A 7-GENE OPERON ESSENTIAL FOR FORMATE-DEPENDENT NITRITE REDUCTION TO AMMONIA BY ENTERIC BACTERIA

The DNA sequence of the regulatory region and the structural gene, nrf A, for cytochrome c552 of Escherichia chia coll K-12 have been reporte d. 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 essenti al for formate-dependent nitrite reduction to ammonia. This operon ter minates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression o f lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, F NR-dependent anaerobic induction, nitrite induction and nitrate repres sion during anaerobic growth, exactly as previously reported for the n rfA promoter. In contrast, expression of the gltP-lac fusion was FNR-i ndependent. The open reading frame immediately downstream of nrfA enco des 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 peptid e for a periplasmic protein: cleavage at the putative signal peptide c leavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24567, contains 1 6 cysteine residues arranged in four clusters typical of the CooF supe r-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and b asic 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 include the PsrC subunit of polysulphide reductas e from Wolnella, but, as seven of the 10 most similar proteins are NAD H-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal compone nts of the Nrf pathway. NrfE, M(r) 60851, is predicted to be another h ydrophobic, integral membrane protein homologous to the Ccl1 protein o f Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14522, is strikingly similar to the Ccl2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQ N. The translation stop codons of nrfE and nrfF overlap the start codo ns of nrfF and nrfG, respectively, suggesting that expression of nrfE, nrfF and nrfG may be translationally coupled. However sequence analys is suggests no apparent role for NrfG, although the sequence shows som e similarities with the RecA protein from Synechococcus. The synthesis of two c-type cytochromes in wild-type bacteria, but not in an nrf de letion mutant, during anaerobic growth in the presence of nitrite was confirmed. Furthermore, we demonstrate overexpression of several Nrf p olypeptides and GalE''Nrf fusion proteins: in each case, the sizes of the products were consistent with the predicted sequence. Two alternat ive proposals for how the components of the Nrf pathway might be organ ized across the cytoplasmic membrane are presented.