Transcription regulation of the nir gene cluster encoding nitrite reductase of Paracoccus denitrificans involves NNR and NirI, a novel type of membrane protein

The nirIX gene cluster of Paracoccus denitrificans is located between the n ir and nor gene clusters encoding nitrite and nitric oxide reductases respe ctively. The NirI sequence corresponds to that of a membrane-bound protein with six transmembrane helices, a large periplasmic domain and cysteine-ric h cytoplasmic domains that resemble the binding sites of [4Fe-4S] clusters in many ferredoxin-like proteins. NirX is soluble and apparently located in the periplasm, as judged by the predicted signal sequence. NirI and NirX a re homologues of NosR and NosX, proteins involved in regulation of the expr ession of the nos gene cluster encoding nitrous oxide reductase in Pseudomo nas stutzeri and Sinorhizobium meliloti, Analysis of a NirI-deficient mutan t strain revealed that NirI is involved in transcription activation of the nir gene cluster in response to oxygen limitation and the presence of N-oxi des, The NirX-deficient mutant transiently accumulated nitrite in the growt h medium, but it had a final growth yield similar to that of the wild type. Transcription of the nirIX gene cluster itself was controlled by NNR, a me mber of the family of FNR-like transcriptional activators. An NNR binding s equence is located in the middle of the intergenic region between the nirI and nirS genes with its centre located at position -41.5 relative to the tr anscription start sites of both genes. Attempts to complement the NirI muta tion via cloning of the nirIX gene cluster on a broad-host-range vector wer e unsuccessful, the ability to express nitrite reductase being restored onl y when the nirIX gene cluster was reintegrated into the chromosome of the N irI-deficient mutant via homologous recombination in such a way that the wi ld-type nirI gene was present directly upstream of the nir operon.