Characterization of the MexC-MexD-OprJ Multidrug Efflux System in ΔmexA-mexB-oprM Mutants of Pseudomonas aeruginosa

Bacterial strains and media.The bacterial strains used in this study are listed in Table 1. Bacterial cells were grown in L broth (1% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, and 0.5% [wt/vol] NaCl) or L agar (L broth plus 1.5% [wt/vol] agar) at 37°C. BM2 minimal medium (3) was used for selection of P. aeruginosa sinceEscherichia coli cannot utilize citrate. The following antibiotics were added to media at the indicated concentrations: ampicillin, 100 μg/ml for E. coli; carbenicillin, 200 μg/ml for P. aeruginosa; streptomycin, 30 μg/ml forE. coli and 100 μg/ml for P. aeruginosa; tetracycline, 10 μg/ml for E. coli and 100 μg/ml forP. aeruginosa; and chloramphenicol, 30 μg/ml for E. coli and 200 μg/ml for P. aeruginosa. L agar was supplemented with 5% (wt/vol) sucrose as required.

Recombinant DNA techniques.Transformation of E. coli, Southern hybridization, isolation of chromosomal DNA and plasmids, and restriction endonuclease digestions were carried out according to standard protocols (28). For PCR amplification of chromosomal DNA sequences (6), a bacterial colony was directly suspended in reaction mixture (6) lacking the primers and Taq polymerase. The mixture was boiled for 10 min and used as the template for amplification. The primer pairs used were as follows: mexR1 (5′-ATGAACTACCCCGTGAATCCC-3′) and mexR2 (5′-TTAAATATCCTCAAGCGGTTGC-3′) for mexR, 1611 and 1612 for mexA, 1613 and 1614 for mexB, and 1615 and 1680 for oprM, all of which have been described previously (6). The sizes of the amplified DNA fragments obtained by using these primer pairs were about 0.7, 1.5, 3.2, and 1.4 kb, respectively.

Cloning of chromosomal regions flanking themexR-mexA-mexB-oprM operon.To clone the chromosomal region located downstream of mexR, the chromosomal DNA of PAO1 was digested completely with XhoI and then partially with PvuII. The restriction fragments with sizes around 20 kb were collected and ligated with pMT5059 (33) that had been treated with XhoI and PvuII. The ligation mixture was used for transformation of E. coli DH5α (1) to select Ap^r clones on L agar plates. ThemexR gene was successfully amplified by PCR (see above) from one of the 48 Ap^r clones, and the plasmid obtained from such a clone was termed pKMM102. This plasmid carried an ∼24-kb chromosomal fragment extending from a PvuII site downstream of mexR to the XhoI site in oprM. The chromosomal region located downstream of oprM was cloned as follows. Since the KG2213 (see below) chromosome carried a part of the pMT5059-derived oriV and bla gene, the chromosomal DNA of KG2213 was digested with SacI, self-ligated, and used to transform DH5α to obtain Ap^rclones. One of the plasmids thus recovered, termed pKMM151, contained an ∼10-kb chromosomal fragment extending from the XhoI site in oprM to a SacI site located downstream ofoprM.

Construction of recombinant plasmids.TheSacI-digested res-Ω cassette from pMT5096 (34) was inserted into the SacI site in themexR gene of pKMM102, followed by insertion of the pMT5071-derived, NotI-flanked Mob cassette (34) into the NotI site to generate pKMM127 (see Fig. 1). The 8.4-kb SacI-HindIII fragment encompassing amexA-mexB-oprM region on pPV20 (25) was inserted into pAK1900 (25), and the resulting plasmid contains a 2.6-kb XhoI-HindIII fragment that covers the 3′ part of oprM and its downstream region. A NotI site in this fragment was disrupted by blunt ending with T4 DNA polymerase, and such a modified fragment was inserted into pMT5059 to construct pKMM089. pKMM096 was constructed by insertion of the pMT5095-derived, XhoI-flanked res-Ω cassette into the XhoI site of pKMM089. The NotI-flankedres-tnpR block from pMT5085 (34) was inserted into the pMT5059-derived NotI sites of pKMM089 and pKMM096 to construct pKMM091 and pKMM157, respectively.

The chromosomal region between the start codon of mexR andmexA was amplified by using primers mexR7 (5′-ATTGTTTGGCCGAGTAAACC-3′) and mexA3 (5′-TAGCGTTGTCCTCATGAGCG-3′) and inserted into the blunt-ended SalI site of pMP190 (31) to yield pKMM301, such that the promoterless lacZ gene was transcribed from the mexA promoter.

Mobilization of recombinant plasmids to P. aeruginosachromosome.An appropriate plasmid residing in an E. coli strain, S17-1 (30), was conjugationally mobilized to P. aeruginosa cells. After mating on L agar at 37°C for 4 h, the cell mixture was suspended in 0.4 ml of physiological saline. Aliquots (0.1 ml) of the 10- or 100-fold-diluted suspensions were plated on BM2 minimal agar plates (3) supplemented with appropriate antibiotics and incubated at 30°C for 2 days. The transconjugants thus obtained were purified once on the same selective plates, and examined for resistance to streptomycin, tetracycline, chloramphenicol, and/or sucrose on L agar plates. Clones indicating appropriate resistance to these selective markers were used in subsequent experiments.

Deletion of chromosomal mexR-mexA-mexB-oprM. Plasmid pKMM127 (Fig. 1 and Table 1) carries ares-Ω cassette in the mexR gene and a Mob cassette, and this plasmid was mobilized from S17-1 to PAO1 (a wild-type strain), KG3052 (a type-A nfxB strain), and KG3056 (a type-B nfxB strain) to select Sm^rtransconjugants. Among the transconjugants, those showing resistance to sucrose (e.g., KG2212, KG2217, and KG2236 from PAO1, KG3052, and KG3056, respectively) were presumed to have been formed by allelic exchange of the wild-type mexR gene with the mutant allele. This was confirmed by the fact that the mexR region could not be amplified by PCR from KG2212, KG2217, or KG2236 chromosome, consistent with an increase in the size of the mexR region in these strains as a result of insertion of the res-Ω cassette (data not shown). The argument for the expected allelic exchange was further supported by Southern hybridization experiments (data not shown).

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Fig. 1.

Schematic models of the TnpR-mediated site-specific deletion based on an improvement of the deletion system developed by Tsuda (34). Specifically, the plasmid carrying an additionalres-Ω cassette and a res-tnpR block (pKMM157) was integrated into the oprM gene of the chromosome containing another res-Ω cassette in the mexRgene. Deletion between the two copies of the res site led to removal of the intervening chromosomal region and the integrated plasmid sequence, leaving one copy of the res site. Abbreviations for restriction endonuclease sites: P, PvuII; S, SacI; X, XhoI; H, HindIII; and N, NotI.

To obtain the chromosomal deletion by using the original TnpR-mediated deletion system (34), pKMM091 (Table 1) carrying a pMT5085-derived res-tnpR block was transferred from E. coli S17-1 to KG2212 (PAO1mexR::res-Ω) to select Tc^r transconjugants on BM2 agar plates. These plasmid integrants were unstable, and their cultivation on L agar plates generated segregants which commonly lost the Sm^r and Tc^r markers but not the sacB gene (e.g., KG2213).

To eliminate, in the process of deletion mutagenesis, the plasmid-derived sequence including the Cb^r marker (bla), we developed a new improved method that involved mobilization of pKMM157 (Fig. 1 and Table 1) from S17-1 to the threemexR::res-Ω mutants of P. aeruginosa (KG2212, KG2217, and KG2236) and selection of Tc^r transconjugants on BM2 agar plates. Phenotypic characterization and Southern analysis indicated that such transconjugants were formed by integration of pKMM157 into the chromosomal oprM gene (Fig. 1) (data not shown). These transconjugants gave rise to Tc^s segregants at high frequencies after single-colony isolation on L agar plates. Each of three segregants obtained from the three crosses were resistant to sucrose and sensitive to streptomycin and carbenicillin. Such segregants derived from KG2212, KG2217, and KG2236 were designated KG2239, KG2240, and KG2259, respectively (Fig. 1).

Design of oligopeptides and development of polyclonal antisera specific to MexA and MexB.To avoid potential problems with the purification of MexA and MexB, oligopeptides based on the deduced amino acid sequences of these proteins (24, 25) were synthesized and used to immunize rabbits. Hydropathy analysis was performed with the Analysis Plot program (the Kyte and Doolittle algorithm [11]) included in the GeneWorks software package (Intelligenetics), and the amino acid sequences predicted to belong to the hydrophilic region of each protein were used for design of the oligopeptides. Multiple antigen peptides (32) composed of these oligopeptides linked to poly-lysin carrier were manually synthesized on a Multiple Peptide Synthesizer (Shimadzu Model PSSM-8) by solid-phase peptide synthesis on TAK08-WTGS resin (Shimadzu). New Zealand White rabbits (female; 10 weeks old) were immunized with 20 μg of the prepared antigen at weeks 1, 3, and 6. The first injection was in Freund’s complete adjuvant, the second and third ones were in Freund’s incomplete adjuvant, and in all cases the antigen was injected subcutaneously. Titers were determined by an enzyme-linked immunosorbent assay using total membranes of the P. aeruginosa OCR1 cells, prepared by sonication and subsequent centrifugation (see below), as the antigen.

Development of murine monoclonal antibody specific to OprM.BALB/c mice were immunized with 20 μg of purified OprM (4) on days 1, 7, 14, and 17 and an anti-OprM antibody-producing clone was prepared as described previously (5). A monoclonal antibody secreted by this clone was termed TM001.

Isolation of total membranes, SDS-PAGE, and immunoblot assay.Cells grown in L broth were harvested by centrifugation at 5,000 × g for 10 min at 4°C. The cells were resuspended in 10 mM Tris-HCl, pH 8.0, and were broken by sonication. After the removal of unbroken cells by centrifugation, total membranes (cell envelopes) were pelleted from the resulting supernatant by centrifugation at 20,000 × g for 30 min. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (4), with 10% (wt/vol) acrylamide in the running gel. Proteins fractionated by SDS-PAGE were electrophoretically transferred to a nitrocellulose membrane (0.45-μm pore size; Bio-Rad) as described previously (5). Binding of the primary antibodies was detected as described previously (4), with alkaline phosphatase-conjugated goat antibodies to rabbit immunoglobulin G (Cappel) or alkaline phosphatase-conjugated goat antibodies to mouse immunoglobulin G (Cappel) as the secondary antibodies and an Ap Conjugate Substrate Kit (Bio-Rad) for color development.

β-Galactosidase assays.Bacteria harboring the plasmid pKMM301 were cultured overnight at 37°C in A medium (28) supplemented with 0.4% (wt/vol) glucose, thiamine (1 μg/ml), 1 mM MgSO₄, and chloramphenicol (12.5 μg/ml for KG2239 or 400 μg/ml for KG2259) and subsequently diluted 50-fold into fresh medium consisting of the same solutes. Following growth to mid-log phase (A ₆₀₀, 0.3 to 0.6), cultures were assayed for β-galactosidase activity as described previously (20).