INTRODUCTION

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Organic solvents with a partition coefficient in an octanol-water mixture (log P_ow) between 1.5 and 3 are extremely toxic to microorganisms (34). Organic solvents cause irreversible lesions to cell membranes (loss of ions, metabolites, lipids, and proteins, dissipation of the pH gradient and electrical potential, etc.) followed by cell lysis and death (5, 36, 37).

Several Pseudomonas putida strains have been isolated as able to grow in the presence of high concentrations of toxic organic solvents, such as toluene, styrene, and p-xylene (4, 10, 11, 31, 40). Tolerance to organic solvents in these P. putida strains is mainly achieved by a series of efflux pumps that actively remove the organic solvent from the cell membranes (10-14, 19, 25, 32, 33, 36). These efflux pumps, which belong to the resistance-nodulation-division (RND) family, are formed by a translocase made of an inner membrane transporter and a periplasmic fusion protein anchored in the inner membrane plus an outer membrane protein that spans the periplasm and forms a tunnel. It has been proposed that this tunnel is contacted by the cytoplasmic membrane pump, presumably assisted by the inner membrane-periplasmic fusion protein to facilitate the direct removal of drugs across the two membranes and the intervening periplasmic space (17). Evidence for this mode of operation has been obtained with antibiotic efflux pumps in Escherichia coli (17, 46) and Pseudomonas aeruginosa (43, 44).

We previously identified two efflux pumps that expel toluene in the solvent-tolerant P. putida DOT-T1E strain (25, 33). The TtgABC pump is synthesized at a high basal level in cells growing in the absence of toluene and to a significant but lower level in cells growing with toluene (6). The TtgABC pump extrudes solvents and several antibiotics (33). The other pump, called TtgDEF, is highly homologous to TtgABC (70% similarity at the protein level) and is able to expel toluene but not antibiotics (25). The ttgDEF genes are expressed in response to aromatic hydrocarbons in the culture medium, and the genes are linked to the chromosomal tod genes (24, 25), which encode the enzymes of the so-called toluene dioxygenase pathway and which allow the strain to grow with toluene as the sole C source.

In the wild-type P. putida DOT-T1E strain, toluene tolerance is influenced by growth conditions. Preexposure of cells to low concentrations of this aromatic hydrocarbon led to survival of almost 100% of the cells after a sudden toluene shock; in contrast, only a fraction (10⁴) of cells that had not been preexposed survived (33). The TtgABC pump seemed to contribute to the innate tolerance of P. putida DOT-T1E to solvents, and both the TtgABC and TtgDEF pumps contributed to solvent tolerance when cells were preinduced (25). P. putida strain DOT-T1E-82 is a derivative of the wild type with knockouts in the ttgB and ttgD genes. Inactivation of the TtgABC and TtgDEF pumps in this strain still allowed the survival of 10⁵ cells if they were preexposed to low concentrations of toluene, although none (<10⁸) survived without induction. This result suggests the presence of at least one undiscovered pump.

We now report the identification of a third toluene efflux pump in P. putida DOT-T1E and show that inactivation of this pump in the double mutant described above produced a strain that was not only unable to stand sudden toluene shock, regardless of the growth conditions, but was also unable to grow in the presence of toluene even when it was supplied via the gas phase.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, plasmids, and culture medium. The bacterial strains and plasmids used in this study are shown in Table 1. Bacterial strains were routinely grown in liquid Luria-Bertani (LB) medium (35). E. coli cultures were incubated at 37°C, while P. putida cultures were incubated at 30°C. Cultures were shaken on an orbital platform operating at 200 strokes per min. When P. putida cultures were supplied with toluene via the gas phase we used culture flasks with a central vessel in which 0.15 ml of toluene was introduced to avoid direct contact with the liquid medium. The flasks were closed tightly with a Teflon screw top. Under these culture conditions the concentration of toluene in the liquid medium was about 2 mM.

DNA techniques. Preparation of chromosomal DNA, digestion with restriction enzymes, electrophoresis, and Southern blotting were carried out using standard methods (3, 35). Hybridizations were done using a digoxigenin (DIG) DNA labeling and detection kit (Boehringer Mannheim) in accordance with the manufacturer's instructions. For plasmid isolation a Qiagen kit was used. Plasmid DNA was sequenced on both strands with universal, reverse, or specifically designed primers using an automatic DNA sequencer (ABI-PRISM 310; Applied Biosystems, Inc.).

Primer extension analysis. P. putida DOT-T1E was grown overnight in LB medium. Cells were then diluted 100-fold in fresh medium, and aliquots were incubated in the absence or presence of toluene supplied via the gas phase until the culture reached a turbidity of about 1.0 at 660 nm. Cells (30 ml) were pelleted and processed for RNA isolation according to the method of Marqués et al. (23). About 20 µg of RNA/ml was treated with RNase-free DNase I and RNase inhibitor (Boehringer Mannheim) to ensure the complete removal of DNA and to maintain the integrity of mRNA. The primer used for extension was complementary to the ttgG gene. This primer was labeled at its 5' end with [-³²P]ATP and T4 polynucleotide kinase as described before (3). About 10⁵ cpm of the labeled primer was hybridized to 20 µg of total RNA, and extension was carried out as described previously (3, 23). Electrophoresis of cDNA products was done in a urea-polyacrylamide sequencing gel to separate the reaction products, and dry gels were exposed to X-ray films and visualized. The relative intensity of the bands was quantitated using the Molecular Imagen System GS 525 (Bio-Rad) with the multianalyst program (3, 23).

Cloning of the pump genes and construction of the mutant strains. To check if a gene homologous to the P. putida srpB gene was present in the genome of P. putida DOT-T1E, a PCR with primers designed based on the srpB gene sequence was performed in 25 µl using 1 µg of DOT-T1E chromosomal DNA/ml as a template. The PCR conditions were 94°C for 1 min, 60°C for 1 min, and 72°C for 30 s for 30 cycles. This yielded a 565-bp fragment that was labeled with DIG by standard procedures and used to hybridize a DOT-T1E genomic library previously generated in our laboratory in pUC18 (33). A positive clone, named pGG1, was obtained with a BamHI insert of 7,674 bp. This clone was digested with EcoRV (which cuts once in the plasmid within the ttgH gene) and ligated to a 2.2-kb SmaI fragment from pHP45-Sm containing the STR resistance cassette to yield pGG2 (Ap^r, Sm^r).

MIC assays. The assays were done in LB medium by the microtiter broth dilution method (2). Microtiter plates were inoculated with 10⁵ CFU/ml and incubated for 20 h at 30°C.

Survival in response to toluene shocks. Cells were grown in 30 ml of LB medium with or without toluene in the gas phase overnight. On the following day the cultures were diluted 1:100 and grown under the same conditions until the culture reached a turbidity of about 0.8 at 660 nm. These cultures contained about 10⁸ CFU/ml. The cultures were divided into two halves; to one we added 0.3% (vol/vol) toluene, and the other was kept as a control. The number of viable cells was determined before toluene was added and 10, 30, and 60 min later.

RT-PCR. P. putida DOT-T1E cells (5 ml) growing exponentially on LB medium were harvested by centrifugation (5,000 × g for 10 min). RNA from P. putida DOT-T1E was isolated with the RNeasy Total RNA kit. About 100 µg of total RNA was treated with RNase-free DNase I (50 U) in the presence of 3 U of an RNase inhibitor (Boehringer Mannheim) to avoid DNA contamination and RNA degradation. Reverse transcriptase (RT)-PCR was performed with 1 µg of RNA/ml in a 20-µl final volume using the Titan OneTube RT-PCR system according to the manufacturer's instructions (Boehringer Mannheim). The annealing temperature used for the PCR was 60°C, and the cycling conditions were as follows: 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min. Positive and negative controls were included in all assays. The primers used to test contiguity in the mRNA of the ttgG and ttgH genes and the ttgH and ttgI genes are available upon request.

Computer analysis. Open reading frames (ORFs) in DNA sequences were predicted with various programs included in the DNA Strider 1.1 package. Sequences were compared with the BlastX programs (1) available from the National Institute for Biotechnology Information server. Protein sequences were aligned with the multiple sequence alignment Clustal W program (38).

Nucleotide sequence accession number. The nucleotide sequences of the efflux system genes and adjacent DNA described here have been submitted to the GenBank/EMBL data bank under accession number AF299253.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Cloning of the ttgGHI genes of P. putida DOT-T1E. SrpABC is an inducible efflux pump involved in solvent tolerance described in P. putida S12 (12, 13, 41). Sequence comparison of the SrpABC proteins with those of the TtgABC and TtgDEF pumps identified in DOT-T1E as involved in toluene tolerance revealed 54 to 70% similarity. Because our previous data pointed towards the involvement of a third inducible pump in solvent tolerance in P. putida DOT-T1E, we tested whether the srpABC genes or their homologues were present in P. putida DOT-T1E. To this end we designed two oligonucleotides based on the srpB gene sequence of P. putida S12 and used them in a PCR with P. putida DOT-T1E chromosomal DNA as the template (see Materials and Methods). A 565-bp fragment was amplified, sequenced, and compared with the srpB sequence. The amplified DNA fragment sequence differed by only one nucleotide among the 565 bp.

Knockout of the ttgH gene in different P. putida DOT-T1E backgrounds resulted in mutants that are extremely sensitive to toluene. To determine the function of the TtgGHI proteins in solvent tolerance we decided to generate a knockout of the ttgH gene by using the suicide plasmid pGG2, which carries an -Sm cassette (30) within the ttgH gene (see Materials and Methods). The mutation was transferred to the chromosome of the wild-type strain as described above, and a mutant strain was selected and called DOT-T1E-PS28. Mutant DOT-T1E-PS28 grew on LB medium with a generation time similar to that of the wild-type strain (50 ± 2 min), but in contrast to the wild type, it did not grow in LB medium supplemented with 0.3% (vol/vol) toluene (log P_ow = 2.5). However, DOT-T1E-PS28 did grow in LB medium supplemented with 0.3% (vol/vol) m-xylene (log P_ow = 3.2), ethylbenzene (log P_ow = 3.4), propylbenzene (log P_ow = 3.6), and heptane (log P_ow = 4.5), which suggests an important role for the TtgGHI efflux pump in toluene tolerance.

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Contribution of efflux pumps to the extrusion of different solvents. P. putida DOT-T1E is able to grow in the presence of a number of organic solvents with a log P_ow between 3.6 and 2.9, i.e., propylbenzene, m-xylene, ethylbenzene, and styrene. The availability of single, double, and triple mutants for the Ttg pumps allowed us to assess the involvement of these efflux pumps in the extrusion of other solvents. Wild-type and mutant cells were grown in LB medium in the absence (noninduced) and presence (induced) of toluene (except for DOT-T1E-PS34, which is not viable when exposed to toluene) and then challenged with 0.3% (vol/vol) concentrations of different solvents; survival was determined after 10 min (Table 2). We found that noninduced cells of the single mutants lacking the TtgABC or TtgGHI pumps were hypersensitive to styrene, and sensitivity was exacerbated in a double ttgABC ttgGHI mutant (Table 2). These results suggest that these two pumps were involved in styrene efflux. The mutant lacking the TtgDEF pump was more sensitive than the wild type to styrene under induced conditions, suggesting that this pump plays a role in styrene efflux as well. In fact, toluene-induced cells of the single ttgABC and ttgGHI mutants were more tolerant to a sudden styrene shock than noninduced ones. Furthermore, double mutants of TtgDEF with either TtgABC or TtgGHI were more susceptible to the styrene shock than the single mutants, an observation which confirms the role of TtgDEF in styrene efflux.

The ttgGHI operon is expressed constitutively, but its expression is enhanced by the presence of toluene in the culture medium. We determined the pattern of expression of the ttgGHI genes in wild-type cells grown on LB medium with and without toluene supplied via the gas phase. Primer extension analysis identified a 213-bp cDNA in cells that were not exposed to toluene, whereas two cDNA bands, of 213 and 218 bp, were found in cells exposed to toluene. It then follows that in the absence of the aromatic hydrocarbon, some basal expression of the ttgGHI operon takes place which increases due to an inducible promoter in cells exposed to toluene. The intensity of the 213-bp cDNA band was three- to fourfold higher in cells grown with toluene than in cells grown in the absence of this aromatic hydrocarbon (Fig. 1A). The intensity of the 218-bp cDNA band was half the intensity of one produced by the 213-bp cDNA in induced cells (Fig. 1A, lane 2). The pattern of expression of the ttgGHI genes explains why the TtgGHI pump is a key element in the innate and inducible efflux of toluene. We observed that the ttgGHI operon was expressed at relatively high levels from a single promoter in noninduced cells and that expression of ttgGHI in induced cells took place at a higher level than in noninduced cells, because in addition to the increase in the levels of expression from the constitutive promoter, expression of this operon also occurred from a second inducible promoter that overlapped the constitutive one. In contrast with our results, Kieboom et al. (13) showed that the srpABC genes were expressed only in response to solvents, with styrene being the best inducer.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Pattern of expression of the ttgGHI operon in wild-type cells under different growth conditions. (A) Transcription initiation mapping of the ttgG gene. Lanes 1 and 2, cells grown on LB medium without and with toluene in the gas phase, respectively. The acgt lanes correspond to the sequencing ladder. (B) DNA sequence at the ttgG promoter region. The transcription initiation points are in bold and the arrows indicate the direction of gene transcription. The ATG start codon of the ttgG gene is also in bold. (C) Proposed 10 and 35 boxes (underlined) for PG1 and PG2.

Contribution of solvent efflux pumps to the extrusion of antibiotics. Several efflux pumps have been described as able to extrude antibiotics as well as solvents. We have previously shown that the TtgABC pump is able to extrude AMP, carbenicillin, nalidixic acid, chloramphenicol, and tetracycline (33; also see Table 3), whereas the TtgDEF pump is specific for organic solvents and does not extrude any of these antibiotics (25).