IN-VIVO CHARACTERIZATION OF THE TYPE-A AND TYPE-B VANCOMYCIN-RESISTANT ENTEROCOCCI (VRE) VANRS 2-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, faecal is have been developed to study the respective VanR-VanS two component regulatory systems. P-vanH-, P-vanRa-, P-vanY-, and P-vanRb-lacZ fusi ons report on expression from the vancomycin resistant enterococci pro moters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. T hese strains also express from single-copy chromosomal genes vanR(a), vanR(b), or vanRS(b) behind their respective promoter (P-vanRa or P-va nRb) or vanS(a) or vanS(b) behind the rhamnose-inducible P-rhaB. Resul ts show that activation (phosphorylation) of the response regulator Va nR(a) by its sensor kinase VanS(a) leads to transcriptional activation of both P-vanH and P-vanRa. Additionally, VanR(b) activates its cogna te promoters P-vanY and P-vanRb, although this occurs only in the abse nce of VanS(b) and presumably is caused by VanRb phosphorylation by an unidentified endogenous E, coli kinase. Thus, VanSb interferes with a ctivation of VanRb, probably by acting as a phospho-VanR(b) phosphatas e, 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 regulato ry switches in vancomycin-resistant enterococci, VanR(a) also is activ ated by the nonpartner kinase PhoR, Because this occurs in the absence of its inducing signal (Pi limitation), PhoR autophosphorylation appa rently is regulated in vivo. Furthermore, the activation of VanR(a) ca used by cross talk from PhoR in the absence of a signal allows distinc tion of cross talk from cross-regulation as the latter, but not the fo rmer, responds to environmental cues.