RegB/RegA, a Highly Conserved Redox-Responding Global Two-Component Regulatory System

Abstract

SUMMARY The Reg regulon from Rhodobacter capsulatus and Rhodobacter sphaeroides encodes proteins involved in numerous energy-generating and energy-utilizing processes such as photosynthesis, carbon fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, electron transport, and aerotaxis. The redox signal that is detected by the membrane-bound sensor kinase, RegB, appears to originate from the aerobic respiratory chain, given that mutations in cytochrome c oxidase result in constitutive RegB autophosphorylation. Regulation of RegB autophosphorylation also involves a redox-active cysteine that is present in the cytosolic region of RegB. Both phosphorylated and unphosphorylated forms of the cognate response regulator RegA are capable of activating or repressing a variety of genes in the regulon. Highly conserved homologues of RegB and RegA have been found in a wide number of photosynthetic and nonphotosynthetic bacteria, with evidence suggesting that RegB/RegA plays a fundamental role in the transcription of redox-regulated genes in many bacterial species.

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

“…In PrrA.BeF 3 -samples the majority of the protein is dimeric, as expected, although a small amount of monomer is observable at protein concentrations of 30 and 100 μM. At 200 μM, only a dimeric form was observed ( Figure 2).…”

“…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). PrrA.BeF 3 -and the purified dimer had the same elution time when injected on gel filtration ( Figure 1A), and very similar 1D 1 H NMR spectra, which are both very different from those of PrrA alone ( Figure 1B).…”

“…Gel permeation chromatography was carried out using Superdex G75 (Pharmacia) in a 20 × 300 mm column, in the standard NMR buffer. 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).…”

“…Velocity analytical centrifugation experiments were performed with PrrA and PrrA.BeF 3 -at different concentrations of protein (30, 100 and 200 μM) and at 10 °C. Analysis of the sedimentation coefficient distributions was performed with dcdt+ (35), and the fitting of the distributions produced PrrA monomer and dimer sedimentation coefficients of about 2.0 S and 3.1 S respectively ( Figure 2 and Table 1).…”

“…To PrrA, PrrA.BeF 3 -and PrrA C (300 μM starting concentration) was added increasing amounts of a 25-bp section of the cycAP2 promoter from R. sphaeroides, with the sequence 5′-TCGTTGTGCGGCAATCCGTCATATA-3′. DNA was prepared as reported (15).…”