MexR Repressor of the mexAB-oprM Multidrug Efflux Operon of Pseudomonas aeruginosa : Identification of MexR Binding Sites in the mexA-mexR Intergenic Region

Abstract

The MexR repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa was purified as a C-terminal histidine-tagged protein by metal chelate affinity chromatography. The purified protein was shown to bind ca. 200 bp upstream of mexA , at two sites, each of which contains a repeat of the nucleotide sequence GTTGA in inverse orientation. DNA sequence analysis identified mexA and mexR promoters within the MexR binding regions, consistent with the previously observed negative regulation of mexR and mexAB-oprM expression by MexR. Transcription of mexA from the promoter originating within the MexR binding site II was confirmed and shown to be markedly enhanced in a nalB (i.e., mexR ) mutant of P. aeruginosa . A second mexA promoter was also identified, ca. 70 bp upstream of mexAB-oprM , and transcription from this promoter appeared to occur in both the wild type and a nalB mutant. Production of MexAB-OprM in wild-type cells may be due to expression from a constitutively expressed proximal promoter, while MexAB-OprM hyperexpression in nalB mutants is due to the additional expression from a MexR-regulated distal promoter.