Involvement of MexR in the transcriptional regulation of MexAB-OprM, a multidrug efflux system in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a significant opportunistic pathogen known for high intrinsic resistance to multiple antibiotics. This resistance is attributable to low outer membrane permeability and multidrug efflux pumps. P. aeruginosa possesses four known efflux pumps, and several others are predicted within the genome sequence by homology. These tripartite efflux pumps include an inner membrane transporter and an outer membrane channel linked by a periplasmic protein. The efflux pumps are encoded by the mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY operons. The objective of this thesis is to demonstrate the regulatory role of MexR on the expression of mexAB-oprM. MexR is proposed to be a repressor of the mexAB-oprM operon based on the homology of the deduced mexR product to a number of repressor proteins and the genetic proximity of mexR to the mexAB-oprM operon. MexAB-OprM is shown to be growth-phase regulated, with expression of mexAB-oprM increasing to a maximum in the mid to late log phase. LasR, AlgU/T, RpoN, and MexR are not responsible for the growth-phase dependent regulation of mexAB-oprM expression. Inactivation of mexR increased expression of both a mexA and mexR reporter fusion, indicating that MexR is a negative regulator of both genes. As a regulatory protein, MexR is proposed to bind to the mexR-mexA intergenic region in the vicinity of the putative mexAB-oprM and mexR promoters. MexR was purified as a C-terminal histidine tagged protein. The DNA-binding activity of the purified MexR-his protein was demonstrated using a gel shift assay and DNase I footprinting. Two MexR-binding sites (site I & II), 28 bp in size were separated by 3 bp and occurred 189 bp upstream of mexA (from the 3′ end of site I to the translational start site of mexA) and 25 bp upstream of mexR (from the 3′ end of site II to the translational start of mexR). MexR-binding site I is downstream of a second putative promoter for mexA (which occurs approximately 120 bp upstream of the originally proposed mexA promoter), whereas site II is downstream of the mexR promoter. In the same position within each binding site, the sequence GTTGA was repeated in an inverse orientation separated by 4 bp. The presence of two mexA promoters, of which only one contains a MexR binding site, and growth-phase dependent expression suggests that MexR is not the sole regulator of mexAB-oprM. The regulation of mexAB-oprM is proposed to involve a complicated network of regulators.