INTRODUCTION

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Many different species of soil- and water-borne bacteria have adapted to the presence of xenobiotic organic molecules in their environment by developing the capacity to use such compounds as carbon sources (7). These microbes have evolved networks of enzymes by which complex organic compounds are broken down into metabolic intermediates. In many instances, transcription of the genes encoding the enzymes that participate in these degradation pathways is regulated so that expression of these catabolic enzymes is dramatically enhanced by the presence of the compounds that they degrade. This type of transcriptional control is achieved by the interaction of transcriptional activator proteins with specific gene promoters. These proteins contain a DNA binding domain, a transcriptional activation domain, and a recognition domain (18). Organic molecules bind to the recognition domain and induce a conformational change in the protein that results in enhanced interaction of the DNA binding domain with specific promoter sequences. This interaction triggers the formation of a complex on the promoter DNA consisting of the transcriptional activator, -factor 54, integration host factor, and the RNA polymerase (19, 20). The complex effectively initiates transcription of the genes encoding the catabolic enzymes that lie directly downstream of the promoter sequence.

One such system is found in the TOL plasmid pWWO carried by the toluene-degrading soil microbe Pseudomonas putida mt-2 (3, 4, 28). In the TOL operon, the binding of toluene and related compounds to the transcriptional activator XylR results in an activating interaction between XylR and a promoter sequence (termed P_u) in the upper region of the operon (reviewed in reference 19). This interaction triggers transcription of the genes involved in converting toluene to benzoate. A second transcriptional activator, XylS, binds benzoate and activates transcription at a second promoter site (P_m) further downstream in the operon. The genes downstream of the P_m promoter encode the enzymes responsible for breakdown of benzoate to Krebs cycle intermediates. Thus, the enzymatic degradation of toluene is greatly increased precisely when the bacteria are exposed to toluene. A number of transcriptional activator proteins, in addition to XylR and XylS, with specificity for other common organic contaminants have been described for other bacterial species. These include DmpR (23, 24), a phenol-specific transcriptional activator, and NahR (21, 29), which is activated by salicylate, a product of naphthalene catabolism.

The discovery of transcriptional activators and their corresponding promoter sequences has made possible the development of bacterial biosensors of organic pollutants. A biosensor of this type can be engineered by placing a reporter gene, such as those encoding -galactosidase or luciferase, under the control of the transcriptional activator. In the presence of an organic contaminant, the transcriptional activator becomes operative and transcription of the reporter gene is enhanced. The resulting increase in reporter gene product is then detected by measuring the activity of the reporter enzyme. Thus, under appropriate conditions, a direct correlation between organic contaminant concentration and reporter enzyme activity can be established. This potential for development of bacterial biosensors has been demonstrated for naphthalene (8, 17). The gene for bacterial luciferase (lux) was randomly inserted by transposon mutagenesis into the nahG gene of a naphthalene catabolic plasmid in Pseudomonas fluorescens. The resulting mutant cells showed a good correlation between naphthalene exposure and bioluminescence. Initial reports have also described biosensors for benzene and benzoate and their derivatives using the P_u and P_m promoters and XylR and XylS transcriptional activators linked to the luciferase or -galactosidase reporter genes (5, 11). Other known transcriptional activators could be used to make biosensors of this type for a wide variety of organic pollutants. Sensitive and accurate biosensors would be useful alternatives to current chemical analysis methods for organic contaminants because of their low cost and their adaptability to field analysis or in situ monitoring.

In this paper, we report the construction, laboratory characterization, and environmental sample testing of a specific biosensor that detects toluene and related compounds when placed in Escherichia coli. A reporter plasmid in which expression of the luc gene for firefly luciferase was placed directly under the control of the P_u promoter and the xylR transcriptional activator of P. putida mt-2 was constructed. Cells harboring this construct detected toluene and specific derivatives with high sensitivity and quantitative accuracy in both contaminated water and soil samples.

	MATERIALS AND METHODS

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Construction of the biosensor plasmid. The starting vector for engineering of the toluene biosensor plasmid was a previously constructed plasmid, pGLTMRS, made for the detection of benzoate (21a). The benzoate plasmid was prepared from the commercially available luciferase reporter plasmid pGL2 (Promega, Madison, Wis.). An E. coli rrnB terminator sequence and the P_m promoter sequence were PCR amplified from E. coli and P. putida mt-2, respectively. They were ligated together at a PstI restriction site. The fused sequence was inserted at the KpnI and XhoI restriction sites in the multiple cloning region of pGL2, just upstream of the promoterless luc gene, with the P_m promoter positioned adjacent to the luc gene. The xylS and xylR genes were PCR amplified as a single amplicon from P. putida mt-2 and inserted at the BamHI digestion site of the pGL2 vector. The resulting pGLTMRS construct was used to prepare the toluene biosensor plasmid, pGLTUR. The P_u promoter was amplified from DNA purified from P. putida mt-2 by PCR using the forward primer 5'-CCAACTGCAGGGAAAGCGCGATGAAC-3' containing a PstI site and a reverse primer, 5'-CCAGCTCGAGGACTCCAGGCGTAACG-3', containing an XhoI site. The P_u sequence and pGLTMRS plasmid DNA were cleaved with PstI and XhoI, ligated, and transformed into competent E. coli DH5 cells (Life Technologies, Gaithersburg, Md.). The xylR gene, including its promoter (P_r), was also PCR amplified using the forward primer 5'-GCGCCAACCTATGGATTTTAATGTGGGCTGCTTGGT-3' containing a PfiMI site and the reverse primer 5'-CGCGGATCCTTTTCACACAACCTGGGGCG-3' containing a BamHI site. The xylR amplicon and the plasmid with the P_u insert were digested with PfiMI plus BamHI. The two were ligated together and then transformed into DH5 cells to produce the final pGLTUR plasmid construct (see Fig. 1).

Induction of luciferase by toluene and its derivatives. Single colonies of DH5 cells harboring the pGLTUR plasmid were grown in 10 ml of Luria-Bertani (LB) medium containing 100 µM ampicillin at 37°C. Cells were allowed to grow in log phase (6 to 8 h) to an optical density at 600 nm (OD₆₀₀) of between 0.2 and 1.0. Similar luminescence responses were observed within this growth density range, but above values of 1.0, luminescence responses began to diminish. After this growth period, the cell culture was diluted to an OD₆₀₀ of 0.2 with LB medium, and luciferase transcription was induced by mixing 0.9 ml of the diluted culture with 0.9 ml of LB medium containing increasing amounts of toluene or its derivative compounds. Incubation was done in 2.0-ml glass vials sealed with Teflon septa to avoid loss of the volatile organic compound. The cultures were incubated at 37°C for 30 min and then cooled on ice for 10 min. Luciferase activity was measured as described previously (22). Briefly, 75 µl of the induced culture was lysed by addition of 25 µl of 4× lysis buffer (100 mM Tris · HCl [pH 7.8], 32 mM NaH₂PO₄, 8 mM dithiothreitol, 8 mM CDTA, 4% [vol/vol] Triton-X 100, and 200 µg of polymyxin B sulfate per ml). Twenty-five microliters of the lysed cell solution was added to 25 µl of a combined 4× concentrate of luciferase substrates A and B (Analytical Luminescence Laboratories, Ann Arbor, Mich.) to give a final 2× concentration of each substrate. The luminescence was read immediately after substrate addition for 45 s in a Turner TD-20e luminometer.

Determination of organic compound concentrations in cell cultures. Saturated solutions of toluene and its derivative compounds were made by mixing 4 ml of LB medium with 4 ml of organic compound for 1 h at room temperature on a platform shaker. The solutions were then centrifuged for 5 min at 3,000 × g to separate the organic and aqueous phases. Dilutions of the saturated LB medium in the aqueous phase were made by addition of fresh LB medium and were used to induce luciferase transcription in the biosensor cells. To determine the concentrations of toluene and its derivatives in the saturated LB media, solutions containing the 1% (wt/vol) NaCl of LB medium, without the tryptone and yeast extract, were saturated with organic compound in parallel with the LB medium solutions. The concentration of the organic compound in the 1% saline solutions was determined by measuring the UV absorption at _max and calculating the concentration from the extinction coefficient for each compound at _max. Extinction coefficients for all the compounds tested were obtained from literature values measured in ethyl alcohol or methanol (9, 26, 27). The saturation concentration of the organic compound in LB medium was taken to be the same as that of the 1% NaCl solution. This assumption was verified by directly measuring the saturation concentration of toluene in LB medium and 1% NaCl by high-pressure liquid chromatography (HPLC) analysis. Saturated concentrations of toluene in each solution were made as described above and were analyzed on an SD-200 HPLC system (Rainin Instrument, Ridgefield, N.J.) using a Rainin Microsorb-mv C₁₈ analytical column. Elution from the column was monitored at 250 nm with a Rainin Dynamax UV-DII absorbance detector. LB medium components were eluted from the column by isocratic flow in 50% acetonitrile-0.1% trifluoroacetic acid in H₂O for 5 min. The mobile phase was then increased to 100% acetonitrile-0.1% trifluoroacetic acid in 1 min and run isocratically at 100% acetonitrile for 7 min. Toluene eluted as a single peak at 9.5 min. By comparing the peak area for toluene in LB medium or 1% NaCl solutions with the peak area for known amounts of toluene in ethanol, the saturation concentrations of toluene (means ± standard deviations) were found to be 5.2 ± 0.1 mM (n = 3) in LB medium and 4.82 ± 0.06 mM (n = 3) in 1% NaCl. Thus, the solubility of toluene is the same, within 10%, in LB medium or 1% NaCl. These values correlate with the toluene saturation concentrations determined by UV spectroscopy (6.1 ± 0.9 mM; n = 19). The values compare well with the published value for toluene saturation in water (5.8 mM [10]).

Testing of contaminated water. Deep aquifer water from well OS-13, known to have BETX (benzene, toluene, and xylene) contamination, from the Baca Street site in Santa Fe, N. Mex., was obtained with permission from the Underground Storage Tank Bureau of the State of New Mexico Environmental Department. Three well volumes of water were purged before samples were taken. Sample water was filtered through a 0.2-µm-pore-size filter and stored in vials with Teflon septa at 4°C. When stored in this manner, no change in toluene contaminant concentration was observed for 1 week. Longer storage periods were not evaluated. Water was tested by adding 850 µl of OS-13 sample to 284 µl of 4× concentrate of LB medium, 216 µl of LB medium, and 450 µl of DH5 cells harboring the pGLTUR plasmid in LB medium at a cell density of 0.4 OD₆₀₀. The cells were incubated for 1 h at 37°C, and then luciferase activity was measured as described above. Samples containing known concentrations of toluene in the 216-µl portion of LB medium were tested in parallel with 850 µl of deionized, distilled laboratory water in place of well water to generate a standard curve. As a control, water from an adjacent well (well USTB-2), which had no BETX contamination, was tested in the same manner. Standard curve data were fitted to the Hill equation, and the concentrations of toluene or toluene equivalents in the well water samples were calculated from the standard curve.

Testing of contaminated soil. Contaminated soil was obtained from the DP road site at Los Alamos National Laboratory. This site, a former vehicle fueling and maintenance area, was known to be contaminated with BETX materials and was in the process of being remediated. The soil was placed in tightly sealing glass bottles immediately after excavation and stored at 4°C until assayed. Similar results were obtained for 1 month when the soil was stored in this manner. Uncontaminated soil from the same site was also obtained and used as a control. Thirty grams of soil samples were extracted with 30 ml of ethyl alcohol by being mixed on a platform shaker for 2 to 3 h at room temperature. Samples were centrifuged at 13,000 × g for 1 min to pellet the soil. Fifty microliters of the ethyl alcohol extraction supernatant was added to 850 µl of LB medium and 900 µl of cells at a cell density of 0.2 OD₆₀₀. Parallel samples containing known toluene concentrations in the 850-µl portion of LB medium were also tested by using 50 µl of pure ethyl alcohol to obtain a standard curve. The data were fitted to the Hill equation, and the concentration of toluene or toluene equivalents in the ethyl alcohol extract was calculated from the standard curve. The 50 µl of ethyl alcohol extract inhibited toluene-induced luminescence by 40% (data not shown). This was caused entirely by the ethyl alcohol and not by any other component of the extract. To account for ethyl alcohol inhibition in the unknown samples, 50 µl of ethyl alcohol was added to the samples used to generate the standard curve. Thus, unknown and control samples contained the same amount of ethyl alcohol, and any error resulting from addition of ethyl alcohol to the biosensor cells was accounted for.

	RESULTS AND DISCUSSION

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Construction of the toluene biosensor. A map of the pGLTUR plasmid is shown in Fig. 1. The position of the P_u promoter was 43 bp upstream of the luc gene at the XhoI site. The promoter fragment spanned 325 bp of the P_u region of the TOL plasmid including the upstream regulatory sequences for XylR binding, the ntr/nif 24 and 12 promoter sequences, the transcription initiation site, and 90 bp of the xylA gene (toluene oxygenase) (12, 16). The xylR structural gene sequence was placed 318 bp directly downstream of the luc gene in the same orientation as luc at the PfiMI site. It consisted of 2,335 bp spanning the autorepressor region upstream of xylR (1, 6, 14) and the entire open reading frame of xylR (13, 15, 25). The rrnB sequence was positioned between the Amp^r gene and the luc gene at the KpnI site to decrease background luminescence. When DH5 cells were transformed with this vector containing the P_u promoter, luciferase reporter, and xylR activator together on the same plasmid, consistent luminescence signals were observed in response to toluene and derivative compounds.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Plasmid map of the pGLTUR biosensor construct. Important features of the toluene biosensor are indicated, including the location and orientation of the Pu promoter, the E. coli rrnB transcription terminator sequence, the luc luciferase gene, the Pr promoter, and the xylR transcriptional activator gene. Restriction sites used to insert Pu and xylR are shown for reference. rbs, ribosome binding site.

Characterization of the toluene biosensor. The time course of luciferase activity in DH5 cells harboring the pGLTUR plasmid is shown in Fig. 2A. Cells were exposed to 250 µM 3-xylene and were assayed for luciferase activity at the times indicated. Luminescence in exposed cells increased rapidly from a relative luminescence of 5.85 U at 5 min to 1,020 U at 120 min. Luminescence from unexposed cells increased much more slowly from 4.68 to 30.9 U over the same period as a result of cell growth. To determine the rate of induction of luciferase activity independently of the contribution of cell growth to that rate, the ratio of luminescence from induced versus uninduced cells was determined (Fig. 2B). The luciferase synthesis rate was treated as a pseudo-first-order reaction, and the induction ratio was fitted to a first-order rate equation. From the fit, the rate constant (mean ± standard deviation) was found to be 0.011 min¹ ± 0.003, yielding a half-life of 64 min and a maximal induction ratio of 50.5 ± 10.0. This rate is about twice that reported previously for xylR-activated transcription from the P_u promoter in E. coli grown in LB medium (1). Thus, relatively rapid induction of luciferase activity occurred in DH5 cells containing the pGLTUR plasmid. Testing of toluene and other xylR effectors was completed with 30-min induction periods in the linear portion of the time response. Testing of environmental samples was completed by using 60-min induction periods to increase sensitivity.

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Kinetics of induction of the toluene biosensor by 3-xylene. (A) Rate of luminescence increase (arbitrary luminescence units) in DH5 cells harboring the toluene biosensor plasmid in nonexposed cells () or after exposure to 250 µM 3-xylene (). Luminescence was measured as described in Materials and Methods. Error bars represent the standard deviations from three replicates of each time point. The rate of cell growth was the same for 3-xylene-exposed and control cell suspensions (data not shown). (B) Kinetics of the ratio of luminescence from 3-xylene-exposed and -unexposed cells in panel A. The line represents the nonlinear least-squares fit of the data to the first-order rate equation y = ymax (1  ekt), in which y is the ratio of luminescence from 3-xylene-exposed versus -unexposed cells at time t, ymax is the maximal value of the induction ratio at infinite time, and k is the rate constant. A ymax value of 50.5 ± 10.0 and a k value of 0.011 ± 0.003 min1 were obtained from the curve fit.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   The toluene concentration dependence of luminescence from toluene biosensor cells. Luminescence from DH5 cells harboring the pGLTUR plasmid was measured at the concentrations of toluene indicated, as described in Materials and Methods. Data from three separate experiments were normalized for maximal luminescence and combined. The error bars represent the standard deviations of three replicates of each sample within the same experiment. The line represents the nonlinear least-squares fit of the data to the Hill equation, y = {(ymax  ymin)[toluene]n/(K1/2n + [toluene]n)} + ymin, in which y is the percentage of maximal luminescence at a given concentration of toluene, ymax is the percentage of maximal luminescence at the saturating concentration of toluene (100%), and ymin is the percentage of maximal luminescence at a toluene concentration of zero. K1/2 is the concentration of toluene at which half-maximal effect is observed, and n is the Hill coefficient, napp. Values of 169 ± 9.6 µM for K1/2 and 2.3 ± 0.3 for napp were obtained from the curve fit.

Testing of contaminated water and soil with the toluene biosensor. To determine the utility of the toluene biosensor in measuring actual environmental contamination, water samples of known contaminant concentration were tested and results of the biosensor assay were compared to the known concentrations. Deep aquifer water was obtained from contaminated and uncontaminated wells at the Baca Street site in Santa Fe, N. Mex. This site has been actively monitored by the Underground Storage Tank Bureau of the State of New Mexico Environmental Department for 3 years. One particular well, designated OS-13, was contaminated principally with benzene, toluene, xylenes, and ethylbenzene. Water from well OS-13 and from an uncontaminated well in the same area (USTB-2) was tested by using the pGLTUR-based biosensor. The results of a typical experiment are shown in Fig. 4A. Water from OS-13 gave a luminescence response of 72 ± 4 (mean ± standard deviation; n = 8) luminescence units. From the graph, one can see that this luminescence corresponded to ~100 µM toluene. By calculating the contaminant concentration from the standard curve, a value of 102 ± 3.3 µM was obtained. Taking the dilution factor of the assay into account (see Materials and Methods), the final concentration of contaminants in the water was 217 ± 7.4 µM (20.0 ± 0.7 ppm) (means ± standard deviations). The average value from three separate tests was 215 ± 23.0 µM (19.8 ± 2.1 ppm). This concentration should be referred to as a toluene equivalent concentration because it could have resulted from 601 µM benzene or 50.1 µM 3-xylene or some combination of those compounds detected by the biosensor. K_1/2 values from Table 1 give the relative sensitivity of the toluene biosensor for these other compounds. The toluene biosensor did not detect any contaminants in the uncontaminated well, USTB-2 (Fig. 4A). Furthermore, no quenching of the luminescence response to spiked samples containing 720 µM toluene was observed up to the highest concentration of OS-13 water added (47%) to the assay mixture.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Testing of contaminated water and soil by using the toluene biosensor. (A) Water from BETX-contaminated well OS-13 and from uncontaminated well USTB-2 at the Baca Street site in Santa Fe, N. Mex., was tested for toluene derivative contamination as described in Materials and Methods. The luminescence responses to water from the contaminated well (black bar) and the uncontaminated well (cross-hatched bar) are shown, as is the luminescence response to different known toluene concentrations (). Error bars represent the standard deviations of three replicates of each sample. The standard curve was fit to the Hill equation as described for Fig. 3 (represented by the line), and the resulting equation was used to calculate the toluene equivalent concentrations of the well water (indicated by the positions of the bars on the x axis). (B) Soil from the DP road site at Los Alamos, N. Mex., was extracted with ethanol and tested for toluene derivative contamination as described in Materials and Methods. The luminescence responses of ethyl alcohol extracts from contaminated (black bar) and uncontaminated (cross-hatched bar) soil collected from the same site are shown, as is the luminescence response to different known toluene concentrations (). Error bars represent the standard deviations of three replicates of each sample. The toluene equivalent concentrations were determined as for panel A.