"Biochemical Characterization of PhoQ, the extracellular Mg2+ Sensor in Salmonella”

MARÍA E. CASTELLI, CESAR R. OLSINA, FERNANDO C. SONCINI, AND ELEONORA GARCÍA VÉSCOVI

Salt Lake City, Utah, USA

Congreso; 102nd General Meeting, American Society for Microbiology; 2002

American Society for Microbiology

Signal Transduction and Bacterial Pathogenesis. Characterization of PhoQ, The Mg2+ Sensor in Salmonella. García Véscovi, Eleonora, Castelli, María Eugenia, Olsina, César, Soncini, Fernando C. IBR-CONICET and School of Biochemistry, Rosario National University, Rosario, ARGENTINA.   The prototypical architecture of the two-component regulatory systems consists of a sensor protein and an associated effector protein.  Two major biochemical activities play an opposite role to balance the phosphorylation status of the response regulator: a) the autokinase activity of the sensor that defines the phosphotransfer availability, and b) a specific phosphatase that dephosphorylates the response regulator.  By modulating these reactions, the sensor component defines the phosphorylation status of the  response regulator, which directs the expression of a specific set of target genes.  This modulation results in the final adaptive response of the bacteria to its primary signal. In Salmonella, the PhoP/PhoQ two-component system governs the adaptation to environmental Mg2+ deprivation and controls the expression of essential virulence factors.  Binding of Mg2+ to the sensor protein PhoQ was identified as the primary event in the signal transduction cascade that results in the down regulation of the PhoP-activated genes. By analyzing the regulatory effect of Mg2+ in each phosphotransfer step, we postulated a model where PhoQ kinase activity remains essentially constant while the interaction of the sensor with the signal promotes the phospho-PhoP phosphatase activity. We analyzed the role of the conserved H277 residue in PhoQ phosphatase activity and the basis for the structure-function relationship. H277 was replaced by different aminoacids and the PhoQ phosphatase activity was determined in vivo. The substitutions of the H277 by asparagine, glutamine, tyrosine and serine preserved the phosphatase activity, while in the mutants where H277 was replaced by arginine or lysine, this activity was completely abolished. We thus conclude that the H277 residue is structurally important but not functionally essential for the phosphatase activity. To dissect the functional domains of the PhoQ sensor, the sensor region, the second transmembrane domain, the linker and the H box have been sequentially deleted preserving the G, N and F conserved motifs. These truncated proteins were examined for their ability to retain the phospho-PhoP phosphatase activity. The analysis in vivo and in vitro of the different PhoQ subdomains, lead us to the conclusion that the phosphatase activity depends on the dimerization-histidine phosphotransfer subdomain (DHp). The identification of the functional domains and the biochemical characterization of the sensor activities of PhoQ provide new clues for understanding the signal transducing mechanism of two-component systems.