Target genes and DNA-binding sites of the response regulator PhoR from Corynebacterium glutamicum

The two-component signal transduction system PhoRS of Corynebacterium glutamicum is involved in the phosphate (P(i)) starvation response. To analyze the binding of unphosphorylated and phosphorylated PhoR to the promoters of phosphate starvation-inducible (psi) genes, this response regulator and the kinase domain of its cognate sensor, PhoS (MBP-PhoSDelta1-246), were overproduced and purified. MBP-PhoSDelta1-246 showed constitutive autophosphorylation activity, and a rapid phosphoryl group transfer from phosphorylated MBP-PhoSDelta1-246 to PhoR was observed. Gel mobility shift assays revealed that phosphorylation increases the DNA-binding affinity of PhoR. The affinity of PhoR approximately P to different promoters varied and decreased in the order pstSCAB > phoRS > phoC > ushA > porB > ugpA > pitA > nucH and phoH1 > glpQ1. The binding sites in front of pstSCAB and phoRS were localized at positions -194 to -176 and -61 to -43 upstream of the transcriptional start sites, respectively. Alignment of these two 19-bp binding sites revealed a high identity in the 5'-terminal part, but not in the 3'-terminal part. As many OmpR-type response regulators bind to direct repeats, the 19-bp sequence might be interpreted as a loosely conserved 8-bp direct repeat separated by 3 bp. This idea was supported by the fact that the highest binding affinity was observed with a perfect 8-bp direct repeat of the sequence CCTGTGAAaatCCTGTGAA. Inspection of the other target promoters revealed sequences with some similarity to this binding motif, which might represent PhoR binding sites. The in vivo relevance of the PhoR-binding site within the phoRS promoter was supported by reporter gene studies.