Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes

Two-component signal-transduction systems are widespread in bacteria. They are usually composed of a transmembrane histidine kinase sensor and a cytoplasmic response regulator. The PhoP/PhoQ two-component system of Salmonella typhimurium contributes to virulence by co-ordinating the adaptation to low concentrations of environmental Mg²⁺. Limiting concentrations of extracellular Mg²⁺ activate the PhoP/PhoQ phosphorylation cascade modulating the transcription of PhoP-regulated genes. In contrast, high concentrations of extracellular Mg²⁺ stimulate the dephosphorylation of the response regulator PhoP by the PhoQ kinase sensor. In the present study, we report the purification and functional reconstitution of PhoQ_His, a PhoQ variant with a C-terminal His tag, into Escherichia coli liposomes. The functionality of PhoQ_His was essentially similar to that of PhoQ as shown in vivo and in vitro. Purified PhoQ_His was inserted into liposomes in a unidirectional orientation, with the sensory domain facing the lumen and the catalytic domain facing the extraluminal environment. Reconstituted PhoQ_His exhibited all the catalytic activities that have been described for histidine kinase sensors. Reconstituted PhoQ_His was capable of autokinase activity when incubated in the presence of Mg²⁺-ATP. The phosphoryl group could be transferred from reconstituted PhoQ_His to PhoP. Reconstituted PhoQ_His catalysed the dephosphorylation of phospho-PhoP and this activity was stimulated by the addition of extraluminal ADP.