A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome C₅₅₂ of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter, in contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M_r of 20714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18kDa. The NrfC polypeptide, M_r 24567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD inciude the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M_r 60851, is predicted to be another hydrophobic, integral membrane protein homologous to the Ccl1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF poiypeptide, M_r 14522, is strikingly similar to the Ccl2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN. The translation stop codons of nrfE and nrfF overlap the start codons of nrfF and nrfG, respectively, suggesting that expression of nrfE, nrfF and nrfG may be translationally coupled. However sequence analysis suggests no apparent role for NrfG, although the sequence shows some similarities with the RecA protein from Synechococcus. The synthesis of two c-type cytochromes in wild-type bacteria, but not in an nrf deletion mutant, during anaerobic growth in the presence of nitrite was confirmed. Furthermore, we demonstrate over-expression of several Nrf polypeptides and GalE Nrf fusion proteins: in each case, the sizes of the products were consistent with the predicted sequence. Two alternative proposals for how the components of the Nrf pathway might be organized across the cytoplasmic membrane are presented.