Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression

Abstract

The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

Structural Insight on the Mechanism of Regulation of the MarR Family of Proteins: High-Resolution Crystal Structure of a Transcriptional Repressor from Methanobacterium thermoautotrophicum

“…Others regulate tissue-specific activities, such as adhesive properties of cells, hemolytic properties, and the regulation of protease expression. 13,[19][20][21][22][23] A number of proteins within the MarR family interact with phenolics, including salicylate, which have been shown to modulate their repressor properties. 11,13,24,25 The crystal structures of proteins in the MarR family have been determined from a number of organisms including MarR from E. coli, SlyA from Enterococcus faecalis, and MexR from P. aeruginosa.…”

Gene-specific silencing induced by parallel complementary RNA in Pseudomonas aeruginosa

“…Pseudomonas aeruginosa K854 is a MexR -and spontaneous streptomycin-resistant derivative of PAO1 wild type (Poole et al 1996). The mexR gene, which encodes the repressor protein of the mexAB-oprM operon, is knocked out in K854 cells, leading to higher expression of the MexAB-OprM efflux pump (Poole et al 1996).…”

“…The mexR gene, which encodes the repressor protein of the mexAB-oprM operon, is knocked out in K854 cells, leading to higher expression of the MexAB-OprM efflux pump (Poole et al 1996).…”

Bacterial antibiotic efflux systems of medical importance

“…Accordingly, inactivation of any of the three genes mexA, mexB, or oprM results in a hypersusceptible strain with MICs of i-lactams, chloramphenicol, and tetracyclines close to those for a wild-type E. coli strain [38,49]. The expression of the mexAB-oprM operon is controlled by a repressor protein MexR [50] ( fig. 2), transcribed in the opposite direction to mexABoprM.…”