Transcriptional activation of the Rhodobacter sphaeroides cytochrome c(2) gene P2 promoter by the response regulator PrrA.
Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the alpha subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome c(2) gene (cycA) promoter, P2, which contained sequences from -73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of cycA P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate cycA P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the prrA gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.
Comolli
James C
JC
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Carl
Audrey J
AJ
Hall
Christine
C
Donohue
Timothy
T
D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D003574:Cytochrome c Group / Q000235:genetics*; D045362:Cytochromes c2; D000071677:Histidine Kinase; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D003574:Cytochrome c Group; C085225:REGA protein, Rhodobacter sphaeroides; D015534:Trans-Activators; D045362:Cytochromes c2; D011494:Protein Kinases; D000071677:Histidine Kinase