Transcriptional Activation of the Rhodobacter sphaeroides Cytochrome c ₂ Gene P2 Promoter by the Response Regulator PrrA

Abstract

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides, a member of the ␣ subclass of the class Proteobacteria, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a twocomponent 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.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

“…Both the PrrB/PrrA system and FnrL are global regulators; in addition to activating PS genes, they control expression of a number of other genes in an oxygen-dependent manner (Comolli et al, 2002;Laratta et al, 2002;.…”

Activation of the Global Gene Regulator PrrA (RegA) from Rhodobacter sphaeroides

“…It is known that PrrA possesses autodephosphorylation activity, and that the phosphorylated form has a half-life of around 330 min (32). We have therefore used BeF 3 -to generate a stable pseudo-phosphorylated PrrA as previously described (15).…”

“…BeF 3 -was generated in situ by adding 2 mM beryllium chloride and 6 mM sodium fluoride. The D63A mutant of PrrA (and a wild-type for comparison purposes) was overexpresed in E. coli and purified using an intein/chitin-binding domain fusion system, as described (32).…”

“…Studies of the influence of phosphorylation on PrrA (and RegA) suggest that PrrA DNAbinding and transcription activation activity are increased upon phosphorylation (32,33). PrrA C in isolation is also capable of significant DNA binding (15).…”

“…The dimeric form of PrrA is thus the fully active form of the response regulator. Because unphosphorylated PrrA has also been shown to be able to activate transcription (32) and bind DNA (21), we have investigated the presence of a dimeric form in the unphosphorylated species by analytical ultracentrifugation (AUC) and NMR diffusion. We could not detect any unphosphorylated dimer.…”