"Molecular characterization of the PhoP/PhoQ regulon in Salmonella enterica"

S. LEJONA, M. E. CASTELLI, A. AGUIRRE, E. GARCIA VÉSCOVI, AND F. C. SONCINI.

Vancouver, Canada

Congreso; 2001 Meeting of International Research Scholars, Howard Hughes Medical Institute; 2001

Howard Hughes Medical Institute

Molecular characterization of the PhoP/PhoQ regulon in Salmonella enterica S. Lejona, M.E. Castelli, A. Aguirre, E. García Véscovi, F.C. Soncini Institute of Molecular and Cellular Biology of Rosario, CONICET, and National University of Rosario, Argentina. The PhoP/PhoQ two-component system controls the expression of essential virulence traits in the pathogenesis bacterium Salmonella typhimurium. The membrane sensor PhoQ is a bifunctional autokinase/PhoP phosphatase that responds to variations in extracytoplasmic Mg to control the level of phosphorylation of the response regulator PhoP. In environments with millimolar concentrations of Mg PhoQ acts as a phosphatase, dephosphorylating PhoP. This results in decreased expression of PhoP-activated genes. Conversely, during Mg limitation PhoQ induces phsosphorylation of PhoP, which in turn induces expression of genes that allow Salmonella to survive under such adverse conditions. We investigated whether phosphorylation of PhoP is essential for the PhoP-activated genes. By overexpressing PhoP in both phoQ+ and phoQ- strains, we found that the response regulator can activate its target genes regardless of its phosphorylation status in a concentration-dependent manner. When overexpressed, the PhoPD52A mutant protein can also activate the target genes. This mutated protein lacks the target residue for phosphorylation and, as a result, it is unable to be “activated” by the sensor protein PhoQ. We analyzed in vitro the interaction of the response regulator PhoP with its target site in the promoter region of different PhoP-activated genes. Electrophoresis mobility shift assays and footprinting analysis revealed the presence of a PhoP- protected sequence 30 to 40 base pairs 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 restriction.