The nitric oxide regulated nor promoter of Paracoccus denitrificans

Abstract

The promoter of the Paracoccus denitrificans nitric oxide reductase operon (norCBQDEF) has been characterized by primer extension and deletion analysis. A major transcript that is detectable only in anaerobically grown cells initiates 43·5 bp downstream of the centre of a putative binding site for the transcription factor NNR (nitrite and nitric oxide reductase regulator, which is known to regulate nor expression). A minor transcript initiates 121 bp upstream of the major transcript and is detectable in cells grown aerobically or anaerobically. Deletion derivatives of the nor promoter region were constructed and analysed in vivo using transcriptional fusions to the reporter gene lacZ. Expression patterns from promoter deletions in a wild-type strain and an nnr mutant confirmed that the minor transcript is NNR independent, and makes a small contribution to nor expression under both aerobic and anaerobic growth conditions. A deletion derivative truncated to within 7 bp of the putative NNR-binding site showed a near wild-type response to anaerobic growth, showing that no upstream DNA sequences are required for activation of the major promoter. Site-directed mutagenesis of the putative NNR-binding site confirmed that this is the major cis-acting sequence mediating the anaerobic inducibility of nor expression.