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.

FIG. 1

Overexpression and purification of MexR-His. E. coli BL21/DE3(pLysE) carrying pKLE1 (lanes 2 and 4) or pET23a (lanes 3 and 5) was cultured as described in the text, and MexR-His was purified from pKLE1-containing E. coli BL21/DE3(pLysE) by metal chelate affinity chromatography (Talon). Lanes 1 and 6, molecular mass markers; lanes 2 and 3, clarified lysate; lanes 4 and 5, eluant from Talon column; lane 7, DTT (1.5% [wt/vol])-treated sample from lane 4. The arrow indicates the location of the MexR-His monomer.

FIG. 2

Interaction of MexR with the mexA-mexR intergenic region. (A) The 183-bp NcoI-BglII fragment of pKLE2 containing the mexA upstream region was incubated without MexR (lane 1) or with 3.7 μM (lane 2), 7.4 μM (lane 3), or 15 μM (lane 4) MexR-His. This fragment was incubated with 15 μM MexR and 0.5 μg of either unlabeled NcoI-BglII fragment (lane 5), NcoI-NotI fragment (lane 6), or calf thymus (lane 7) DNA as competitor DNA. (B) The 168-bp NcoI-NotI fragment containing the mexR upstream region was incubated without MexR (lane 1) or with 3.7 nM (lane 2), 7.4 nM (lane 3), 15 nM (lane 4), 30 nM (lane 5), or 7.4 μM (lane 6) MexR-His. This fragment was incubated with 30 nM MexR-His and 0.5 μg of either unlabeled NcoI-NotI fragment (lane 7) or calf thymus (lane 8) DNA as competitor DNA. MexR-DNA complexes (C1 to C3) and free DNA (F) are highlighted.

FIG. 3

DNase I footprinting of MexR-DNA interactions on the mexA-mexR intergenic region. DNase I footprinting was conducted with the 407-bp NotI-SacI fragment from pKLE2 (which labels the mexR coding strand) (A) or the 328-bp NcoI-BglII fragment from pKLE2 (which labels the mexA coding strand) (B) in the presence of no MexR (lane 1) or 30 μM (lane 2), 60 μM (lane 3), 120 μM (lane 4), or 180 μM (lane 5) MexR-His. The protected regions are bracketed at the right and labeled I and II. The nucleotide sequence of the protected region is indicated at the left, with bases identified in the G+A sequencing reaction (lane G+A) shown in uppercase. (C) Nucleotide sequence of the mexA-mexR intergenic region highlighting the MexR binding sites (shaded) and 5′-GTTGA-3′ inverted repeat sequences (arrows) within each binding site. Putative mexA and mexR -35/-10 promoter sequences are highlighted in bold italics, and the +1 site (as determined by the RACE method [Fig. 5]) for the more distal of the two mexA promoters is underlined and in boldface.

FIG. 4

Schematic showing the positions of promoters and MexR binding sites within the mexA-mexR intergenic region. The relative positions and orientations of P_mexR, P1_mexA, P2_mexA, and MexR binding sites I and II (from Fig. 3) are highlighted. Restriction sites marked with an asterisk do not occur within the mexA-mexR coding coding or intergenic sequence but instead flank the cloned intergenic region in plasmid pKLE2. They are indicated here to illustrate the specific portion of the mexA-mexR intergenic region that was encompassed by the restriction fragments used in the gel shift experiments in Fig. 2. The relative locations of RT-PCR and RACE primers (Fig. 5) as well as the predicted amplification products are also highlighted. Placement of the RACE anchor primers corresponds with the predicted transcription starts sites for mexA.

FIG. 5

Identification and mapping of mexA transcripts. (A) RT-PCR of total RNA from P. aeruginosa strains OCR1 (nalB) (lane 2) and PAO1 (K767) (lane 3) using primers K14 and LA19 (Fig. 4). The 564-bp λ HindIII fragment is shown in lane 1. (B) 5′ RACE products from P. aeruginosa strains OCR1 (nalB) (lane 2) and PAO1 (K767) (lane 3) prepared with the mexA-specific LA20 primer and the PCR anchor primer provided with the RACE kit (Roche). A 293-bp RACE product produced using control template and primers provided with the RACE kit is shown in lane 1. The migration position of the 564-bp λ HindIII fragment is shown at the right.