ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation

A two-component signal transduction system composed of a sensor kinase, Res E, and a response regulator, ResD, encoded by resD and resE genes of the re s operon (resABCDE), has a regulatory role in both aerobic and anaerobic re spiration. In terms of aerobic respiration, resD functions upstream of ctaA , a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases. Although ResD is probably a tra nscription factor, there was no direct evidence that ResD protein, either p hosphorylated or unphosphorylated, interacts directly with regulatory regio ns of ResD-controlled genes. Here, we report the overexpression and purific ation of ResD and ResE and their role in gene activation. ResD can be phosp horylated by ResE in vitro and is a monomer in solution in either the phosp horylated or unphosphorylated state. The binding activity of ResD to the ct aA promoter was examined by gel shift assays and DNase I footprinting assay s. DNase I footprinting showed both unphosphorylated and phosphorylated Res D binding to the cfaA promoter and showed that there are three binding site s (A1, A2 and A3), two (A1 and A2) upstream of the -35 promoter region and one (A3) downstream of the -10 of the promoter. The role of each site in ct aA promoter activity and ResD binding was characterized using deletion anal ysis, followed by the DNase I footprinting and in vivo transcription assays of promoter-lacZ fusions. Our results showed that the concentration of Res D required to hind at each site is different and that ResD binding at the A 1 site is independent of the other two ResD binding sites, but that the con centration of ResD similar to P required to protect site A2 is reduced when site A3 is present. in vivo transcription assays from promoter-lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential f or promoter activity and that promoter constructs containing both binding s ites A2 and A3 were sufficient for full promoter activity.