Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen Agricultural University, PO Box 8129, 6700 EV Wageningen, The Netherlands1
Author for correspondence: Jasper Kieboom. Tel: +31 317 484412. Fax: +31 317 484978. e-mail: jasper.kieboom{at}imb.ftns.wau.nl
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ABSTRACT |
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Keywords: solvent tolerance, multidrug resistance, efflux
Abbreviations: RND family, resistance/nodulation/cell division family
The GenBank accession number for the arp sequence is AF183959.
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INTRODUCTION |
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In the solvent-tolerant P. putida strain S12 it was shown that the energy-dependent efflux of organic solvents was the key factor in organic solvent tolerance via the organic solvent transporter SrpABC (Kieboom et al., 1998a ). Similar efflux systems for the active removal of organic solvents have been found in other P. putida strains. These RND-type efflux systems, encoded by ttgABC (Ramos et al., 1998
), ttgDEF (Mosqueda & Ramos, 2000
) and mepABC (Fukumori et al., 1998
), are involved in the active efflux of toxic compounds such as toluene, p-xylene and styrene. In the case of the multidrug-resistant P. aeruginosa four efflux systems have been described: MexAB-OprM (Poole et al., 1993
), MexCD-OprJ (Poole et al., 1996
), MexEF-OprM (Kohler et al., 1997
) and AmrAB (Westbrock-Wadman et al., 1999
). These systems contribute to the energy-dependent efflux of a wide variety of antimicrobial agents such as ß-lactams, tetracycline, fluoroquinolones and chloramphenicol. The interesting question now arises whether the RND-type transporters are able to export both antibiotics and solvents. Indications so far confirm that these pumps have dual pumping capacity. The mex-encoded efflux systems have recently been shown to be involved in the efflux of organic solvent in P. aeruginosa (Li et al., 1998
). Moreover, solvent-sensitive mutants of P. putida DOT-T1E and P. putida KT2442 became more sensitive to antibiotics such as tetracycline, chloramphenicol and ampicillin, suggesting the active removal of multiple antibiotics by these efflux systems (Ramos et al., 1998
; Fukumori et al., 1998
).
We have now studied this aspect at the molecular level and have found a new efflux system in P. putida S12 that is involved in the intrinsic resistance of this strain to a wide variety of structurally unrelated antibiotics. Sequence analysis showed that this efflux system is highly homologous to the solvent/antibiotic transporters MepABC and TtgABC. Moreover, we demonstrate that ArpABC in P. putida S12 is only involved in multidrug resistance and not in tolerance towards organic solvents.
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METHODS |
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DNA techniques.
Total genomic DNA from P. putida strains was prepared by the CTAB procedure (Ausubel et al., 1991 ). Plasmid DNA was isolated by the alkaline SDS lysis method of Birnboim & Doly (1979)
. DNA was digested with restriction enzymes and ligated with T4 ligase as recommended by the supplier (Life Technologies). DNA restriction fragment and PCR products were visualized by 0·8% agarose gel electrophoresis in 45 mM Tris/borate, 1 mM EDTA buffer. DNA from agarose gels was isolated using the method of Vogelstein & Gillespie (1979)
. Southern analyses of chromosomal DNA and colony hybridizations were carried out according to Maniatis et al. (1982)
. Probes were labelled with DIG-dUTP using the PCR DIG Probe Synthesis Kit (Boehringer Mannheim) with the appropriate primers. Southern blot hybridizations were carried out by chemiluminescent detection under high-stringency conditions as described by the supplier (Boehringer Mannheim). Plasmid DNA was introduced into either E. coli DH5
or P. putida cells by electroporation (Dennis & Sokol, 1995
) using a Gene Pulser (Bio-Rad).
DNA sequences of both strands of the arpABC operon were determined by a combination of subcloning and primer walking. All sequencing and PCR reactions were performed using a Gene Amp PCR System 9700 (Perkin-Elmer). Nucleotide sequencing reactions were performed with purified double-strand plasmid DNA using AmpliTaq FS DNA polymerase fluorescent dye terminator reactions (Perkin-Elmer) as recommended by the supplier. Sequencing products were detected using an Applied Biosystems 373A stretch automated DNA sequencer. Nucleotide sequence analysis was performed either with the Lasergene analysis package (DNASTAR) or with the National Centre for Biotechnology Information BLAST server (Altschul et al., 1990 ).
Construction of pCMC2 for complementation.
For the complementation experiments we reconstructed the arpABC operon since the clones obtained from P. putida CM1 and CM2 contain the transposon-mutagenized DNA. Therefore, a 2·2 kb BamHIPstI fragment from pCM1B was cloned into the broad-host-range vector pUCP22; the resulting plasmid was designated pCMC1. A 2·3 kb PstIEcoRI fragment from pCM2P and a 2·0 kb EcoRIPstI fragment from pCM1P were ligated into PstI-digested and alkaline-dephosphatase-treated pCMC1. The resulting plasmid, pCMC2, contained the arpABC genes in the same orientation as the lac promoter and was transferred to the mutant strains CM1 and CM2.
Construction of suicide plasmid pSC1.
We amplified a 1289 bp fragment containing the tetracycline-resistance gene tetA from pACYC184. The primers 5'-CGGAATTCTCATGTTTGACAGCT-3' and 5'-GCGGTACCTCAGGTCGAGGTGG-3' contain added recognition sites for EcoRI and KpnI, respectively. The reaction mixture (50 µl) was treated for 10 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 2 min at 55 °C and 1 min at 72 °C before finishing for 10 min at 72 °C. This 1·3 kb EcoRIKpnI fragment and a 0·7 kb KpnISstI fragment from TnMod-KmO, containing the ColE1 origin of replication, were ligated in EcoRI- and SstI-digested pBBR1MCS, resulting in pTO1. The suicide plasmid pSC1 was constructed by ligating a 2·0 kb EcoRISstI fragment from pTO1, a 3·0 kb PstISstI fragment from pCM1P and a 3·0 kb PstIEcoRI fragment from pCMPE.
Construction of pKRZ-arp and determination of ß-galactosidase activity.
PCR reactions for amplifying the region of genomic P. putida S12 DNA containing the arp promoter were performed using Super Taq DNA polymerase (SphearoQ). The reaction mixture (50 µl) was treated for 10 min at 94 °C followed by 35 cycles of 1 min at 94 °C, 1 min at 58 °C and 1 min at 72 °C before finishing for 10 min at 72 °C. Primers for this reaction were 5'-CCGCTCGAGTACAACCTCATCTGGCCC-3' and 5'-CGCTCTAGAATTGCATGAGGATCCTCG-3', which amplified a 276 bp fragment corresponding to the region immediately upstream of the arpABC genes. The primers contain added recognition sites for XhoI and XbaI, respectively. ß-Galactosidase activity in P. putida S12(pKRZ-arp) was determined by growing the cells to late-exponential phase at 30 °C in 10 ml LB broth supplemented with inducer. The ß-galactosidase activity in these experiments was determined in triplicate by the method of Miller (1972) using chloroform and SDS to permeabilize the cells.
Determination of solvent tolerance and the MIC of antibiotics.
Solvent tolerance of the P. putida strains was determined in duplicate by growing the cells in 10 ml liquid LB medium in 100 ml flasks supplemented with 1 mM magnesium chloride. Toluene and p-xylene (1% final concentration) were separately added to identical subcultures in LB/MgCl2 medium during the early exponential growth phase. The maximal aqueous benzene concentration was determined by adding increasing amounts of benzene. Growth of the cultures was measured 24 h after solvent addition with no continued growth indicating solvent sensitivity. The MIC for various antibiotics was determined in duplicate by threefold serial dilution in LB broth in microtitre plates. The inoculum was 1% of an overnight culture and growth was determined by measuring the OD600 after 36 h at 30 °C.
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RESULTS |
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Cloning and analysis of the genes for chloramphenicol resistance
To characterize the genes for chloramphenicol resistance in P. putida S12, the regions of the genome containing the transposon insertion in mutants CM1 and CM2 (both 5·8 kb) were cloned. From both mutants a clone was obtained from PstI-digested chromosomal DNA; the clones were designated pCM1P and pCM2P, respectively. Genomic DNA cut with BamHI from strain CM1 was used to construct the 14·0 kb plasmid pCM1B. Plasmid pCM1B was shown to contain a complete operon, including the inserted TnMod-KmO (Fig. 1). The nucleotide sequence of the operon was determined and screening for similar nucleotide sequences in the GenBank database revealed a significant match with genes encoding multidrug and solvent export pumps. We labelled the genes arp, for antibiotic resistance pump. A diagram of the nucleotide sequence obtained is presented in Fig. 1
to show the relationship of the open reading frames with the three clones pCM1P, pCM2P and pCM1B. The deduced nucleotide sequences of arpA, arpB and arpC encode proteins of 371, 1050 and 484 amino acid residues with calculated molecular masses of 40·3, 112·8 and 52·8 kDa, respectively. Putative ribosome-binding sites precede the arpABC genes and a stable stem-loop structure was found downstream of arpC that may function as transcriptional terminator (Fig. 2
).
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Complementation of P. putida mutants
Complementation experiments were performed to prove that the TnMod-KmO-inserted open reading frames detected in P. putida CM1 and CM2 are actually responsible for chloramphenicol resistance. These complemented strains regained chloramphenicol resistance similar to the wild-type levels (Table 2). These results are consistent with the chloramphenicol-resistance phenotype being dependent on expression of the arpABC genes. We tested a variety of structurally unrelated antibiotics, which are known substrates for homologous efflux systems of the RND family of transporters (Nikaido, 1996
; Paulsen et al., 1996
). Mutants CM1 and CM2 were not only sensitive to chloramphenicol but also to a number of other antibiotics (Table 2
), demonstrating that a single genetic trait in P. putida S12 is responsible for resistance to the antibiotics tested.
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DISCUSSION |
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Proton-dependent efflux systems also play an important role in organic solvent tolerance in P. putida strains (Kieboom et al., 1998a ; Ramos et al., 1998
; Fukumori et al., 1998
). The first RND-type efflux system for toluene was isolated by Kieboom et al. (1998)
and was shown to be responsible for organic solvent tolerance in P. putida S12 (Kieboom et al., 1998a
). The involvement of efflux systems in solvent tolerance was confirmed with the isolation of the ttgABC, ttgDEF genes from P. putida DOT-T1E (Ramos et al., 1998
; Mosqueda & Ramos, 2000
) and the mepABC genes from P. putida KT2442 (Fukumori et al., 1998
). Moreover, Kim et al. (1998)
reported that a transposon insertion in a protein of the RND family resulted in a P. putida mutant with a solvent-sensitive phenotype. Surprisingly, other studies suggested that the isolated solvent transporter in P. putida was also involved in the active efflux of multiple antibiotics (Ramos et al., 1998
; Fukumori et al., 1998
). A toluene-sensitive mepB
Tn5 mutant of a P. putida KT2442 was also sensitive to ampicillin, penicillin G, erythromycin, novobiocin and tetracycline (Fukumori et al., 1998
); and a toluene-sensitive ttgB
mini-Tn5'phoA-Kmr mutant of P. putida DOT-T1E was sensitive to chloramphenicol, ampicillin and tetracycline (Ramos et al., 1998
). With the construction of a srp-arp double mutant we were able to demonstrate that the ArpABC efflux system was not involved in organic solvent tolerance in P. putida S12.
An interesting, but as yet not completely clear, picture is now emerging from studies on the efflux pumps of pseudomonads with regard to both their substrate recognition and their induction patterns. The inherent problem in studying these aspects is that responses to doses of antibiotics or organic solvents must be monitored at the whole-cell level. Both wild-type cells and mutants may or may not contain additional, and often unknown, pumps. Moreover, the rate of influx of compounds will depend on their chemical structure, while the cell may alter the composition of its cell envelope, thus further obscuring the explanation of results. Nevertheless, on the basis of this type of experimentation it is now possible to distinguish three types of proton-dependent efflux systems in pseudomonads on the basis of compounds expelled from the cell. To date two efflux pumps have been described that are solely involved in the efflux of solvents and do not appear to be able to export antibiotics. This type of efflux pump includes SrpABC in P. putida S12 and TtgDEF in P. putida DOT-T1E and was shown to be induced by organic solvents (Kieboom et al., 1998a ; Mosqueda & Ramos, 2000
). Most of the efflux systems characterized in pseudomonads, however, export both antibiotics and solvents. These systems include the constitutive TtgABC pump in P. putida DOT-T1E (Ramos et al., 1998
), the MepABC pump in P. putida KT2442 (Fukumori et al., 1998
) and the Mex efflux systems in P. aeruginosa (Li et al., 1998
). The third type is the ArpABC system reported here, which is involved in antibiotic resistance but not in solvent tolerance as shown by the phenotype of the srp-arp double mutant. However, a note of caution in identifying ArpABC as an antibiotic-removing pump and not as a solvent-removing system is appropriate. A solvent pump has to operate at a higher speed than an antibiotic pump due to the considerably higher influx of hydrophobic solvents compared to the influx of antibiotics. Toluene may be present at 5 mM in the aqueous phase, while antibiotics usually remain below 1 mM and it is to be expected that antibiotics will diffuse more slowly into the cell than do uncharged, small solvent molecules. Consequently, solvent efflux pumps may have to generate an efflux that is probably 10100 times higher than the efflux created by the antibiotic pumps. If the expression level is not affected by the presence of solvent as substrate, as is the case for the ArpABC system, then the solvent-pumping ability may not be registered at the whole-cell level. We have tried to overcome this pitfall by using benzene as test solvent and by employing it at different concentrations. This method should be relatively suitable for detecting minor contributions to solvent pumping, but nevertheless, no effect of the presence of ArpABC on benzene sensitivity was observed.
The grouping of the known Pseudomonas efflux pumps according to their pumping activity for either solvent and/or antibiotics is not supported by the molecular structure of these pumps. The ArpB, TtgB and MepB proteins are almost identical at the amino acid level, which is quite surprising because they have been identified in strains isolated from three very different locations. Also, their A and C components are almost identical. ArpB differs only in one amino acid from TtgB (Ala-544 to Gly-544), while compared to MepB it differs in three amino acids (Ala-533 to Thr-533, Ala-544 to Gly-544 and Glu-692 to Ala-692). On the basis of the MexB membrane topology (Guan et al., 1999 ) it is expected that Ala-544 is located in a putative transmembrane segment, whereas Ala-544 and Glu-692 are periplasmic. These observations would lead to the conclusion that Gly-544 might be a key amino acid in interactions with antibiotics. However, as yet the substrate-binding domain of RND-type proteins has not been identified.
Alternatively, it might be argued that MepABC and TtgABC are present at higher levels in their respective hosts. For P. putida KT2442 it was anticipated that the mep operon was overexpressed in the toluene-tolerant variant TOL. Possibly a similar adaptation phenomenon has taken place already during the isolation of strain P. putida DOT-T1E. Therefore, in both strains, a high transcription level of the efflux operons may result in a high solvent efflux, required for the cell to survive in the presence of toluene. Similar results were reported for the mexAB-oprM efflux system in P. aeruginosa. In this strain a single substitution in the mexR regulator resulted in the overexpression of MexAB-OprM resulting in an increased multidrug resistance and organic solvent tolerance (Poole et al., 1996 ; Li et al., 1998
; Li & Poole, 1999
).
In summary, the intrinsic resistance of P. putida S12 to multiple antibiotics is due to efflux of these components by ArpABC. Whether ArpABC in P. putida S12 is unable to transport solvent due to an amino acid substitution or due to the lack of overexpression, the extrusion of organic solvents by this efflux system is too low to prevent their influx but sufficiently high to combat the antibiotic influx. P. putida S12 does not have to rely on ArpABC-mediated solvent efflux for its solvent-tolerant phenotype, since it possesses the SrpABC efflux system that is induced under solvent stress.
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Received 17 May 2000;
revised 25 August 2000;
accepted 19 September 2000.