In vivo and in vitro analysis of RegA response regulator mutants of Rhodobacter capsulatus.

Abstract

In the facultative photosynthetic bacterium Rhodobacter capsulatus, the transcription of genes encoding pigment binding proteins is tightly regulated in response to the oxygen partial pressure by the RegB/ RegA two component system. After a shift from high to low oxygen tension, the response regulator RegA enhances transcription of the puf and puc operon coding for the reaction center, light-harvesting complex I (LHI), and LHII proteins. Various regA mutant strains were analyzed in this study. In a RegA deficient strain, activation of puf and puc transcription is severely impaired which consequently leads to the synthesis of only a few photosynthetic complexes. Strains carrying a mutation in the helix-turn-helix domain of RegA or a mutation of the phosphorylation site, Asp63, show a phenotype like the RegA deficient mutant, although the RegA(D63K) mutant protein showed the same DNA binding behavior as the wild type protein. In contrast, the puf and puc mRNAs still reach about 50-70 % of the wild type level after reduction of oxygen tension in strains which synthesize the C-terminal RegA activator domain only or a hybrid protein composed of the RegA activator and the FixJ receiver domain, while both mutant proteins are impaired in DNA binding. Our data suggest that phosphorylation is not required for DNA binding but rather plays a role for efficient initiation of transcription.