The induction of Vancomycin resistance and crosstalk among bacterial two-component systems

Over the past several years, it has become increasingly clear that bacteria use two-component systems as the mode of choice to meet their signal transduction requirements. The typical bacterial two-component system consists of a transmembrane sensor kinase (SK) and a cytoplasmic response regulator (RR). Each two-component system governs the physiological response to a specific environmental parameter. Bacteria can transmit information among different two-component systems through the mechanism of crosstalk. Crosstalk is a phenomenon where the sensor kinase of one two-component system can activate the response regulator of another. One issue of pressing importance is the delineation of the determinants of interaction specificity between sensor kinases and their cognate and heterologous response regulator partners.;We have attempted to probe the determinants of interaction specificity among bacterial two-component systems using crosstalk between the VanS/VanR system and the PhoR/PhoB system as a model. VanS and VanR constitute a two-component system in Enterococcus faecium that mediates resistance to the glycopeptide antibiotic Vancomycin. In E. coli, the levels of free phosphate are modulated by the sensor kinase PhoR and its cognate response regulator, PhoB. The expression of VanS in E. coli leads to the high-level activation of PhoB in vivo.;Several lines of experimentation were pursued to isolate mutants of PhoB with enhanced recognition properties for VanS. The initial failure of structure-based approaches led to the development of a genetic screen for PhoB mutants with altered recognition (AR) properties. To date, we have isolated several mutants of PhoB that show greatly enhanced phosphotransfer activity from VanS in vivo. Two of these mutants have been purified to homogeneity and have been characterized in vitro with VanS and PhoR. Finally, the complete in vitro kinetic characterization of the cognate PhoR/PhoB system is described, as are PhoR/PhoB mutant interactions. Type B Vancomycin resistance is governed by yet another two-component system. VanS...