Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa

Several nalB-type multidrug-resistant mutants of Pseudomonas aeruginosa overexpressed MexAB-OprM and carried mutations in the local regulatory gene, mexR. Others, dubbed nalC types, carried mutations elsewhere and overexpressed MexAB-OprM less extensively than the nalB strains. Available evidence showed that MexR acted solely as repressor. Disruption of the mexR gene at various places suggested that the 5' end of mexR may be a part of the mexAB-oprM promoter.     

Organic solvent-tolerant mutants of Pseudomonas aeruginosa display multiple antibiotic resistance.

Organic solvent-tolerant mutants of Pseudomonas aeruginosa selected in the presence of hexane exhibited increased resistance to a variety of structurally unrelated antimicrobial agents, including beta-lactams, fluoroquinolones, chloramphenicol, tetracycline, and novobiocin, a phenotype typical of nalB multidrug-resistant mutants. Western immunoblotting with antibodies specific to components of the three known multidrug efflux systems in P. aeruginosa demonstrated that the solvent-tolerant mutants displayed increased expression of the MexAB-OprM system and decreased expression of the MexEF-OprN system. Sequence analysis of mexR, the repressor gene of mexAB-oprM efflux operon, identified a nonsense mutation and a point mutation in the mexR genes of two solvent-tolerant mutants. These results emphasize the importance of the MexAB-OprM efflux system in organic solvent tolerance and the ability of environmental pollutants to select bacteria with a medically relevant antibiotic-resistant phenotype.     

The mexR repressor of the mexAB-oprM multidrug efflux operon in Pseudomonas aeruginosa: characterization of mutations compromising activity

Mutations in mexR yield a multidrug resistance phenotype in nalB mutants of Pseudomonas aeruginosa as a result of derepression of the mexAB-oprM multidrug efflux operon. MexR produced by several nalB strains carried single amino acid changes that compromised MexR stability or its ability to dimerize. Changes at residues L95 and R21, however, produced a stable MexR protein capable of dimerization and, thus, likely compromised DNA binding.     

High-level fluoroquinolone resistance in Pseudomonas aeruginosa due to interplay of the MexAB-OprM efflux pump and the DNA gyrase mutation

Fluoroquinolone resistance in Pseudomonas aeruginosa is mainly attributable to the constitutive expression of the xenobiotic efflux pump and mutation in DNA gyrase or topoisomerase IV. We constructed cells with a double-mutation in gyrA and mexR encoding DNA gyrase and repressor for the mexAB-oprM operon, respectively. The mutant showed 1,024 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher fluoroquinolone resistance than cells lacking the MexAB-OprM. In contrast, a single mutation in gyrA or mexR caused only 4 and 64 times higher resistance, respectively. These findings manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance.     

Contribution of multidrug efflux pumps to multiple antibiotic resistance in veterinary clinical isolates of Pseudomonas aeruginosa

The contribution of efflux pumps to multidrug resistance in 12 Pseudomonas aeruginosa isolates from various animal sources was assessed. Western immunoblot analyses demonstrated that all twelve isolates expressed significant levels of the MexAB OprM efflux system whereas two isolates simultaneously expressed the MexEF OprN or MexXY systems, respectively. One strain contained a single mutation in mexR, a regulator of mexAB-oprM expression, that did not adversely affect the MexR amino acid sequence, and three isolates contained the same, single base change in the mexA-mexR intergenic region. The MexXY-expressing strain contained two base substitutions in its mexZ regulatory gene which did not alter the MexR sequence.     

Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance

The self‐assembling MexA‐MexB‐OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR‐wt as well as a selected set of MDR single mutants distant from the proposed DNA‐binding helix. Although DNA affinity and MexA‐MexB‐OprM repression were both drastically impaired in the selected MexR‐MDR mutants, MexR‐wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR‐MDR mutants, secondary structure content and oligomerization properties were very similar to MexR‐wt despite their lack of DNA binding. Despite this, the MexR‐MDR mutants showed highly varying stabilities compared with MexR‐wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA‐binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR‐wt in both free and DNA‐bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations—stability, domain interactions, and internal hydrophobic surfaces—are also critical for the regulation of MexR DNA binding.     

Mutation in mexR-gene leading to drug resistance in corneal keratitis in human

The present study deals with the genetic polymorphism of the mexR gene which is involved in the resistance to drugs like ciprofloxacin. Mutations in mexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system. The MexR product contains 147 amino acids with a molecular mass of 16,964 Da. We detected 28 point mutations in 14 samples from corneal scraping infected with Pseudomonas aeruginosa, which were screened for ciprofloxacin resistance. Twenty four were silent mutations and four missense mutations. Mapping these mutations was done by using in silico methods on the protein 3D- structure obtained from PDB database, localized at 3 specific sites. Single amino acid changes (mutations) may influence MexR stability or its ability to dimerise, and thus result in the conformation changes at the DNA-binding domain, of the structure. Hence it is concluded that the mutations in the DNA binding domain of mexR gene could be one of the factors contributing to the possible drug resistance in these patients.     

Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product

The MexXY components of the MexXY-OprM multidrug efflux system of Pseudomonas aeruginosa are encoded by a MexZ repressor-regulated operon that is inducible by antibiotics that target the ribosome. Mutant strains disrupted in a gene, PA5471, were shown to be compromised for drug-inducible mexXY expression and, therefore, MexXY-OprM-mediated antimicrobial resistance. The PA5471 gene was inducible by the same ribosome-targeting agents that induce mexXY expression. Moreover, vector-driven expression of cloned PA5471 was sufficient to promote mexXY expression and MexXY-mediated resistance in the absence of antibiotic exposure, consistent with PA5471 directly or indirectly activating mexXY expression following its own upregulation in response to antibiotics. The requirement for PA5471 for mexXY expression and antimicrobial resistance was, however, obviated in mutants lacking the MexZ repressor of mexXY expression, suggesting that PA5471 directly or indirectly modulates MexZ activity in effecting mexXY expression. While the recruitment of PA5471 and MexXY in response to ribosome disruption by antimicrobials is consistent with their genes playing a role in protecting cells from the adverse consequences of disrupting the translation process, reminiscent of trans-translation, these genes appear to operate independently in their contribution to resistance: mutants defective in trans-translation showed a much more modest (twofold) decrease in resistance to ribosome-targeting agents than those lacking PA5471 or MexXY, and this decrease was observed whether functional PA5471/MexXY was present or not.     

Characterization of the RND-type multidrug efflux pump MexAB-OprM of the plant pathogen Pseudomonas syringae

In gram-negative bacteria, transporters belonging to the RND family are the transporters most relevant for resistance to antimicrobial compounds. In Pseudomonas aeruginosa, a clinically important pathogen, the RND-type pump MexAB-OprM has been recognized as one of the major multidrug efflux systems. Here, homologues of MexAB-OprM in the plant pathogens Pseudomonas syringae pv. phaseolicola 1448A, P. syringae pv. syringae B728a, and P. syringae pv. tomato DC3000 were identified, and mexAB-oprM-deficient mutants were generated. Determination of MICs revealed that mutation of MexAB-OprM dramatically reduced the tolerance to a broad range of antimicrobials. Moreover, the ability of the mexAB-oprM-deficient mutants to multiply in planta was reduced. RNA dot blot hybridization revealed growth-dependent regulation of the mexAB-oprM operon in P. syringae; the expression of this operon was maximal in early exponential phase and decreased gradually during further growth.     

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.