In vivo characterization of the type A and B vancomycin-resistant enterococci (VRE) VanRS two-component systems in Escherichia coli : A nonpathogenic model for studying the VRE signal transduction pathways

Escherichia coli reporter strains modeling the high-level type A and B vancomycin resistances ofEnterococcus faecium BM4147 andEnt. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems.P vanH -,P vanRa -,P vanY -, andP vanRb -lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type AvanRSHAXYZ and type BvanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genesvanR a ,vanR b , orvanRS b behind their respective promoter (P vanRa orP vanRb ) orvanS a orvanS b behind the rhamnose-inducibleP rhaB . Results show that activation (phosphorylation) of the response regulator VanRa by its sensor kinase VanSa leads to transcriptional activation of bothP vanH andP vanRa . Additionally, VanRb activates its cognate promotersP vanY andP vanRb , although this occurs only in the absence of VanSb and presumably is caused by VanRb phosphorylation by an unidentified endogenousE. coli kinase. Thus, VanSb interferes with activation of VanRb , probably by acting as a phospho-VanRb phosphatase. Although both VanRa and VanRb activate their cognate promoters, neither activates the heterologousP vanR ,P vanH , orP vanY , arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. VanRa also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal (Pi limitation), PhoR autophosphorylation apparently is regulatedin vivo . Furthermore, the activation of VanRa caused by cross talk from PhoR in the absence of a signal allows distinction of cross talk from crossregulation as the latter, but not the former, responds to environmental cues.