title: Mutational analysis of the residue at position 48 in the Salmonella enterica Serovar Typhimurium PhoQ sensor kinase.
abstract: The PhoP/PhoQ two-component regulatory system of Salmonella enterica serovar Typhimurium plays an essential role in controlling virulence by mediating the adaptation to Mg(2+) depletion. The pho-24 allele of phoQ harbors a single amino acid substitution (T48I) in the periplasmic domain of the PhoQ histidine kinase sensor. This mutation has been shown to increase net phosphorylation of the PhoP response regulator. We analyzed the effect on signaling by PhoP/PhoQ of various amino acid substitutions at this position (PhoQ-T48X [X = A, S, V, I, or L]). Mutations T48V, T48I, and T48L were found to affect signaling by PhoP/PhoQ both in vivo and in vitro. Mutations PhoQ-T48V and PhoQ-T48I increased both the expression of the mgtA::lacZ transcriptional fusion and the net phosphorylation of PhoP, conferring to cells a PhoP constitutively active phenotype. In contrast, mutation PhoQ-T48L barely responded to changes in the concentration of external Mg(2+), in vivo and in vitro, conferring to cells a PhoP constitutively inactive phenotype. By analyzing in vitro the individual catalytic activities of the PhoQ-T48X sensors, we found that the PhoP constitutively active phenotype observed for the PhoQ-T48V and PhoQ-T48I proteins is solely due to decreased phosphatase activity. In contrast, the PhoP constitutively inactive phenotype observed for the PhoQ-T48L mutant resulted from both decreased autokinase activity and increased phosphatase activity. Our data are consistent with a model in which the residue at position 48 of PhoQ contributes to a conformational switch between kinase- and phosphatase-dominant states.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada H3A 2B4.
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanowar
authors.forename: Alexandre
authors.initials: A
authors.lastname: Martel
authors.forename: Hervé Le
authors.initials: HL
authors.lastname: Moual
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; C079792:PhoQ protein, Bacteria; C061064:PhoP protein, Bacteria; D011494:Protein Kinases; D000071677:Histidine Kinase; D010744:Phosphoric Monoester Hydrolases; D008274:Magnesium