The third genes, opmE, opmD and opmB, of multidrug efflux operons deduced from the Pseudomonas aeruginosa PAO1 genome data were cloned by polymerase chain reaction. The opmB gene product showed functional cooperation with inner membrane-associated components, MexAB, MexCD and MexXY, of the previously characterized multidrug efflux systems responsible for resistance to antimicrobial agents and extrusion of ethidium. The opmE and opmD gene products did not show functional cooperation. Immunoblots using a specific rabbit antiserum demonstrated, through exponential to stationary phases, constant expression of opmB and growth phase-dependent expression of opmD.
Outer membrane protein
Multidrug efflux system
Tripartite efflux systems are composed of an inner membrane protein called a resistance–nodulation–cell division efflux protein (RND), a periplasmic protein referred to as a membrane fusion protein (MFP), and an outer membrane protein called an outer membrane efflux protein (OEP). A channel is formed through both the inner and outer membrane which can mediate the removal of xenobiotics including antibiotics, thereby providing resistance to these agents .
Pseudomonas aeruginosa is a clinically important Gram-negative bacterium which can cause refractory infections in immunocompromised patients. One cause of the refractory infections is that this bacterium shows highly intrinsic and acquired resistance to a variety of antimicrobial agents. The tripartite efflux operons such as mexAB-oprM[2,3], mexCD-oprJ, and mexEF-oprN, which encode all three components, and dipartite efflux operons such as mexXY[6,7], which lack OEP genes, have been cloned from the P. aeruginosa chromosome. Characterization of these operons demonstrated that expression of these efflux operons contributes to intrinsic and acquired multidrug resistance of this bacterium. MexX (MFP) and MexY (RND) of the dipartite efflux operon mexXY are able to function as an effective efflux system by associating with OprM (OEP) from the mexA-mexB-oprM operon [7,8]. Furthermore, experiments designed to swap the OEPs expressed from mexA-mexB-oprM, mexC-mexD-oprJ and mexE-mexF-oprN have demonstrated that substrate specificities of these efflux systems are not altered, although the rate of efflux may be reduced [9–11].
In silico analysis of sequencing data from the P. aeruginosa PAO1 genome have suggested the existence of a further six multicomponent efflux operons such as PA3677-PA3676, PA4206-PA4207-PA4208, PA3523-PA3522-PA3521, PA1435-PA1436, PA4374-PA4375 and PA2528-PA2527/PA2526-PA2525 implicated in multidrug resistance . Characterization of these operons as well as the previously identified efflux operons is important in understanding antibiotic resistance mechanisms in this bacterium. In this paper, we describe the cloning and characterization of OEP-coding genes such as PA4208, PA3521 and PA2525, and to development of rabbit antisera specific to the gene products. For clarity, we use the nomenclature OpmD, OpmE and OpmB for the gene products of opmD (PA4208), opmE (PA3521) and opmB (PA2525) respectively, as proposed by Hancock (http://cmdr.ubc.ca/bobh/omps/).
2 Materials and methods
2.1 Bacterial strains and media
The P. aeruginosa strains used in this study are isogenic mutants of PAO1 and listed in Table 1. Bacterial cells were grown in L broth (1% w/v tryptone, 0.5% w/v yeast extract and 0.5% w/v NaCl) or L agar (L broth plus 1.5% w/v agar) at 37°C. BM2 minimal medium  was used for selection of P. aeruginosa since Escherichia coli cannot utilize citrate. Antibiotics were added to the media at the following concentrations: gentamicin, 15 µg ml−1 for E. coli and P. aeruginosa; chloramphenicol, 30 µg ml−1 for E. coli. L agar was supplemented with 10% w/v sucrose as required.
P. aeruginosa strains and plasmids used in this study
Strain or plasmid
ΔnfxB-mexCD-oprJ derivative of PAO1 constructed with pKMJ196
mexR::ΩSm derivative of KG3104 constructed with pKMM193; Smr
ΔoprM derivative of KG2260 constructed with pKMM200; Smr
ΔnfxB-mexCD-oprJ mexR::ΩSm ΔoprMΔmexXY; ΔmexXY derivative of KG2263 constructed with pNS003; Smr
nfxBΔmexRAB-oprM derivative of PAO1
ΔoprJ derivative of KG2259 constructed with pKMJ185
nfxBΔmexRAB-oprMΔoprJΔmexXY; ΔmexXY derivative of KG3107 constructed with pNS003
MexXY-overproducing, nalBΔmexAB; formerly N127
MexXY-overproducing, nalBΔmexAB oprM:: ΩSm; oprM::ΩSm derivative of KG5005 constructed with pMK1; Smr
pMT5059  carrying ΩSmr in mexR of mexRAB-oprM and Mob cassette from pMT5071  in Not I; Cbr, Smr, Cmr
pMT5059  carrying 10-kb Bam HI fragment lacked nfxB of nfxB-mexCD-oprJ from pKMJ002  and Mob cassette from pMT5071  at Not I; Cbr Cmr
pMT5059  carrying mexAB-oprM fragment lacked 1256-bp Pvu II-Sac II region in oprM and Mob cassette from pMT5071  at Not I; Cbr Cmr
pMT5059  carrying 1.2-kb PCR fragment with 3′ flanking region of mexY in Spe I-Hin dIII and 1.0-kb PCR fragment with 5′ flanking region of mexX in Mlu I-Nhe I in MCS, and Mob cassette from pMT5071  in Not I; Cbr Cmr
pMT5059  carrying a 5654-bp Eco RI-Bam HI fragment lacked 660-bp Eco RV region in oprJ of 6314-bp Eco RI-Bam HI fragment containing a part of mexD and a complete oprJ from pKMJ002  and Mob cassette from pMT5071  at Not I site; Cbr Cmr
pMT5059  carrying oprM::ΩSm in MCS and Mob cassette from pMT5071  in Not I site; Cbr, Smr, Cmr
pME6031  carrying 1.8-kb Nru I-Eco RI fragment from pJF118EH ; Tetr
pME6032 carrying 1.6-kb Hin dIII fragment encompassing ΩGm cassette from pGMΩ1  in tet gene; Gmr
pTO001 carrying 948-bp Apa I-Hin dIII fragment containing Xba I site; Gmr
pTO001A carrying His-tag linker in Xba I-Xho I region; Gmr
pTO001-His carrying 1476-bp PA3521 (opmE) in Xba I and Hin dIII region; Gmr
pTO001-His carrying 1464-bp PA4208 (opmD) in Xba I and Hin dIII region; Gmr
pTO001-His carrying 1497-bp PA2525 (opmB) in Xba I and Hin dIII region; Gmr
2.2 Construction of His-tagged recombinant (r-) OpmE, OpmD and OpmB expression plasmids
His-tagged recombinant protein was produced using the expression vector pTO001-His. The region between the Apa I site (in lac Iq) and Eco RI site (in the multicloning site) on pTO001 (Table 1) was exchanged by a PCR product amplified on pTO001 as a template using the primer pair P1 (5′-AATGGGCCCGCTAACAGCGCGATTTGCTG-3′) and P2 (5′-TCTAGAGCCAAGCTTTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACAC-3′) which contain a recognition site (underlined) for restriction endonucleases, Apa I, Xba I and Hin dIII, respectively, to yield pTO001A. A His-tag linker (sense: 5′-CTAGACACCACCACCACCACCACTAAC-3′; antisense: 5′-TCGAGTTAGTGGTGGTGGTGGTGGTGT-3′) added a respective cohesive end for Xba I and Xho I sites, and a stop codon was inserted into the Xba I–Xho I site in pTO001A to yield pTO001-His.
PCR was performed to amplify OpmD (PA4208), OpmE (PA3521) and OpmB (PA2525) genes using P. aeruginosa PAO1 chromosomal DNA as a template. The primer pair PA4208F (5′-GTAAAGCTTATGAAGCGCTCCTACCCGAA-3′) and PA4208R (5′-GCATCTAGAACGGTTGGCCCCGGCGGT-3′), PA3521F (5′-GTAAAGCTTATG AAGCCATACCTGCGGTCT-3′) and PA3521R (5′-AGGTCTAGAGGAGCGACTGTCGCGCTG-3′), PA2525F (5′-GTAAAGCTTATGAAACACACCCCCTCGTTG-3′) and PA2525R (5′-GGGTCTAGAGGGCGAAGGCGGCAGGC-3′), contain a newly added restriction endonuclease recognition site (underlined). The PCR product was ligated into the Hin dIII–Xba I site of pTO001-His to yield pOpmE, pOpmD and pOpmB. The nucleotide sequence of the OpmE, OpmD and OpmB genes was checked to ensure no mistakes had been introduced during amplification. These sequences are deposited at the DDBJ under accession numbers AB085583, AB085582 and AB085581, respectively.
2.3 Construction of knockout mutants of efflux protein genes
P. aeruginosa mutants, KG4510, KG4506 and KG4520, lacking the efflux protein genes and the regulatory genes were constructed by allele exchanges using plasmids listed in Table 1 as described previously . Construction of KG4510 followed the order: PAO1 to KG3104 with pKMJ196 (for deletion of nfxB-mexCD-oprJ), KG3104 to KG2260 with pKMM193 (for insertion of ΩSm cassette to mexR), KG2260 to KG2263 with pKMM200 (for deletion of oprM), and KG2263 to KG4510 with pNS003 (for deletion of mexXY). Construction of KG4506 followed the order: KG2259  to KG3107 with pKMJ185 (for deletion of oprJ); KG3107 to KG4506 with pNS003 (for deletion of mexXY). KG4520 was constructed with pMK1 (for insertion ΩSm cassette to oprM) from KG5005 .
2.4 Preparation of rabbit antisera
Three oligopeptides were designed to each OEP gene, based on the translation product of the published sequences . A cysteine residue was incorporated at the N-terminus of each oligopeptide (shown in brackets): (C)SEHLGEFSGERREA (aa 42–55), (C)QQLDYDGEPRRRLA (aa 117–130), and (C)LGSGLPEDVENAQA (aa 215–228) for OpmD; (C)TPAGQQRDIETYRG (aa 116–129), (C)LDVLEAERSDYLSR (aa 425–438), (C)TSPGVARQRDSRS (aa 479–491) for OpmE; and (C)AEFKEAEGWRRAEPRDV (aa 34–50), (C)LDEESGVQREALES (aa 404–417), and (C)GRVEEGLPPSP (aa 488–498) for OpmB. Oligopeptides were synthesized by F-moc solid phase methodology, and purified by HPLC. Following conjugation to keyhole limpet hemocyanin, antibodies were raised in rabbit (Asahi Techno Glass Co., Chiba, Japan).
2.5 Western immunoblot analysis
Membrane preparation and SDS–PAGE were performed as described previously [13,15]. The fractionated proteins were subjected to immunoblot analysis using either His-tag antibody (C-term) (Invitrogen, Carlsbad, CA, USA) or rabbit antisera developed in this study as the primary antibody and horseradish peroxidase-linked whole antibody to rabbit IgG (Amersham Pharmacia Biotech, Little Chalfont Buckinghamshire, UK) as the secondary antibody. The binding antibodies were detected using ECL plus Western blotting detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
2.6 Accumulation of ethidium bromide
A previously described method  was used, with slight modifications. Briefly, bacteria in exponential growth were collected by centrifugation, and resuspended to an A650 of 0.2 in sodium phosphate buffer (pH 7.0) in the presence of 0.05% of glycerol. Ethidium bromide was added to a final concentration of 10 µM and the change in fluorescence intensity (excitation and emission wavelengths were 520 nm and 590 nm, respectively) was recorded.
2.7 Other procedures
Preparation of plasmid DNA and related in vitro manipulation, agarose gel electrophoresis, transformation, restriction endonuclease digestion, ligation and PCR were performed as described previously . The MIC (minimum inhibitory concentration) was determined by the two-fold agar dilution technique  with L agar containing 0.5 mM IPTG with an inoculum size of 104 cells.
3 Results and discussion
3.1 Expression of recombinant OEPs
The His-tagged recombinant (r-) protein expression plasmids pOpmE, pOpmD and pOpmB encoding opmE, opmD and opmB genes, respectively, were introduced into a series of isogenic mutants of P. aeruginosa which lack the corresponding OEP gene: KG4510 (MexAB+OprM−), KG4506 (MexCD+OprJ−) and KG4520 (MexXY+OprM−). Immunoblot analysis using the anti-His-tag monoclonal antibody of membrane fractions from IPTG-induced cells (Fig. 1A) detected a band of approximately 50-kDa corresponding to a molecular size of the respective r-OpmE (52.8 kDa), r-OpmD (52.9 kDa) and r-OpmB (53.8 kDa) polypeptides. The level of detected r-OpmE, r-OpmD and r-OpmB varied between 100 and 130% for r-OpmB, 30 and 50% for r-OpmD and 10 and 20% for r-OpmE, compared with a expression level of r-OpmB in KG4520 (Fig. 1B).
A: Detection of r-OpmE, r-OpmD and r-OpmB in P. aeruginosa KG4510, KG4506 and KG4520 using the anti-hexa-His-tag murine monoclonal antibody. Preparation of total membranes (5 µg protein for all lanes) and analysis by SDS–PAGE and immunoblot assay were performed as described in Section 2. B: Band intensity was quantified using the Scion Image program (Scion corporation) based on NIH image for Macintosh by NIH. Data is expressed as a percentage of the quantity of r-OpmB in KG4520. Each bar represents the mean±S.D. of the intensity levels of the three experiments.
3.2 Functional cooperation with inner membrane associated components
An extrusion activity for the chimeric efflux systems composed of the inner membrane associated components MexAB, MexCD or MexXY, and the deduced OEPs r-OpmE, r-OpmD and r-OpmB were examined by susceptibility tests to antimicrobial agents and accumulation experiments of ethidium bromide. KG4510, KG4506 and KG4520 showed high susceptibilities to most of the tested antimicrobial agents due to loss of the OEPs OprM or OprJ (Table 2). These results are consistent with previous findings [3,6,8–10] that OprM and OprJ are essential components for function of MexAB-OprM, MexCD-OprJ and MexXY-OprM. Intrinsic resistance of KG4506 to sparfloxacin, norfloxacin, tetracycline, erythromycin and ethidium bromide was higher than for KG4510 and KG4520, though none of MexAB-OprM, MexCD-OprJ and MexXY-OprM are functional in these strains (Table 2). This suggests an outer membrane protein other than oprJ is cooperating with MexCD in KG4506.
Expression of r-OpmB in KG4510 (MexAB+OprM−), KG4506 (MexCD+OprJ−) and KG4520 (MexXY+OprM−) resulted in an increase in resistance to sparfloxacin, norfloxacin, tetracycline, chloramphenicol, erythromycin, cefpirome and ethidium bromide (Table 2). Furthermore, in r-OpmB-expressing KG4510 and KG4520, resistance to novobiocin, aztreonam and carbenicillin associated with the MexAB-OprM efflux system , and aminoglycosides such as tobramycin and amikacin associated with MexXY-OprM efflux system  was observed (Table 2), in spite of replacement of OprM by r-OpmB. These results are consistent with previous findings [9–11] that the outer membrane efflux component is not a determinant of substrate specificity. Expression of r-OpmB caused a decrease in ethidium accumulation in KG4510 or KG4506 cells (Fig. 2), indicating that enhanced resistance to the antimicrobial agents including ethidium bromide in both r-OpmB-expressing KG4510 and KG4506 is due to extrusion by the chimera MexAB and r-OpmB, and by MexCD and r-OpmB.
Accumulation of ethidium in host cells and in transformed cells. P. aeruginosa KG4510 and KG4506 cells were grown in L broth. Ethidium bromide was added to cell suspensions of KG4510 and KG4506 cells transformed with pOpmE, pOpmD and pOpmB at a final concentration of 10 µM. Accumulation of ethidium was monitored continuously by measuring the fluorescence of ethidium in cells, at the excitation and emission wavelengths of 520 and 590 nm, respectively.
Expression of r-OpmE or r-OpmD (Fig. 1) did not increase resistance to the agents tested (data not shown), nor cause a corresponding decrease of ethidium accumulation (Fig. 2). The apparent lack of cooperation of either r-OpmE or r-OpmD with the inner membrane associated components may be due to the low level of expression and/or the presence of a His-tag sequence of the recombinant proteins (Fig. 1). However, structural incompatibility between the recombinant proteins and the membrane components may be a more likely explanation for this result. Cooperation of OprN homologous to OpmE and OpmD with MexAB was not observed even by expression of a native OprN . The significant degree of homology (approximately 30% identity; 45% similarity) between the OpmB, OpmE and OpmD polypeptides suggests that all three are involved in the efflux of xenobiotics. Further studies such as the construction of a genetic system for high-level expression of native OpmE and OpmD is necessary for further characterization.
3.3 Development of antisera specific to OpmE, OpmD and OpmB
For characterization of three species of multidrug efflux operons containing opmE, opmD and opmB, we developed rabbit antisera by immunization of oligopeptides based on the respective gene sequences. Immunoblot analysis using these developed antisera detected a band (Fig. 3, lanes 2, 3 and 4) of about 50 kDa corresponding to r-OpmE, r-OpmD and r-OpmB recognized by the anti-His-tag murine monoclonal antibody (Fig. 3, lanes 1). These developed antisera did not show cross-reactivity between r-OpmE, r-OpmD and r-OpmB (data not shown). The corresponding antisera did not display reactivity with OprM, OprJ and OprN in cell membranes of KG5004 (formerly N116; MexAB−OprM+) , KG5008 (formerly N119; MexCD−OprJ+)  and KG4001 (MexEF−OprN+) , respectively (data not shown). Immunoblot analyses using these antisera in PAO1 cells grown in L broth showed constitutive high level expression of OpmB, whereas the low level of expression of OpmD at mid-exponential phase increased substantially from late exponential to early stationary phase (Fig. 4). Expression of OpmE was undetectable during all phases tested (Fig. 4). The efflux operon containing opmE may function to remove xenobiotics, which exist in other environments such as human infection sites rather than in a laboratory medium. Constant expression of OpmB suggested that an efflux operon PA2528-PA2527/PA2526-PA2525 including opmB is constitutively expressed as mexA-mexB-oprM system [2,3]. On the other hand, expression of an efflux operon PA4206-PA4207-PA4208 including opmD appears to be dependent on the growth phase.
Detection of r-OpmE, r-OpmD and r-OpmB in P. aeruginosa KG4510 using the anti-His-tag murine monoclonal antibody and the developed rabbit antisera. Preparation of total membranes (5 µg protein for all lanes) and analysis by SDS–PAGE and immunoblot assay were performed as described in Materials and Methods. OpmE-, OpmD- or OpmB-specific rabbit antisera were prepared as described in Section 2. Lane 1, anti-His-tag antibody; 2, anti-OpmE antiserum; 3, anti-OpmD antiserum; 4, anti-OpmB antiserum. Molecular sizes are indicated in kilodaltons on the left side of the panels.
Immunodetection using the specific rabbit antisera of OpmE, OpmD, and OpmB in P. aeruginosa PAO1 cells grown in L broth. An overnight culture was diluted at 1000-fold with prewarmed L broth and incubated aerobically at 37°C. At 2, 4, 6, 12, and 16 h, an optical density (A650) of the culture was measured. Total membranes, SDS–PAGE and the immunoblot assay were performed as described in Section 2. A: Immunodetection of OpmE, OpmD and OpmB. Lane 1, 2 h; 2, 4 h; 3, 6 h; 4, 12 h; 5, 16 h. B: Growth of PAO1 cells (− — -) and expression levels of OpmD and OpmB. Band intensity was quantified using the Scion Image program (Scion corporation) based on NIH image for Macintosh by NIH. Each bar represents the mean±S.D. of the intensity levels of the four experiments.
Increased expression of opmD at late-exponential phase suggests that expression may be related to the cell density, controlled by a quorum sensing mechanism . During the preparation of this manuscript (August, 2002), a paper appeared in the literature  describing a mutation of opmD which markedly down-regulates production of elastase, rhamnolipids, pyocyanine, pyoverdine and other pathogenic factors that are known to be regulated by a quorum sensing mechanism in P. aeruginosa. Taken together, it is likely that the efflux operon PA4206-PA4207-PA4208 including opmD is regulated by a quorum-sensing mechanism, although further investigation such as detection of expression of the operon in a quorum-sensing mutant is required to confirm this. Thus, our developed antisera will be useful for investigating expression of the efflux operons in P. aeruginosa under different growth conditions.
We thank Stephan Heeb and Dieter Haas (Universite de Lausanne, Switzerland) for providing pME6032 and Takahumi Satou for help with the ethidium bromide accumulation experiment. This research was supported by grants for scientific research to N.G. from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan and from the Ministry of Health, Labor and Welfare of Japan. T.M. was the recipient of a scholarship of from The Japan Scholarship Foundation.