Magnesium control of the PhoP/PhoQ virulence regulon in Salmonella

MARIA E. CASTELLI, SERGIO LEJONA, ANDRÉS AGUIRRE, ELEONORA GARCÍA VÉSCOVI AND FERNANDO SONCINI.

Cancun, Mexico

Congreso; 10th anniversary Alumni Reunion; 2000

Pew

Magnesium control of the PhoP/PhoQ virulence regulon in Salmonella Maria E. Castelli, Sergio Lejona, Andrés Aguirre, Eleonora García Véscovi and Fernando Soncini. Facultad de Ciencias Bioquímicas y Farmaceúticas, Universidad Nacional de Rosario. Suipacha 531 (2000) Rosario. Bacteria are exposed to all types of molecules from their surrounding environment and to which they mount an appropriate response. This response is usually controlled by two-component signal transduction systems and involves the transcriptional regulation of genes whose products specifically cope with a given environmental change. In Salmonella, The PhoP/PhoQ two component system controls the expression of essential virulence traits that allow this pathogenic bacterium to survive and multiply within the host’s tissues. Environmental deprivation of Mg activates the PhoP/PhoQ signal transduction cascade that results in an increased expression of genes necessary for survival inside the macrophages. It was previously demonstrated that the interaction of Mg with the periplasmic domain of PhoQ promotes a conformational change in the sensor protein that leads to the down regulation of PhoP-activated genes. We have now 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 sensing 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 strain PhoQ down regulated the PhoP-activated genes in the presence of millimolar concentration of Mg in the culture medium. These findings reveal that the sensor 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 (PhoP box) located upstream the transcription start site of these genes. In vitro experiments showed that phosphorylation of the response regulator increased its affinity for the PhoP-box.