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J. Bacteriol., 09 1997, 5903-5913, Vol 179, No. 18
Copyright © 1997, American Society for Microbiology

Transcriptional regulation of the Enterococcus faecium BM4147 vancomycin resistance gene cluster by the VanS-VanR two-component regulatory system in Escherichia coli K-12

A Haldimann, SL Fisher, LL Daniels, CT Walsh and BL Wanner
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.

An Escherichia coli K-12 model system was developed for studying the VanS-VanR two-component regulatory system required for high-level inducible vancomycin resistance in Enterococcus faecium BM4147. Our model system is based on the use of reporter strains with lacZ transcriptional and translational fusions to the PvanR or PvanH promoter of the vanRSHAX gene cluster. These strains also express vanR and vanS behind the native PvanR promoter, the arabinose-inducible ParaB promoter, or the rhamnose-inducible PrhaB promoter. Our reporter strains have the respective fusions stably recombined onto the chromosome in single copy, thereby avoiding aberrant regulatory effects that may occur with plasmid-bearing strains. They were constructed by using allele replacement methods or a conditionally replicative attP plasmid. Using these reporter strains, we demonstrated that (i) the response regulator VanR activates PvanH, but not PvanR, expression upon activation (phosphorylation) by the partner kinase VanS, the noncognate kinase PhoR, or acetyl phosphate, indicating that phospho-VanR (P-VanR) is a transcriptional activator; (ii) VanS interferes with activation of VanR by PhoR or acetyl phosphate, indicating that VanS also acts as a P- VanR phosphatase; and (iii) the conserved, phosphate-accepting histidine (H164) of VanS is required for activation (phosphorylation) of VanR but not for deactivation (dephosphorylation) of P-VanR. Similar reporter strains may be useful in new studies on these and other interactions of the VanS-VanR system (and other systems), screening for inhibitors of these interactions, and deciphering the molecular logic of the signal(s) responsible for activation of the VanS-VanR system in vivo. Advantages of using an E. coli model system for in vivo studies on VanS and VanR are also discussed.


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