Interaction of CbbR and RegA* transcription regulators with the Rhodobacter sphaeroides cbb(I) promoter-operator region

Abstract

The form I (cbb(I)) Calvin-Benson-Bassham (CBB) reductive pentose phosphate cycle operon of Rhodobacter sphaeroides is regulated by both the transcriptional activator CbbR and the RegA/PrrA (RegB/PrrB) two-component signal transduction system. DNase I footprint analyses indicated that R. sphaeroides CbbR binds to the cbb(I) promoter between -10 and -70 base pairs (bp) relative to the cbb(I) transcription start. A cosmid carrying the R. capsulatus reg locus was capable of complementing an R. sphaeroides regA- deficient mutant to phototrophic growth with restored regulated synthesis of both photopigments and ribulose-bisphosphate carboxylase/oxygenase (Rubisco). DNase I footprint analyses, using R. capsulatus RegA*, a constitutively active mutant version of RegA, detected four RegA* binding sites within the cbb(I) promoter. Two sites were found within a previously identified cbb(I) promoter proximal regulatory region from -61 to -110 bp. One of these proximal RegA* binding sites overlapped that of CbbR. Two sites were within a previously identified promoter distal positive regulatory region between - 301 and -415 bp. Expression from promoter insertion mutants showed that the function of the promoter distal regulatory region was helical phase- dependent. These results indicated that RegA exerts its regulatory affect on cbb(I) expression through direct interaction with the cbb(I) promoter.