cis-acting sequences required for NtcB-dependent, nitrite-responsive positive regulation of the nitrate assimilation...

There are three binding sites for NtcA (nirI, nirII, and nirIII), the global nitrogen regulator of cyanobacteria, in the DNA region between the two divergently transcribed operons (nirA and nirB operons) involved in nitrate assimilation in Synechococcus sp. strain PCC 7942. Using the luxAB reporter system, we showed that nirI and nirIII, which are located 23 bp upstream from the -10 promoter element of nirA and nirB, respectively, are required for induction by nitrogen depletion of the nirA and nirB operons, respectively. The induction of nirA operon transcription was a prerequisite for the nitrite-responsive positive regulation of the transcription by NtcB, a LysR-type protein. The NtcA-binding site nirII, located in the middle of the nirA-nirB intergenic region, and a potential binding site for a LysR-type protein (TGCAN5TGCA; designated L1), located between nirI and nirII, were required for the nitrite-responsive, NtcB-dependent enhancement of nirA operon transcription. Although the requirement for the L1 site was consistent with the involvement of the LysR family protein NtcB in transcriptional regulation, NtcB did not bind to the nirA regulatory region in vitro in the presence of nitrite and NtcA, suggesting the involvement of some additional factor(s) in the regulation. An L1-like inverted repeat with the consensus sequence TGCN7GCA was conserved in the nirA promoter region of cyanobacteria, being centered at position -23 with respect to the NtcA-binding site corresponding to nirI, which suggested the common occurrence of nitrite-responsive regulation of the nitrate assimilation operon among cyanobacteria.