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

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The National Academy of Sciences

RESUMO

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. PvanH-, PvanRa-, PvanY-, and PvanRb-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 vanRa, vanRb, or vanRSb behind their respective promoter (PvanRa or PvanRb) or vanSa or vanSb behind the rhamnose-inducible PrhaB. Results show that activation (phosphorylation) of the response regulator VanRa by its sensor kinase VanSa leads to transcriptional activation of both PvanH and PvanRa. Additionally, VanRb activates its cognate promoters PvanY and PvanRb, although this occurs only in the absence of VanSb and presumably is caused by VanRb phosphorylation by an unidentified endogenous E. 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 heterologous PvanR, PvanH, or PvanY, 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 regulated in 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.

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