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.
Highlights
The nonsulfur purple bacterium Rhodobacter sphaeroides is capable of both dark aerobic chemoheterotrophic growth and anoxic photosynthetic growth
Complementation Studies—Purified RegA, RegA*, is only available from R. capsulatus. To use this protein for in vitro DNA binding studies with the R. sphaeroides cbb system, it was necessary to establish that the R. capsulatus regA gene could complement a R. sphaeroides regA mutant
R. sphaeroides CAC::regA⍀ was unable to grow under phototrophic conditions, the strain complemented with the R. capsulatus regA gene, R. sphaeroides CAC⍀-C, regained the ability to grow under both photoheterotrophic and photoautotrophic conditions
Summary
The nonsulfur purple bacterium Rhodobacter sphaeroides is capable of both dark aerobic chemoheterotrophic growth and anoxic photosynthetic growth. Superimposed on the requirement for cbbR is the regA-regB (prrA-prrB) twocomponent regulatory system, encoding sensor kinase RegB (PrrB) and response regulator RegA (PrrA) This system has been shown to play a role in cbb regulation, based primarily on genetic studies in which a R. sphaeroides regB insertion mutant was found to exhibit reduced cbbI and cbbII expression during photoautotrophic growth in a 1.5% CO2/98.5% H2 atmosphere [15]. Because the interaction of RegA with the cbb system has not been investigated beyond the physiological and genetic studies previously described [15], the potential to use purified RegA* for detailed in vitro studies was attractive In this communication, we first identified binding sites for CbbR within the cbbI promoter proximal regulatory domain using DNase I footprinting experiments.
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