Transcriptional regulation of cytochrome d in nitrogen-fixing Azotobacter vinelandii. Evidence that up-regulation during N2 fixation is independent of nifA but dependent on ntrA

Abstract

Cytochrome d has been postulated to be the "respiratory protection" oxidase of Azotobacter vinelandii, allowing this organism to fix nitrogen under aerobic growth conditions. We have previously cloned and characterized the structural genes for the A. vinelandii cytochrome d (cydA and cydB). The cyd genes are co-transcribed, yielding an mRNA of approximately 3.6 kilobase pairs. The level of the cyd message was 2-3-fold higher in cells that were fixing nitrogen, as compared with non-nitrogen-fixing cells. RNase protection analysis was used to determine the transcriptional start site at 275 bases upstream of the initiator ATG of cydA, and this start site was the same for nitrogen-fixing and non-nitrogen-fixing cells. The cyd promoter has sequence similarities to the canonical Escherichia coli promoters, which are transcribed by the major sigma 70 form of RNA polymerase. Plasmid-borne lacZ transcriptional fusions were constructed, using approximately 650 base pairs of 5'-upstream sequences of the cyd structural genes. This region had a strong promoter activity which was further up-regulated 1.5-2.5-fold upon the induction of nitrogen fixation. The cyd-lacZ fusions were characterized in a nifA- as well as an ntrA- background. Mutations in neither of these nif regulatory genes affected the constitutive expression of cyd under non-nitrogen-fixing conditions. However, the up-regulation of this promoter during the induction of nitrogen fixation was abolished only in the ntrA- background. Based on these results, the cytochrome d promoter of A. vinelandii belongs to a new class of nitrogen-regulated promoters which, unlike the authentic nif genes, does not require the ntrA gene product for its expression. The up-regulation of this promoter during nitrogen fixation, however, requires the ntrA gene product.