Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram-negative bacteria, including Vibrio cholerae non-O1. There are six operons for putative RND-type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non-O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (ΔacrAB, ΔydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC-deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolC_Vc of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolC_Vc showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF-TolC_Vc, and the activity required Na⁺. Thus, VexEF-TolC_Vc is either a Na⁺-activated or a Na⁺-coupled transporter. To our knowledge, this is the first report on the requirement of Na⁺ for an RND-type efflux transporter.