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J Bacteriol. Nov 2003; 185(21): 6233–6240.
PMCID: PMC219392

Identification and Characterization of the emhABC Efflux System for Polycyclic Aromatic Hydrocarbons in Pseudomonas fluorescens cLP6a

Abstract

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.

Efflux pumps are prevalent in gram-negative bacteria, in which they contribute to antibiotic resistance and organic solvent tolerance. In pseudomonads, the major efflux pumps belong to the resistance-nodulation-cell division (RND) permease superfamily (22) found in the Bacteria, Archaea, and Eucarya (29). The RND protein, a secondary transporter located in the inner membrane, forms a complex with a membrane fusion protein in the periplasm and an outer membrane channel to effect transport from the cell directly to the extracellular medium (33). Several RND efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM, have been characterized functionally in Pseudomonas aeruginosa, in which they are involved in the transport of and resistance to antibiotics, hydrophobic dyes, and detergents (22).

Recently, similar efflux systems were discovered in bacteria capable of growing in the presence of high concentrations of toxic organic solvents. Both Pseudomonas putida S12 and P. putida DOT-T1E tolerate high concentrations of toluene because they possess RND efflux systems that remove toluene from the cell (12, 19, 23, 24). Toluene and other organic solvents that have octanol-water partition coefficients (log Kow) between 1.5 and 3.5 accumulate in cell membranes, where they increase membrane permeability, disrupt the membrane potential, and cause cell lysis and death (27). In contrast, polycyclic aromatic hydrocarbons with three or more rings, such as phenanthrene, anthracene, and fluoranthene, have log Kow values greater than 3.5 and are not toxic to bacterial cells. Thus, the discovery by Bugg et al. (3) of an active efflux mechanism for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens LP6a was surprising.

P. fluorescens LP6a was isolated from petroleum-contaminated soil for its ability to degrade naphthalene, as well as the polycyclic aromatic hydrocarbons phenanthrene and anthracene (7), which makes it an interesting candidate for studying the movement of hydrocarbons across the cell membrane. Not only did the efflux system in P. fluorescens LP6a transport nontoxic growth substrates, but it also displayed unusual hydrocarbon substrate selectivity. While phenanthrene, anthracene, and fluoranthene were exported from the cell, toluene and naphthalene were not (3). This unexpected selectivity prompted further examination of the efflux mechanism.

In this study, an RND efflux system was identified in P. fluorescens LP6a. Disruption of the efflux pump gene demonstrated that this system is involved in the efflux of polycyclic aromatic hydrocarbons and in resistance to certain antibiotics. The efflux genes were designated emhABC (efflux of multicyclic hydrocarbons). A survey of selected pseudomonads indicated that the ability to transport polycyclic aromatic hydrocarbons is widespread and is probably due to the presence of homologous efflux pumps.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

The bacterial strains and plasmids used in this study are listed in Table Table1.1. The cured strain P. fluorescens cLP6a, which lacks the 63-kb catabolic plasmid pLP6a carrying the polycyclic aromatic hydrocarbon degradation genes found in the parent strain P. fluorescens LP6a (7), was used to facilitate analysis of hydrocarbon transport in the absence of metabolism. P. fluorescens, P. aeruginosa, P. putida, and Burkholderia cepacia strains were grown in tryptic soy broth (Difco Laboratories, Detroit, Mich.) at 28°C and were maintained on plate count agar (Difco) or Luria-Bertani (LB) agar supplemented with the appropriate antibiotics. Azotobacter vinelandii UW was grown at 28°C in Burke's buffer with glucose and nitrogen as described previously (3). Escherichia coli strains were cultured in LB medium at 37°C. Ampicillin (100 μg ml−1), kanamycin (50 μg ml−1 for E. coli and 25 μg ml−1 for P. fluorescens), tetracycline (10 μg ml−1), and streptomycin (25 μg ml−1) were added as required.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA techniques.

Genomic DNA from P. fluorescens strains was prepared as described by Maloy (16). Plasmid DNA was isolated either by the alkaline lysis method (25) or with a QIAprep Spin Miniprep kit (Qiagen, Mississauga, Ontario, Canada). Restriction digestion and ligation reactions were performed according to the supplier's directions (Roche Diagnostics, Laval, Quebec, Canada). DNA fragments separated on agarose gels were purified by using a QIAquick gel extraction kit (Qiagen) for DNA fragments less than 6 kb long and a QIAEX II gel extraction kit (Qiagen) for DNA fragments more than 6 kb long. Plasmid DNA was introduced into E. coli by heat shock at 42°C and into P. fluorescens by electroporation with a Gene Pulser (Bio-Rad Laboratories, Mississauga, Ontario, Canada).

PCR was performed with a Flexigene thermal cycler (Techne, Princeton, N.J.). The PCR products were cloned into E. coli TOP10F′ by using a TOPO TA cloning kit (Invitrogen Canada, Burlington, Ontario, Canada).

For Southern blotting and hybridization, DNA from agarose gels was transferred to Hybond N membranes (Amersham Biosciences, Baie d'Urfe, Quebec, Canada), and hybridization was performed according to the manufacturer's protocol (Amersham Biosciences). The DNA fragments used as probes were labeled with [α-32P]dCTP (3,000 Ci mmol−1; Amersham Biosciences) by random primer labeling (Roche Diagnostics).

Nucleotide sequencing reactions were performed with a DYEnamic ET terminator cycling sequencing kit (Amersham Biosciences), and the results were analyzed with a model 373A automated DNA sequencer (Applied Biosystems Inc., Foster City, Calif.) by the Molecular Biology Services Unit, University of Alberta, Edmonton, Alberta, Canada. Sequence data were analyzed with the GeneTool 1.0 software package (BioTools Inc., Edmonton, Alberta, Canada). The BLAST program (1) was used for sequence homology searches in the National Center for Biotechnology Information GenBank database (http://www.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed by using the neighbor-joining method in ClustalX (28).

Detection and inactivation of emhB.

PCR was performed with P. fluorescens cLP6a genomic DNA by using the following degenerate oligonucleotide primers corresponding to conserved regions of RND efflux pumps: forward primer 5′-CGGA(C/T)GG(C/T)TCICAGGT(A/G)CG-3′ and reverse primer 5′-A(A/G)G(A/G)TGAAIGC(G/C)AGIGAGGTC-3′, where the nucleotides in parentheses represent degenerate sites. The PCR mixture (100 μl) contained 500 ng of genomic DNA from P. fluorescens cLP6a, 75 mM Tris-HCl, 20 mM (NH4)2SO4, 2 mM MgCl2, 0.1% (vol/vol) Tween 20, each deoxynucleoside triphosphate at a concentration of 0.2 mM, each primer at a concentration of 0.6 μM, and 5 U of Taq polymerase. The PCR program consisted of initial denaturation at 95°C for 5 min, followed by 25 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 90 s and a final extension step at 72°C for 5 min. A 2.2-kb PCR product was isolated and cloned into pCR2.1 by TA cloning, and the resulting plasmid, pCR17, was introduced into P. fluorescens cLP6a by electroporation. Transformants, in which the plasmid was integrated into the chromosome by homologous recombination, were selected on LB agar containing kanamycin. One transformant, designated P. fluorescens cLP6a-1, was selected for further study. Insertion of pCR17 into the emhB gene in P. fluorescens cLP6a-1 was confirmed by Southern blotting and hybridization by using the 2.2-kb efflux gene fragment and plasmid pCR2.1 as probes.

Sequencing the emh genes.

To locate the chromosomal region containing the efflux pump gene, restriction digests of P. fluorescens cLP6a genomic DNA were analyzed by Southern blotting and hybridization by using the 2.2-kb emhB gene fragment as a probe. The emhB gene hybridized to a 9.7-kb BamHI fragment. Genomic DNA from P. fluorescens cLP6a was digested with BamHI, and the 7- to 14-kb fragments were ligated to BamHI-linearized pUC19 and transformed into E. coli DH5α. About 300 ampicillin-resistant transformants were screened by colony PCR by using primers specific for the emhB gene sequence. One positive colony, harboring plasmid p251 carrying a 9,653-bp insert, was identified, and the nucleotide sequence of the insert was determined.

Complementation of emhB in P. fluorescens cLP6a-1.

A 6,789-bp fragment containing the emhABC genes and their upstream promoter region was amplified from p251 by using the Expand Long Template PCR system (Roche Diagnostics). The primers, corresponding to nucleotide positions 767 to 787 (forward) and 6731 to 6751 (reverse) in the nucleotide sequence (accession number AY349612), contained BamHI sites for cloning the PCR product into the broad-host-range vector pUCP26. The resulting plasmid, pBH5, contained the emhABC genes in the same orientation as the lacZ promoter. Plasmid pBH5 was introduced into P. fluorescens cLP6a-1 by electroporation, and one transformant, designated P. fluorescens cLP6a-1(pBH5), was selected for analysis. As a vector control, pUCP26 was electroporated into P. fluorescens cLP6a-1 to obtain P. fluorescens strain cLP6a-1(pUCP26).

Transport assays.

Time course experiments to measure the accumulation of radiolabeled substrate in the P. fluorescens cLP6a strains were performed by using the rapid centrifugation method described by Bugg et al. (3). The substrates tested included [9-14C]phenanthrene (96.5% pure; 19.3 mCi mmol−1; Amersham, Arlington Heights, Ill.), [side ring U-14C]anthracene (98% pure; 11.2 mCi mmol−1; Amersham), and [3-14C]fluoranthene (97% pure; 45 mCi mmol−1; Sigma Chemical Co., St. Louis, Mo.). The radiolabeled substrates were mixed with unlabeled compound and added to the assay medium at final concentrations of 6.4 μM for phenanthrene, 0.26 μM for anthracene, and 1.2 μM for fluoranthene, which corresponded to 90% of the aqueous solubility limit for each compound. Sodium azide (30 mM) was added to inhibit active transport at appropriate times.

Antibiotic sensitivity tests.

The MICs of chloramphenicol, nalidixic acid, tetracycline, erythromycin, trimethoprim, and streptomycin were determined for the various strains in tryptic soy broth by the microtiter broth dilution method (10).

Determination of emh promoter activity.

The region upstream of the emhA gene was amplified by PCR by using primers corresponding to positions 656 to 680 (forward) and 918 to 940 (reverse) in the nucleotide sequence (accession number AY349612) with added HindIII and BamHI sites. The PCR product was cloned upstream of the promoterless lacZ gene in plasmid pTZ110, and a streptomycin resistance cassette from plasmid p34S-Sm3 was inserted into the unique PstI site to generate the promoter-reporter plasmid pTZ110-emh. A control plasmid, pTZ110-str, was also constructed by inserting the streptomycin resistance cassette into the PstI site of pTZ110. The promoter-reporter plasmid and the control plasmid were electroporated into P. fluorescens cLP6a to generate strains cLP6a(pTZ110-emh) and cLP6a(pTZ110-str), respectively.

To determine the effects of various hydrocarbons on expression of the lacZ reporter gene, P. fluorescens strains cLP6a(pTZ110-emh) and cLP6a(pTZ110-str) were grown in tryptic soy broth in the presence of 0.5 g of phenanthrene per liter, 0.5 g of anthracene per liter, 0.5 g of fluoranthene per liter, 0.5 g of naphthalene per liter, or 0.05% (vol/vol) toluene. Samples of the cultures were collected after 4 h of growth (early exponential phase), and the cells were permeabilized according to the Miller assay (18). The β-galactosidase activities of the samples were determined by measuring the hydrolysis of o-nitrophenyl-β-d-galactoside by the microplate assay method described by Griffith and Wolf (8). The absorbance at 420 nm, the absorbance at 550 nm, and the optical density at 600 nm were measured with a SpectraMax Plus384 microplate spectrophotometer (Molecular Devices Corporation).

Nucleotide sequence accession number.

The nucleotide sequence reported here has been deposited in the GenBank database under accession number AY349612.

RESULTS

Detection and insertional inactivation of the efflux pump gene.

Degenerate PCR primers designed from conserved sequences in the antibiotic and toluene efflux pumps MexB in P. aeruginosa, SrpB in P. putida S12, and TtgB and TtgE in P. putida DOT-T1E were used to amplify homologous genes from P. fluorescens cLP6a genomic DNA. The PCR product corresponding to the expected size (2.2 kb) was cloned, and 6 of 20 recombinant plasmids were selected randomly for partial DNA sequencing. The partial sequences of the six inserts were identical, indicating that only one pump gene had been amplified and cloned. A BLAST search of the translated sequence revealed a high degree of identity (60 to 80%) to gram-negative bacterial RND pumps in the GenBank database.

Plasmid pCR17, containing the 2.2-kb PCR product, was introduced into P. fluorescens cLP6a by electroporation so that integration of the plasmid into the chromosome by homologous recombination would disrupt the efflux pump gene. A disruption mutant, P. fluorescens cLP6a-1, was obtained, and the presence of the insertion was confirmed by Southern blotting and hybridization by using the 2.2-kb efflux gene fragment and plasmid pCR2.1 as probes (data not shown).

To test the role of the gene product in the efflux of polycyclic aromatic hydrocarbons, the cellular accumulation of [14C]phenanthrene by P. fluorescens cLP6a and by the insertion mutant cLP6a-1 was measured before and after addition of the energy inhibitor azide (Fig. (Fig.1).1). The cellular accumulation, which represented both the intracellular and membrane-associated hydrocarbon, was the amount of phenanthrene in the cell pellet after centrifugation and was expressed as the fractional amount of the total 14C added. For P. fluorescens cLP6a, the fractional amount of [14C]phenanthrene in the cells rapidly reached a steady-state level of 0.12 ± 0.07 in the absence of azide. Upon addition of the energy inhibitor azide, the fraction of phenanthrene in the cells increased to 0.32 ± 0.01. This significant increase (P < 0.005) is consistent with inhibition of an active efflux process. In contrast, the insertion mutant P. fluorescens cLP6a-1 contained a significantly higher steady-state level of phenanthrene (0.29 ± 0.02; P < 0.01) prior to azide addition than the cured cLP6a strain. The fraction of phenanthrene in the mutant cells did not increase significantly above the steady-state level when azide was added and was equivalent to the final cellular level in the azide-treated cured strain. These results indicate that the gene for polycyclic aromatic hydrocarbon efflux had been disrupted. This gene was designated emhB.

FIG. 1.
Accumulation of [9-14C]phenanthrene in cell pellets of P. fluorescens cLP6a (•) and the emhB disruption mutant cLP6a-1 (○) over time. The fractional amount of radiolabeled phenanthrene in the cells was measured by a rapid centrifugation ...

Cloning and sequence analysis of the Emh efflux system.

A 9.7-kb BamHI fragment of P. fluorescens cLP6a genomic DNA, which hybridized to the emhB gene fragment, was cloned into pUC19, and its nucleotide sequence was determined. Seven complete open reading frames (ORFs) were identified on the 9.7-kb BamHI fragment (Fig. (Fig.2).2). ORF3 (nucleotides 2083 to 5229) corresponded to the emhB gene. The deduced protein sequence encoded by emhB exhibited a high degree of identity to the RND proteins TtgB (85%) in P. putida DOT-T1E, ArpB (85%) in P. putida S12, and MexB (80%) in P. aeruginosa, and EmhB clustered with these proteins on the phylogenetic tree (Fig. (Fig.3).3). Adjacent to emhB, ORF2 (nucleotides 922 to 2079) and ORF4 (nucleotides 5229 to 6689) encoded the putative membrane fusion protein and the outer membrane channel of the efflux system; these genes were designated emhA and emhC, which is consistent with the nomenclature for three-component efflux systems. The deduced amino acid sequence encoded by ORF1 (nucleotides 23 to 655), which was located upstream of emhABC but in the opposite orientation, exhibited homology to sequences of transcriptional repressors belonging to the TetR family, including TtgR (73%) in P. putida DOT-T1E and ArpR (73%) in P. putida S12. Although the role of ORF1 has not been determined, it is believed to regulate the emhABC genes and has been designated emhR.

FIG. 2.
Schematic representation of the emh gene cluster in P. fluorescens cLP6a and cLP6a-1. In pCR17, the sequence of plasmid pCR2.1, which confers kanamycin resistance (Kmr), is cross-hatched, while the flanking regions homologous to emhB are not.
FIG. 3.
Phylogenetic relationship of EmhB to other RND efflux pumps. The tree was generated by using the neighbor-joining method in ClustalX, and the scale bar represents 0.1 substitution per amino acid. The accession numbers are as follows: MexF, ...

Three additional ORFs were identified on the 9.7-kb fragment. The deduced protein sequence encoded by ORF5 (nucleotides 7041 to 8300) showed homology to porins that are selective for specific substrates, such as BenF (44% identical to ORF5) involved in benzoate uptake (4) and PhaK (38% identical) required for phenylacetic acid uptake (20). Finally, ORF6 (nucleotides 8368 to 8760) and ORF7 (nucleotides 8772 to 9458) encode putative proteins similar to carboxymuconolactone decarboxylases and 3-oxoadipate enol-lactone hydrolases involved in metabolism of protocatechuate, an aromatic intermediate in phenanthrene degradation. Interestingly, genes similar to ORF5, ORF6, and ORF7 are located in the same arrangement adjacent to the toluene efflux operon ttgABC (nucleotide accession number AF031417) in P. putida DOT-T1E.

Phenanthrene efflux is restored by complementation.

To confirm the role of the Emh efflux system, plasmid pBH5 was constructed by ligating the emhABC genes and their upstream promoter region into the broad-host-range vector pUCP26 and transforming it into P. fluorescens cLP6a-1. The resulting transformant, P. fluorescens cLP6a-1(pBH5), accumulated levels of phenanthrene similar to the levels accumulated by the wild-type strain in both the absence and the presence of azide (Fig. (Fig.4).4). As a control, pUCP26 was transformed into P. fluorescens cLP6a-1 to ensure that the tetracycline resistance gene for tetracycline efflux on the vector did not affect the complementation results. There was not a change in the amount of cell-associated phenanthrene in P. fluorescens cLP6a-1(pUCP26) after azide addition (Fig. (Fig.4),4), and the levels of phenanthrene were comparable to those in mutant cLP6a-1, within the error typically observed for the assay. Thus, the increased accumulation of phenanthrene observed in P. fluorescens cLP6a-1(pBH5) upon azide addition resulted from inhibition of the restored Emh efflux system.

FIG. 4.
Distribution of radiolabeled substrates in the cell pellets of P. fluorescens cLP6a strains at steady state before (open bars) and after (solid bars) addition of 30 mM azide. The fractional amounts of phenanthrene, anthracene, and fluoranthene in the ...

EmhABC efflux system exhibits substrate specificity.

Since it was shown previously that anthracene and fluoranthene are also subject to active efflux in P. fluorescens cLP6a (3), the involvement of the emhABC genes in the efflux of these polycyclic aromatic hydrocarbons was investigated. Transport assays were performed by using radiolabeled anthracene or fluoranthene and P. fluorescens cLP6a, cLP6a-1, cLP6a-1(pBH5), or cLP6a-1(pUCP26). Figure Figure44 compares the steady-state levels of these substrates for the four strains in the absence and in the presence of azide. As observed with phenanthrene, inactivation of the emhB gene in P. fluorescens cLP6a-1 caused accumulation of cellular levels of anthracene and fluoranthene before azide addition that were higher than the levels in the P. fluorescens cLP6a cells (P < 0.025). However, the fractional amounts of anthracene and fluoranthene increased significantly after azide treatment of cLP6a-1 cells, suggesting that there are additional active efflux mechanisms for these hydrocarbons. Complementation of the emhB disruption mutant with pBH5 restored the level of accumulated hydrocarbon to the levels in unmodified strain cLP6a. As a control, transport of anthracene and fluoranthene was measured in P. fluorescens cLP6a-1(pUCP26) and was expected to be comparable to transport in the mutant cLP6a-1. Although the fractional amounts of anthracene and fluoranthene after azide addition were similar for the two strains, a higher level of hydrocarbon accumulated prior to azide addition in the vector control cLP6a-1(pUCP26) than in cLP6a-1 (Fig. (Fig.4).4). The presence of plasmid pUCP26 seemed to affect the residual anthracene and fluoranthene efflux activity observed in mutant cLP6a-1. However, these results do not contradict the hypothesis that EmhABC is involved in anthracene and fluoranthene efflux, as indicated by the data obtained with P. fluorescens cLP6a-1 and cLP6a-1(pBH5).

Bugg et al. (3) found that P. fluorescens cLP6a did not possess an active efflux mechanism for naphthalene; however, the limitations of the transport assay in which radiolabeled toluene was used precluded a definitive conclusion regarding the active efflux of toluene. In the present study, the maximum concentration of toluene that allowed growth of both P. fluorescens cLP6a and cLP6a-1 was found to be 0.08% (vol/vol) or approximately one-third of the aqueous solubility limit. P. fluorescens cLP6a, therefore, is not a solvent-tolerant bacterium, and the lack of solvent tolerance, combined with the data of Bugg et al. (3), provides strong evidence that P. fluorescens cLP6a does not have an active efflux system for toluene.

The possibility that the EmhABC system might also export antibiotics was evaluated by comparing the antibiotic susceptibilities of the four strains. Antibiotics that are known substrates of efflux systems in P. aeruginosa were chosen for testing. Table Table22 shows that sensitivity to chloramphenicol and nalidixic acid was affected by disruption of the emhB gene in P. fluorescens cLP6a-1, since the MICs were eight times higher for P. fluorescens cLP6a than for the disruption mutant cLP6a-1. Complementation of the emhB disruption restored resistance to chloramphenicol and nalidixic acid. In contrast, MICs of tetracycline, erythromycin, trimethoprim, and streptomycin for cLP6a and cLP6a-1 were comparable.

TABLE 2.
Antibiotic sensitivities of P. fluorescens strains

emh promoter region is not activated by aromatic hydrocarbons.

To determine if the emhABC efflux genes are induced by aromatic hydrocarbons, the reporter plasmid pTZ110-emh was constructed by inserting the putative promoter region between the emhA and emhR genes adjacent to the lacZ gene. P. fluorescens cLP6a strains carrying either the promoter-reporter plasmid pTZ110-emh or the control plasmid pTZ110-str were grown in the presence of aromatic hydrocarbons. β-Galactosidase activity measurements (Table (Table3)3) revealed that there was no significant induction of the lacZ reporter gene either by the efflux pump substrates phenanthrene, anthracene, and fluoranthene or by the nonsubstrates toluene and naphthalene (P > 0.2 for a comparison with the control cells grown in the absence of hydrocarbon).

TABLE 3.
Effects of hydrocarbons on expression of β-galactosidase activity in P. fluorescens cLP6a(pTZ110-emh) and cLP6a(pTZ110-str)

Polycyclic aromatic hydrocarbon efflux is found in other bacteria.

Table Table44 shows that the ability to transport phenanthrene with an active efflux system is not unique to P. fluorescens cLP6a. Several P. putida strains, including P. putida G7, which harbors the NAH7 plasmid encoding naphthalene degradation genes, P. putida mt-2, which carries the TOL plasmid pWW0 encoding toluene degradation genes, P. putida KT2440, a cured derivative of P. putida mt-2 lacking the TOL plasmid, and P. putida S12, a solvent-tolerant strain, showed increases in the cellular accumulation of phenanthrene upon addition of azide, which was indicative of inhibition of an active efflux process. Three P. aeruginosa strains, including type strain PAO1 and two clinical isolates, and two B. cepacia strains, an environmental isolate and a clinical isolate, also have mechanisms for phenanthrene efflux. As shown previously (3), only A. vinelandii UW did not show evidence of an active efflux mechanism for phenanthrene when it was grown in Burke's buffer under iron-rich conditions (Table (Table4),4), in Burke's buffer under iron-limited conditions, or in tryptic soy broth (data not shown).

TABLE 4.
Accumulation of phenanthrene in bacterial strains before and after azide addition, as a test for efflux

DISCUSSION

When studying the transport of polycyclic aromatic hydrocarbons by the environmental isolate P. fluorescens cLP6a, Bugg et al. (3) observed an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. The unexpected selectivity of this transport system prompted identification of the emhABC gene cluster described here (Fig. (Fig.2).2). Disruption of the emhB gene in P. fluorescens cLP6a-1 proved that the gene product was responsible for the proton-dependent efflux of phenanthrene (Fig. (Fig.11 and and4).4). The EmhB protein also is involved in the active efflux of anthracene and fluoranthene, although additional active efflux mechanisms may be present in P. fluorescens cLP6a to transport these hydrocarbons since disruption of emhB did not completely eliminate the efflux (Fig. (Fig.4).4). Multiple RND efflux pumps have been identified in a draft analysis of the P. fluorescens PfO-1 genome sequence (http://www.jgi.doe.gov/JGI_microbial/html/pseudomonas/pseudo_homepage.html); therefore, it is probable that P. fluorescens cLP6a also possesses more than one efflux system.

The deduced protein sequences encoded by the emhA, emhB, and emhC genes showed a high degree of homology to membrane fusion proteins, RND efflux pumps, and outer membrane proteins of three-component efflux systems in gram-negative bacteria. Although it has not been verified, the homology of the emh genes to the ttgABC and mexAB-OprM efflux operons strongly suggests that the emhA, emhB, and emhC genes form an operon. EmhB is closely related to the TtgB efflux pump, which transports antibiotics and solvents in P. putida DOT-T1E (23), and to the ArpB antibiotic efflux pump in P. putida S12 (11) (Fig. (Fig.3).3). The RND pump MexB in P. aeruginosa, which is involved in the efflux of tetracycline, chloramphenicol, β-lactams, quinolones, and macrolides (17) and in tolerance to the toxic organic solvents hexane and xylene (14), is also highly homologous to EmhB (Fig. (Fig.3).3). Based on the protein sequence homology, it was hypothesized that EmhABC might be involved in antibiotic efflux. The EmhABC system seems to contribute to chloramphenicol and nalidixic acid resistance in P. fluorescens cLP6a (Table (Table2),2), but it does not show the broad substrate specificity for antibiotics that is characteristic of MexAB-OprM, ArpABC, and TtgABC. Multidrug efflux pumps in pseudomonads and other organisms are notorious for transporting structurally unrelated compounds. Therefore, it is not surprising that the EmhABC system in P. fluorescens cLP6a recognizes polycyclic aromatic hydrocarbons as well as chloramphenicol and nalidixic acid, but its limited substrate range is significant. In addition, the inability of polycyclic aromatic hydrocarbons to activate the promoter region of the emhABC genes (Table (Table3)3) is unusual and suggests that the EmhABC efflux system may play another role in P. fluorescens cLP6a. The narrow substrate specificity of the EmhABC efflux pump, coupled with the sensitivity of P. fluorescens cLP6a to typical substrates of multidrug efflux pumps, should be advantageous for studying the substrate-binding and transport mechanism of this RND system.

Polycyclic aromatic hydrocarbons represent a new class of substrates that are recognized and transported by members of the RND superfamily. The presence of an efflux system for polycyclic aromatic hydrocarbons, however, is not unique to P. fluorescens cLP6a. Phenanthrene is actively exported by P. putida, P. aeruginosa, and B. cepacia strains (Table (Table4).4). Phenanthrene efflux is independent of the ability to degrade hydrocarbons, since it has been observed in toluene, naphthalene, and polycyclic aromatic hydrocarbon degraders, as well as in traditionally nondegrading species. Additionally, the location of the emhABC genes, on the chromosome rather than on the plasmid carrying the hydrocarbon degradation genes, in P. fluorescens LP6a strengthens the argument that hydrocarbon efflux and degradation are not linked. Interestingly, A. vinelandii did not show active efflux of phenanthrene despite having an RND pump gene (nucleotide accession number NZ_AAAD01000072) whose protein sequence is 77% identical to that of EmhB. The efflux system in A. vinelandii may recognize a different spectrum of substrates than EmhABC, or it may not have been expressed in A. vinelandii under the growth conditions tested. Since the efflux system and its regulation in A. vinelandii have not been characterized, it is difficult to draw conclusions about the lack of observed phenanthrene efflux in A. vinelandii.

The polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene recognized by the EmhABC efflux system differ from the compounds typically exported by multidrug efflux pumps in that they are not toxic to bacterial cells. While MexAB-OprM has been found to participate in the efflux of some homoserine lactones involved in quorum sensing (21), the major role of efflux systems in pseudomonads appears to be the removal of toxic compounds from the cell. It has been shown that expression of the toluene and antibiotic efflux pumps is induced by their substrates (6, 13), which provides support for the hypothesis that their primary function is detoxification. The presence of the adjacent emhR gene suggests that expression of the emhABC genes is regulated, but the results obtained with the promoter-lacZ reporter plasmid indicated that expression of the efflux pump genes is not activated by aromatic hydrocarbons. Further analysis of the regulation and substrate specificity of EmhABC should provide insight into the role of this efflux system in P. fluorescens cLP6a, as well as into the function of this important family of transport proteins.

Acknowledgments

We thank P. Y. Law for her assistance in constructing the promoter-reporter plasmids.

This work was supported by the Natural Sciences and Engineering Research Council.

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