Activation of bacterial response regulators

In Salmonella typhimurium, PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. Unphosphorylated PhOPHis (a mutant harboring a histidine tag at the C-terminus) was mostly monomeric, whereas PhoP His~P existed as a mixture of monomer and dimer. Binding of PhoP to the PhoP box was barely influenced by phosphorylation as determined by surface plasmon resonance. In contrast, phosphorylation of PhoPHis induced a 200% increase in PhoPHis binding to target DNA. Fluorescence analysis revealed that the conformation of PhOPHis is greatly altered compared to that of wild-type PhoP. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation