TRANSCRIPTIONAL REGULATION OF THE ENTEROCOCCUS-FAECIUM BM4147 VANCOMYCIN RESISTANCE GENE-CLUSTER BY THE VANS-VANR 2-COMPONENT REGULATORY SYSTEM IN ESCHERICHIA-COLI K-12

An Escherichia coli K-12 model system was developed for studying the V anS-VanR two-component regulatory system required for high-level induc ible vancomycin resistance in Enterococcus faecium BM4147. Our model s ystem is based on the use of reporter strains with lacZ transcriptiona l and translational fusions to the P-vanR or P-vanH promoter of the va nRSHAX gene cluster, These strains also express vanR and vanS behind t he native P-vanR promoter, the arabinose-inducible P-araB promoter, or the rhamnose-inducible P-rhaB promoter, Our reporter strains have the respective fusions stably recombined onto the chromosome in single co py, thereby avoiding aberrant regulatory effects that may occur with p lasmid-bearing strains, They, were constructed by using allele replace ment methods or a conditionally replicative attP plasmid, Using these reporter strains, we demonstrated that (i) the response regulator VanR activates P-vanH, but not P-vanR, expression upon activation (phospho rylation) by the partner kinase VanS, the noncognate kinase PhoR, or a cetyl phosphate, indicating that phospho-VanR (P-VanR) is a transcript ional activator; (ii) VanS interferes with activation of VanR by PhoR or acetyl phosphate, indicating that VanS also acts as a P-VanR phosph atase; and (iii) the conserved, phosphate-accepting histidine (H164) o f VanS is required for activation (phosphorylation) of VanR but not fo r deactivation (dephosphorylation) of P-VanR, Similar reporter strains may be useful in new studies on these and other interactions of the V anS-VanR system (and other systems), screening for inhibitors of these interactions, and deciphering the molecular logic of the signal(s) re sponsible for activation of the VanS-VanR system in vivo, Advantages o f using an E, coli model system for in vivo studies on VanS and VanR a re also discussed.