"Magnesium controls the phosphatase activity of the two-component sensor protein PhoQ in Salmonella enterica"

M. E. CASTELLI, S. L. LEJONA, E. G. VÉSCOVI, F. C. SONCINI,

Chevy Chase, USA

Congreso; 2000 Meeting of International Research Scholars, Howard Hughes Medical Institute; 2000

Howard Hughes Medical Institute

Magnesium controls the phosphatase activity of the two-component sensor PhoQ in Salmonella enterica M.E. Castelli, S. Lejona, E.G. Véscovi, F.C. Soncini, National University of Rosario, Rosario, Argentina. The PhoP/PhoQ two-componet system controls the exression of essential virulence traits in the pathogenic bacterium Salmonella enterica serovar Typhimurium. PhoQ is a membrane-bound sensor protein that during environmental limitation of Mg activates by phosphorylation the transcriptional regulator PhoP, resulting in an increased expression of genes necessary for survival inside the host. We previously demonstrated that the interaction of Mg with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein, leading to the down regulation of PhoP-activated genes. We have examined the regulatory effect of Mg on the putative activities of the membrane bound PhoQ. We demonstrated that Mg promotes a phosphorylated PhoP (PhoP-P) phosphatase activity in the sensor protein. The integrity of the N-terminal domain of PhoQ was essential for the induction of the phosphatase activity, since Mg did not stimulate the release of inorganic phosphate from PhoP-P in a fusion protein that lacks this domain. We set up an in vivo experiment using a strain that harbours a PhoP mutant protein (PhoP*) that accepts phosphate from endogenous acetyl phosphate. This mutant is capable of inducing the expression of PhoP-activated genes in a PhoQ-independent manner. In this mutant, PhoQ down regulated the PhoP-activated genes in the presence of millimolar concentration of Mg in the culture medium. These findings reveal that PhoQ harbours a PhoP-P phosphatase activity, and that this phosphatase activity is the target of the extracellular Mg triggered regulation of the PhoP/PhoQ system. We also investigated The interaction of the response regulator PhoP with its target site in the promoter region of different PhoP-activated genes. DNA-shift and footprinting analysis revealed the presence of a PhoP protected sequence (GTTTAT-5bp-TTTA(T/A)) located 30-40 bp upstream of the transcription start site of these genes. This sequence would replace the -35 consensus region and allow selective expression of the PhoP-activated genes during Mg limitation.