Regulated expression of a highly conserved regulatory gene cluster is necessary for controlling photosynthesis gene expression in response to anaerobiosis in Rhodobacter capsulatus.

We utilized primer extension analysis to demonstrate that the divergently transcribed regB and senC-regA-hvrA transcripts contain stable 5' ends 43 nucleotides apart within the regB-senC intergenic region. DNA sequence analysis indicates that this region contains two divergent promoters with overlapping sigma70 type -35 and -10 promoter recognition sequences. In vivo analysis of expression patterns of regB::lacZ and senC-regA-hvrA::lacZ reporter gene fusions demonstrates that the regB and senC-regA-hvrA transcripts are both negatively regulated by the phosphorylated form of the global response regulator RegA. DNase I protection assays with a constitutively active variant of RegA indicate that RegA binds between regB and senC overlapping -10 and -35 promoter recognition sequences. Two mutations were also isolated in a regB-deficient background that increased expression of the senC-regA-hvrA operon 10- and 5-fold, respectively. As a consequence of increased RegA expression, these mutants exhibited elevated aerobic and anaerobic photosynthesis (puf) gene expression, even in the absence of the sensor kinase RegB. These results indicate that autoregulation by RegA is a factor contributing to the maintenance of an optimal low level of RegA expression that allows responsiveness to activation by phosphorylation.

Isolation and in vitro phosphorylation of sensory transduction components controlling anaerobic induction of light harvesting and reaction center gene expression in Rhodobacter capsulatus.

Anaerobic induction of light harvesting and reaction center gene expression involves two transacting factors termed RegA and RegB. Sequence and mutational analysis has indicated that RegA and RegB constitute cognate components of a prokaryotic sensory transduction cascade with RegB comprising a membrane-spanning sensor kinase and RegA a cytosolic response regulator. In this study we have purified RegA, as well as a truncated portion of RegB (RegB') and undertaken an in vitro analysis of autophosphorylation and phosphotransfer activities. Incubation of RegB' with [gamma-32P]ATP and MgCl2 resulted in phosphorylation of RegB' (RegB' approximately P) over a 20-min incubation period. Incubation of RegB' approximately P with RegA resulted in rapid transfer of the phosphate from RegB' to RegA. In analogy to other characterized prokaryotic sensory transduction components, mutational and chemical stability studies also indicate that RegB' is autophosphorylated at a conserved histidine and that RegA accepts the phosphate from RegB at a conserved aspartate.