Cloning and nucleotide sequence of the nitric oxide reductase locus in Paracoccus denitrificans IFO 12442

Abstract

A 5.2-kb DNA fragment containing the upstream region of the nitrite reductase gene (nirS) was cloned from Paracoccus denitrificans IFO 12442 and its DNA sequence was determined. In this fragment, four open reading frames (ORFs) were observed. Among these, two ORFs, located 3 kb upstream of the nirS gene were found to encode nitric oxide reductase (norC, norB) by homology analysis. The norC and norB encoded cytochrome c and b subunits of the enzyme, and the predicted molecular weights of the protein products were 17 kDa (150 amino acid residues) and 52.5 kDa (462 amino acid residues), respectively. The other two open reading frames, designated ORF1 (73 kDa, 681 amino acid residues) and ORF2 (32.5 kDa, 304 amino acid residues), were found between the nir and nor genes. The deduced amino acid sequence of ORF1 showed high similarity (35% identity) to that of NosR which is known to be a transcriptional regulatory protein for the N₂O reductase gene cluster of Pseudomonas stutzeri. Since the ORF1 protein has a well conserved cysteine cluster and 5 hydrophobic transmembrane repeats similar to those of the NosR protein, ORF1 probably functions as a membrane bound sensor for denitrification. Northern blot analysis indicated that the ORF1–2 and norCB regions are probably transcribed as independent operons.