Analysis of a complete library of putative drug transporter genes in Escherichia coli

Abstract

The complete sequencing of bacterial genomes has revealed a large number of drug transporter genes. In Escherichia coli, there are 37 open reading frames (ORFs) assumed to be drug transporter genes on the basis of sequence similarities, although the transport capabilities of most of them have not been established yet. We cloned all 37 putative drug transporter genes in E. coli and investigated their drug resistance phenotypes using an E. coli drug-sensitive mutant as a host. E. coli cells transformed with a plasmid carrying one of 20 ORFs, i.e., fsr, mdfA, yceE, yceL, bcr, emrKY, emrAB, emrD, yidY, yjiO, ydhE, acrAB, cusA (formerly ybdE), yegMNO, acrD, acrEF, yhiUV, emrE, ydgFE, and ybjYZ, exhibited increased resistance to some of the 26 representative antimicrobial agents and chemical compounds tested in this study. Of these 20 ORFs, cusA, yegMNO, ydgFE, yceE, yceL, yidY, and ybjYZ are novel drug resistance genes. The fsr, bcr, yjiO, ydhE, acrD, and yhiUV genes gave broader resistance spectra than previously reported.

FIG. 1

Expression of putative drug transporter ORFs. Twenty-eight putative drug transporter ORFs were cloned into pTrc6His expression vectors to produce hexahistidine-tagged proteins under control of the trc (trp/lac hybrid) promoter. E. coli KAM3 cells harboring the constructed plasmids were cultured in 2×YT medium in the presence of 0.1 mM IPTG for induction of protein expression. The cells were harvested and then disrupted by sonication. Total cell proteins were separated on an SDS-polyacrylamide gel, and protein expression was detected by Western blot analysis with an antipolyhistidine antibody.