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

Abstract

PrrA is a global trancription regulator activated upon phosphorylation by its cognate kinase PrrB in response to low oxygen conditions in Rhodobacter sphaeroides. Here we show by gel filtration, analytical ultracentrifugation and NMR diffusion measurements that treatment of PrrA by a phosphate analogue, BeF 3 -, results in dimerization of the protein, producing a protein that binds DNA. No dimeric species was observed in the absence of BeF 3 -. On addition of BeF 3 -, the inhibitory activity of the N-terminal domain on the C-terminal DNA-binding domain is relieved, after which PrrA becomes capable of binding DNA as a dimer. The interaction surface of the DNA-binding domain with the regulatory domain of PrrA is identified by NMR as being a well conserved region centered on helix α6, which is on the opposite face to the DNA recognition helix. This suggests that there is no direct blockage of DNA binding in the inactive state, but rather that PrrA dimerization promotes a correct arrangement of two adjacent DNA binding domains to recognise specific DNA binding sequences. KeywordsTwo-Component System; PrrA; RegA; dimerization; phosphorylation; DNA recognitionThe purple, non-sulfur bacterium Rhodobacter sphaeroides has very versatile metabolic activities including aerobic and anaerobic respiration, photosynthesis, and carbon and nitrogen assimilation. The R. sphaeroides global regulator PrrA coordinately controls a large number of genes involved in the complex switch between aerobic and anaerobic lifestyles and the optimum use of reducing power. It regulates genes necessary for the synthesis of the photosynthetic apparatus, electron transport, nitrogen and carbon fixation, anaerobic respiration, [NiFe] hydrogenase and aerotaxis, as well as the expression of the Prr gene cluster itself (1-6). † This work was supported by an equipment grant from the Wellcome Trust and by grants from the NIH (GM37509) and DOE (ER63232-1018220-0007203 The Prr system is a bacterial two-component signal transduction system (TCS) (7), consisting of the two proteins PrrB and PrrA. Two-component systems homologous to Prr have been found in other proteobacteria including other photosynthetic species, like the very similar and well-studied Reg system from R. capsulatus, but also in non-photosynthetic bacteria, and suggest a very conserved transduction mechanism, despite different in vivo functions (8,9). PrrB is a membrane-bound histidine kinase and is activated under low oxygen conditions, probably via a third member of the pathway, PrrC, through formation of an intermolecular disulfide bond using a conserved cysteine (10,11). It then autophosphorylates on a conserved histidine and the phosphate is transferred to the response regulator (RR) PrrA on a conserved aspartate residue.PrrA is a two-domain protein. The N-terminal receiver domain (residues 1-130) is a CheY-like domain common to all bacterial TCS response regulators, and there are a number of structures of these domains including CheY (12), FixJ (13) and NtrC (14). The phosphory...

The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1

“…The PrrBA two-component redox sensing pathway plays a critical role in the formation of the photosynthetic apparatus and also serves as a global regulator of gene expression when the oxygen tension decreases (11,21). The cbb 3 cytochrome c oxidase regulates PS gene expression via the PrrBA two-component system by monitoring O 2 levels through sensing the rate of transfer and volume of electrons that travel through the oxidase on their way to O 2 (24,25,(37)(38)(39)41). The same redox flow has been proposed to play a role in the control of carotenoid accumulation (35,36,40).…”

Regulation of the Rhodobacter sphaeroides 2.4.1 hemA Gene by PrrA and FnrL

“…These results suggest either that unphosphorylated PrrA can dimerize and bind DNA or, far less likely, that monomeric PrrA binds to site I with affinities comparable to those of dimeric phosphorylated PrrA. Since the PrrA protein used in our in vitro studies differed from the protein used by Laguri et al (22) in that the protein of Laguri et al was a PrrA protein in which 21 amino acids, including a polyhistidine tag, were added to the N terminus, while the purified protein used here had a Pro-Gly extension at its C terminus (4), and since the importance of the cytoplasmic environment for PrrA or phosphorylated PrrA dimer formation is not known, we used a genetic approach with R. sphaeroides in order to evaluate PrrA dimerization in the cell.…”

“…Previously, phosphorylation of PrrA was achieved by incubation of the purified protein with acetyl phosphate (30). However, it has been reported that the efficiency of modification of PrrA with BeF 3 Ϫ , which emulates phosphorylation of the D63 residue (22), is estimated to be 90%, compared to the approximately 20% efficiency of acetyl phosphate treatment (22). Therefore, we expected BeF 3 Ϫ modification of PrrA would increase the sensitivity of the EMSAs.…”

“…While in vitro transcription assays involving both hemA (30) and cycA (the P2 promoter [4]) have demonstrated that unphosphorylated PrrA can bind DNA and activate transcription, other studies were unable to detect DNA binding of unmodified PrrA to the cycA sequences in the absence of RNA polymerase (22 Ϫ -PrrA for its target within the cycA upstream sequences has been reported to be approximately 5 M, while the K d of unmodified PrrA for the same sequence is approximately 1 mM (22). Thus, while the affinity of unphosphorylated PrrA for one of its target sequences, hemA binding site I, is greater than the affinity of phosphorylated PrrA, the affinity of unphosphorylated PrrA for another target sequence, the cycA binding site, is 200-fold less than the affinity of phosphorylated PrrA.…”

“…Further, multicopy analysis of a prrA gene coding for the D63K-PrrA protein revealed that it behaves in a dominant-negative fashion in wild-type cells. Laguri et al (22) demonstrated that the monomer-dimer equilibrium is shifted toward the dimeric form for the phosphoprotein, while the amount of dimeric unphosphorylated PrrA was below the level that could be detected using their methods. We have not examined the relative concentrations of the dimer and the monomer.…”