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 of Enterococcus faecium BM4147 and Ent. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems. P(vanH)-, P(vanRa)-, P(vanY)-, and P(vanRb)-lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genes vanR(a), vanR(b), or vanRS(b) behind their respective promoter (P(vanRa) or P(vanRb)) or vanS(a) or vanS(b) behind the rhamnose-inducible P(rhaB). Results show that activation (phosphorylation) of the response regulator VanR(a) by its sensor kinase VanS(a) leads to transcriptional activation of both P(vanH) and P(vanRa). Additionally, VanR(b) activates its cognate promoters P(vanY) and P(vanRb), although this occurs only in the absence of VanS(b) and presumably is caused by VanR(b) phosphorylation by an unidentified endogenous E. coli kinase. Thus, VanS(b) interferes with activation of VanR(b), probably by acting as a phospho-VanR(b) phosphatase. Although both VanR(a) and VanR(b) activate their cognate promoters, neither activates the heterologous P(vanR), P(vanH), or P(vanY), arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. VanR(a) also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal (P(i) limitation), PhoR autophosphorylation apparently is regulated in vivo. Furthermore, the activation of VanR(a) 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.