Autophosphorylation, phosphotransfer, and DNA-binding properties of the RegB RegA two-component regulatory system in Rhodobacter capsulatus

In the purple, photosynthetic bacterium, Rhodobacter capsulatus, the RegB/R egA two-component system is required for activation of several anaerobic pr ocesses, such as synthesis of the photosynthetic apparatus and assimilation of CO2 and N-2. It is believed that RegB is an integral membrane histidine kinase that monitors the external environment. Under anaerobic growth cond itions, it transduces a signal through phosphorylation of the response regu lator, RegA, which then induces target gene expression. We used an in vitro assay to characterize the phosphorylation of wild-type RegA and a mutant v ariant (RegA*) that is responsible for abnormally high photosynthesis gene expression under both aerobic and anaerobic growth conditions. Phosphorylat ion assays indicate that phosphorylated RegA* (RegA*similar to P) is much m ore stable than RegA similar to P, indicating that it may be locked in a co nformation that is resistant to dephosphorylation, DNase I footprint assays also indicate that unphosphorylated RegA* has a much higher affinity for s pecific DNA binding sites than the wild-type protein. Phosphorylation of Re gA* increases DNA binding 2,5-fold, whereas phosphorylation of RegA increas es DNA binding more than 16-fold. Collectively, these results support the h ypothesis that RegA* is a constitutively active variant that does not requi re phosphorylation to assume a structural conformation required to bind DNA .