Phosphorylation, dephosphorylation and DNA‐binding of the Bradyrhizobium japonicum RegSR two‐component regulatory proteins

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Abstract
Under low oxygen conditions, induction of many genes required for nitrogen fixation in Bradyrhizobium japonicum depends on the redox‐responsive transcriptional activator NifA which is encoded in the fixR–nifA operon. Basal expression of this operon depends on the response regulator RegR and a DNA element located around position −68 in the fixR–nifA promoter region. To investigate the functional properties of RegR and the interaction with its putative cognate kinase, RegS, we overproduced and affinity‐purified RegR and a truncated soluble variant of RegS (RegSC), both as N‐terminally His6‐tagged proteins. RegSC autophosphorylated when incubated with [γ‐32P]ATP, and it catalyzed the transfer of the phosphoryl label to RegR. The phosphorylated form of RegSC exhibited phosphatase activity on RegR‐phosphate. Chemical stability tests and site‐specific mutagenesis identified amino acids H219 and D63 of RegS and RegR, respectively, as the phosphorylated residues. Competition experiments with isolated domains demonstrated that the N‐terminal but not the C‐terminal domain of RegR interacts with RegSC. Band‐shift experiments revealed that phosphorylated RegR had at least eightfold enhanced DNA‐binding activity compared with dephosphorylated RegR or the mutant protein RegR‐D63N, which cannot be phosphorylated. In conclusion, the RegSR proteins of B. japonicum exhibit functional properties in vitro that are typical of two‐component regulatory systems.