title: Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada H3A 2B4.
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanowar
authors.forename: Hervé
authors.initials: H
authors.lastname: Le Moual
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D015636:Magnesium Chloride; D010766:Phosphorylation; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C079792:PhoQ protein, Bacteria; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D015636:Magnesium Chloride; C061064:PhoP protein, Bacteria; D000244:Adenosine Diphosphate