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

Abstract

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.