. The Fnr and ArcA proteins were not required for this anaerobic induction.     

Chromate reductase activity of Enterobacter aerogenes is induced by nitrite

Abstract A chromate resistant mutant of Enterobacter aerogenes manifested its chromate resistance only under aerobic conditions. Both parent and mutant showed substantial levels of anaerobic chromate reductase activity when grown on glycerol plus fumarate. The chromate reductase was further induced by growth in the presence of nitrite but was repressed by nitrate. The chromate reductase activity paralleled that of the formate-linked nitrite reductase. There was no significant difference in chromate reductase levels between the parent and its chromate resistant mutant, indicating that this enzyme activity is not, in fact responsible for chromate resistance as was suggested previously by others.     

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.     

Characterisation of Escherichia coli K-12 mutants defective in formate-dependent nitrite reduction: essential roles for hemN and the menFDBCE operon

Three Escherichia coli mutants defective in formate-dependent nitrite reduction (Nrf activity) were characterised. Two of the mutants, JCB354 and JCB356, synthesized all five c-type cytochromes previously characterised in anaerobic cultures of E. coli. The third mutant, JCB355, was defective for both cytochrome b and cytochrome c synthesis, but only during anaerobic growth. The insertion sites of the transposon in strains JCB354 and JCB356 mapped to the menFDBCE operon; the hemN gene was disrupted in strain JCB355. The mutation in strain JCB354 was complemented by a plasmid encoding only menD; strain JCB356 was complemented by a plasmid encoding only menBCE. A mutant defective in the methyltransferase activity involved in both ubiquinone synthesis and conversion of demethylmenaquinone to menaquinone expressed the same Nrf activity as the parental strain. The effects of men, ubiA and ubiE mutations on other cytochrome-c-dependent electron transfer pathways were also determined. The combined data establish that menaquinones are essential for cytochrome-c-dependent trimethylamine-N-oxide reductase (Tor) and Nrf activity, but that either menaquinone or ubiquinone, but not demethylmenaquinone, can transfer electrons to a third cytochrome-c-dependent electron transfer chain, the periplasmic nitrate reductase. Received: 9 December 1996 / Accepted: 11 June 1997