Reporter Gene System

In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme β-galactosidase. When bacteria contai...
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Anal. Chem. 2000, 72, 2423-2427

Chlorocatechol Detection Based on a clc Operon/ Reporter Gene System X. Guan, S. Ramanathan, J. P. Garris, R. S. Shetty, M. Ensor, L. G. Bachas,* and S. Daunert*

Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055

A sensitive and selective sensing system for chlorocatechols (3-chlorocatechol and 4-chlorocatechol) was developed based on Pseudomonas putida bacteria harboring the plasmid pSMM50R-B′. In this plasmid, the regulatory protein of the clc operon, ClcR, controls the expression of the reporter enzyme β-galactosidase. When bacteria containing components of the clc operon are grown in the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream from the β-galactosidase gene. Thus, the concentration of chlorocatechols can be related to the production of β-galactosidase in the bacteria. The concentration of β-galactosidase expressed in the bacteria was determined by measuring the chemiluminescence signal emitted with the use of a 1,2-dioxetane substrate. ClcR has a high specificity for chlorocatechols and provides the sensing system with high selectivity. This was demonstrated by evaluating several structurally related organic compounds as potential interfering agents. Both 3-chlorocatechol and 4-chlorocatechol can be detected with this sensing system at concentrations as low as 8 × 10-10 and 2 × 10-9 M, respectively, using a 2-h induction period. In the case of 3-chlorocatechol, a highly selective sensing system was developed that can detect this species at concentrations as low as 6 × 10-8 M after a 5-min induction period; the presence of 4-chlorocatechol at concentrations as high as 2 × 10-4 M did not interfere with this system. Catechols are widely used as antiseptics and as antioxidants in the rubber, dye, fat, oil, and photographic industries. Further, catechols (including chlorocatechols) are important starting materials in the synthesis of cosmetics, pharmaceuticals, inks, and insecticides.1 These compounds have been proven to be strong skin, eye, and respiratory tract irritants and to cause DNA damage.2 Exposure to high concentrations of catechols can cause convulsions.1,3 Chlorocatechols are known to be central intermediates in the biodegradation pathway of a large array of chlorinated compounds,4 including polychlorinated aromatic compounds * Corresponding authors. Phone: (606) 257-7060. Fax: (606) 323-1069. E-mail: [email protected] or [email protected]. (1) Kroschwitz, J. I.; Howe-Grant, M. Encyclopedia of Chemical Technology; John Wiley: New York, 1995; Vol. 13, pp 96-1014. (2) Schweigert, N.; Belkin, S.; Leong-Morgenthaler, P.; Zehnder, A. J. B.; Eggen, R. I. L. Environ. Mol. Mutagen. 1999, 33, 202-210. (3) Genium’s Handbook of Safety, Health, and Environmental Data for Common Hazardous Substances; Genium: Schenectady, NY, 1999. (4) Neilson, A. H. J. Appl. Bacteriol. 1990, 69, 445-470. 10.1021/ac9913917 CCC: $19.00 Published on Web 05/04/2000

© 2000 American Chemical Society

(PCBs). Several microorganisms, such as Pseudomonas putida,5 Pseudomonas sp. B13,6 and Ralstonia eutropha (formerly Alcaligenes eutrophus),7,8 are capable of degrading these compounds. In addition, chlorocatechols are produced during chlorine bleaching of wood pulp and have been found in the aquatic environment and sediments close to pulp mills.9 Chlorocatechols have also been found in the urine of individuals that have been exposed to chlorobenzene and, thus, can serve as a biomarker of exposure to these compounds.10,11 Therefore, it is desirable to develop sensitive sensing systems to detect chlorocatechols. Bacteria-based sensing systems have been developed for metal ions, oxoanions, sugars, and toxic organic compounds.12-21 While organic compounds (toxic and nontoxic) are typically metabolized through different enzyme-catalyzed catabolic pathways and further used as carbon sources, metal ions and oxoanions are removed from the bacteria through active transport, chelation, or chemical action to neutralize their toxicity. The expression of the enzymes and proteins involved in these pathways is typically controlled by a regulatory protein or proteins. In this respect, sensing systems can be developed by employing genetic constructs in which the regulatory proteins have been designed to control the expression (5) Frantz, B.; Chakrabarty, A. M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 44604464. (6) van der Meer, J. R.; Frijters, A. C. J.; Leveau, J. H. J.; Eggen, R. I. L.; Zehnder, A. J. B.; de Vos, W. M. J. Bacteriol. 1991, 173, 3700-3708. (7) Perkins, E. J.; Gordon, M. P.; Caceres, O.; Lurquin, P. F. J. Bacteriol. 1990, 172, 2351-2359. (8) Ogawa, N.; McFall, S. M.; Klem, T. J.; Miyashita, K.; Chakrabarty, A. M. J. Bacteriol. 1999, 181, 6697-6705. (9) van Leeuwen, J. A.; Nicholson, B. C.; Hayes, K. P.; Mulcahy, D. E. Mar. Freshwater Res. 1996, 47, 929-936. (10) Kumagai, S.; Matsunaga, I. Int. Arch. Occup. Environ. Health 1995, 67, 207-209. (11) Kumagai, S.; Matsunaga, I. Occup. Environ. Med. 1995, 52, 65-70. (12) Heitzer, A.; Webb, O. F.; Thonnard, J. E.; Sayler, G. S. Appl. Environ. Microbiol. 1992, 58, 1839-1846. (13) Heitzer, A.; Malachowsky, K.; Thonnard, J. E.; Bienkowski, P. R.; Sayler, G. S. Appl. Environ. Microbiol. 1994, 60, 1487-1494. (14) Virta, M.; Lampinen, J.; Karp, M. Anal. Chem. 1995, 67, 667-669. (15) Ramanathan, S.; Shi, W.; Rosen, B. P.; Daunert, S. Anal. Chem. 1997, 69, 3380-3384. (16) Ramanathan, S.; Shi, W.; Rosen, B. P.; Daunert, S. Anal. Chim. Acta 1998, 369, 189-195. (17) Scott, D. L.; Ramanathan, S.; Shi, W.; Rosen, B. P.; Daunert, S. Anal. Chem. 1997, 69, 16-20. (18) Sticher, P.; Jaspers, M. C. M.; Stemmler, K.; Harms, H.; Zehnder, A. J. B.; van der Meer, J. R. Appl. Environ. Microbiol. 1997, 63, 4053-4060. (19) Ikariyama, Y.; Nishiguchi, S.; Koyama, T.; Kobatake, E.; Aizawa, M.; Tsuda, M.; Nakazawa, T. Anal. Chem. 1997, 69, 2600-2605. (20) Willardson, B. M.; Wilkins, J. F.; Rand, T. A.; Schupp, J. M.; Hill, K. K.; Keim, P.; Jackson, P. J. Appl. Environ. Microbiol. 1998, 64, 1006-1012. (21) Applegate, B. M.; Kehrmeyer, S. R.; Sayler, G. S. Appl. Environ. Microbiol. 1998, 64, 2730-2735.

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of a reporter protein (typically an enzyme). In this manner, the highly selective recognition of the target analyte by the regulatory protein is amplified severalfold by the transcription and translation processes that occur during expression of the reporter enzyme/ protein. When highly sensitive luminescence methods are employed to monitor the reporter enzyme activity, it is possible to detect certain analytes at concentrations as low as 10-15 M.16 The bacterial β-galactosidase gene (lacZ) is widely used as a reporter for the study of gene regulation, protein-protein interactions, and cell fusion.22-24 Chemiluminescent 1,2-dioxetane substrates for β-galactosidase provide highly sensitive enzyme detection25,26 and have been utilized in reporter gene assays.27-33 Because monitoring of β-galactosidase activity with these substrates is sensitive and has a wide dynamic range, the lacZ reporter gene is well suited for the development of bacteria-based chemiluminescent sensing systems. This work describes a sensing system for chlorocatechols, which takes advantage of the high recognition provided by the ClcR regulatory protein of the clc operon along with the sensitivity provided by the chemiluminescence detection of β-galactosidase activity. This strategy permits for the detection of chlorocatechols either in cells or in cell lysates. EXPERIMENTAL SECTION Reagents. 3-Chlorocatechol and 4-chlorocatechol were obtained from TCI (Portland, OR). The chemiluminescent substrate, Galacton-light plus, and light emission accelerator-II, a luminescence enhancer solution, were purchased from Tropix (Bedford, MA). Luria Bertani (LB) broth was obtained from BIO 101 (Vista, CA). Catechol, 2-chlorophenol, 4-chlorophenol, biphenyl, HEPES, tris(hydroxymethyl)aminomethane (Tris), Triton X-100, DMSO (ACS Reagent grade), and all other reagents were obtained from Sigma (St. Louis, MO). All chemicals were reagent grade or better and were used as received. All solutions were prepared using deionized (Milli-Q Water Purification system, Millipore, Bedford, MA) distilled water. The following buffers were used: TBS buffer (24.8 mM Tris, 1.4 mM Na2HPO4, 5.1 mM KCl, 137 mM NaCl, 1.36 mM CaCl2, 1.05 mM MgCl2, pH 7.5), HEPES buffer (100 mM HEPES, 1 mM MgCl2, pH 8.0), Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM β-mercaptoethanol, pH 7.0), and phosphate buffer (100 mM phosphate, pH 7.8). The substrate, Galacton-light plus, was diluted with a 100 mM phosphate buffer, pH 8.0, containing 1 mM MgCl2 (reaction buffer) and stored at 4 °C as suggested by the manufacturer. All (22) Young, D. C.; Kingsley, S. D.; Ryan, K. A.; Dutko, F. J. Anal. Biochem. 1993, 215, 24-30. (23) Alam, J.; Cook, J. L. Anal. Biochem. 1990, 188, 245-254. (24) Martin, C. S.; Wight, P. A.; Dobretsova, A.; Bronstein, I. BioTechniques 1996, 21, 520-524. (25) Bronstein, I.; Fortin, J.; Stanley, P. E.; Stewart, G. S. A. B.; Kricka, L. J. Anal. Biochem. 1994, 219, 169-181. (26) Bronstein, I.; Martin, C. S.; Fortin, J.; Olesen, C. E. M.; Voyta, J. C. Clin. Chem. 1996, 42, 1542-1546. (27) Jian, V. K.; Magrath, I. T. Anal. Biochem. 1991, 199, 119-124. (28) Hu, M.; Bigger, C. B.; Gardner, P. D. J. Biol. Chem. 1995, 270, 44974502. (29) McMillan, J. P.; Singer, M. F. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1153311537. (30) Ng, D.; Su, M. J.; Kim, R.; Bikle, D. Front. Biosci. 1996, 1, 16-24. (31) Reuss, F. U.; Coffin, J. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 92939297. (32) DiSepio, D.; Jones, A.; Longley, M. A.; Bundman, D.; Rothnagel, J. A.; Roop, D. R. J. Biol. Chem. 1995, 270, 10792-10799. (33) O’Connor, K. L.; Culp, L. A. BioTechniques 1994, 17, 502-509.

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stock solutions (0.01 M) of the chlorinated organic compounds employed in this study were freshly prepared in DMSO. Standards were made by serial dilution using a solution of 10% (v/v) DMSO in deionized distilled water. Apparatus. Chemiluminescence measurements were performed on an Optocomp I luminometer from GEM Biomedical (Carrboro, NC) using a 100-µL fixed-volume injector. All experiments were conducted at room temperature unless specified otherwise. All luminescence intensities reported are the averages of at least three replicates. Plasmid and Bacterium. The plasmid pSMM50R-B′ (which contains the lacZ gene) encoding for β-galactosidase under the control of the ClcR protein was kindly provided by Dr. A. M. Chakrabarty, University of Illinois College of Medicine (Chicago, IL).34 Plasmid pSMM50R-B′ was transformed into the P. putida PRS4020 Ben- catR knockout strain and was used in the development of the bacteria-based sensing system for chlorocatechols. Kinetic Study of Chemiluminescence Emission. A single colony of P. putida containing the pSMM50R-B′ plasmid was selected and grown overnight at 30 °C in LB media containing 80 µg/mL ampicillin. Then, the bacteria were inoculated at a 1:20 volume ratio into a 500-mL flask with LB media containing 80 µg/ mL ampicillin. The bacteria were allowed to grow until they reached a cell density corresponding to an absorbance of 0.80.9 at 600 nm. A 1-mL quantity of the bacterial suspension was incubated with 112 µL of a 3-chlorocatechol standard solution at 30 °C for a fixed time period (5 min-2 h); the blank corresponded to 112 µL of deionized distilled water incubated with 1 mL of the bacterial suspension. Then, the bacterial solutions were centrifuged at 10000g for 5 min at 4 °C. The supernatant was removed, and the bacteria were washed twice with 1 mL of TBS buffer. The bacteria were then resuspended in 1 mL of phosphate buffer containing 0.2% (w/v) Triton X-100, and the suspension was placed in a shaker at 37 °C for 1 h. A 20-µL sample of the bacterial cells was added to 100 µL of the substrate solution. The substrate and bacteria were vortexed for 10 s to ensure good mixing and were then incubated in a shaker at 37 °C for a fixed period of time (5-90 min). The chemiluminescence signal was measured by injecting 100 µL of the light emission accelerator-II solution into the substrate-bacteria mixture. After a delay of 20 s, which was necessary to reach maximal chemiluminescence, the emitted chemiluminescence signal was collected over a period of 3 s. Dose-Response Curves for 3-Chlorocatechol and 4Chlorocatechol. A substrate-bacteria mixture was obtained using the protocol described above. The substrate and bacteria were incubated in a shaker at 37 °C for 45 min. The chemiluminescence was measured by injecting 100 µL of the light emission accelerator-II solution into the substrate-bacteria mixture. After a delay of 20 s, the emitted chemiluminescence signal was collected over a period of 3 s. Selectivity Studies. The induction of the sensing system by a variety of structurally related compounds, such as catechol, 2-chlorophenol, 4-chlorophenol, biphenyl, and 4-chlorobiphenyl, was studied by measuring the chemiluminescence produced. The standard solutions of all the organic compounds tested were prepared daily from a fresh 1 × 10-2 M stock solution. A 112-µL (34) McFall, S. M.; Parsek, M. R.; Chakrabarty, A. M. J. Bacteriol. 1997, 179, 3655-3663.

Figure 1. Top: Schematic representation of the clcABD operon of P. putida. Bottom: Partial representation of the chlorocatechol biodegradative pathway involving enzymes ClcA, ClcB, and ClcD.

Figure 2. Schematic representation of plasmid pSMM50R-B′, showing the genes for β-lactamase (ampr), clcR, clcA, and β-galactosidase (lacZ). clcB′ denotes the part of the clcB gene that remained on the plasmid after it was fused with lacZ.

sample of the standard solution was incubated for 2 h with 1 mL of the bacterial suspension (as above) to induce protein expression. The bacterial cell walls were made permeable by employing Triton X-100, and the chemiluminescence was measured by using the protocol described above. RESULTS AND DISCUSSION Degradation of chlorocatechols in bacteria proceeds through the chlorocatechol degradative pathway. Unlike the meta-cleavage pathway encoded by the TOL plasmid,19 four plasmid-encoded ortho-cleavage chlorocatechol pathways have been identified thus far: the clcABD operon,5 the tcbCDEF operon,6 the tfdCDEF operon,7 and the cbnABCD operon.8 It has been shown that there is a strong homology among the tcbCDEF, clcABD, and tfdCDEF gene clusters.35,36 Because these chlorocatechol degradative pathways utilize highly homologous enzymes and proceed through very similar intermediates, we chose the clcABD operon in our research. There are three enzymes involved in the catabolism of chlorocatechols in P. putida that are encoded by the clcABD operon (Figure 1).5 The action of these enzymes results in the conversion of 3-chlorocatechol to 5-carboxy-3-oxo-4-pentenoic acid. This acid is converted to β-ketoadipic acid, which is further metabolized to intermediates of the Krebs cycle. The ClcR protein regulates the expression of the clcABD operon. The plasmid pSMM50R-B′ (Figure 2), in which the ClcR protein regulates the expression of the reporter enzyme β-galactosidase, was employed in this sensing system. In this plasmid, clcR, the gene encoding for the ClcR regulatory protein, is positioned immediately upstream from the clcA gene. The lacZ gene, the gene encoding for (35) Daubaras, D. L.; Chakrabarty, A. M. Biodegradation 1992, 3, 125-135. (36) van der Meer, J. R.; de Vos, W. M.; Harayama, S.; Zehnder, J. B. Microbiol. Rev. 1992, 56, 677-694.

the reporter enzyme β-galactosidase, is fused to the clcB gene. This gene fusion is in frame, so that the expressed protein contains the first 134 amino acids of ClcB fused to the N-terminus of β-galactosidase. Fusing to a portion of a gene in a plasmid is a common strategy used to ensure that the reporter lacZ gene is in frame.16 It has been demonstrated that the inducer of the clcABD operon is 2-chloromuconate,37,38 which is produced in a reaction catalyzed by the ClcA enzyme. Therefore, clcA needs to be present in the plasmid to produce induction of the designed sensing system. The expression of β-galactosidase in P. putida strain PRS4020 bearing pSMM50R-B′ is controlled by the ClcR regulatory protein. In the absence of chlorocatechols, the ClcR protein binds to the operator/promoter (O/P) region of the clcABD operon. This prevents the transcription and translation of the clcA and lacZ genes. In the presence of chlorocatechols, ClcR activates the clcA promoter, which is located upstream of the clcA gene, and ClcA and β-galactosidase are expressed. In our studies, this plasmid was introduced into P. putida and a bacterial-based sensing system for chlorocatechols was developed by correlating the β-galactosidase signal with the concentration of chlorocatechol present in the sample. The activity of β-galactosidase was monitored using Galacton-plus as the substrate of the chemiluminescence reaction. Several methods to lyse the cells were compared, such as using Triton X-100, Polymyxin B sulfate, and sonication. Triton X-100 makes bacteria permeable by dissolution of the inner bacterial plasma membrane, while sonication disrupts the cells through mechanical shearing of the cell wall. Polymyxin B sulfate breaks the cell wall by interaction of the amphipathic polymyxin molecules with lipopolysaccharides in the bacterial outer membrane. The data obtained with the three different methods shown in Table 1, suggest that sonication in phosphate buffer for 30 s was the least efficient method. Although addition of Polymyxin B sulfate (1 mg/mL, in phosphate buffer) resulted in rapid cell lysis (15 min), Triton X-100 (0.2% w/v, in phosphate buffer) gave the highest signal and lowest RSD. To increase the sensitivity of this sensing system, a kinetic study of the emitted chemiluminescence was performed. After induction with 100 µM 3-chlorocatechol for 2 h, the cells were lysed by using the Triton X-100 method. Then, the substrate was added and incubated with the cell lysate for time periods ranging from 5 to 90 min. After injection of 100 µL of the light emission accelerator-II solution to the substrate-bacteria mixture, the emitted chemiluminescence signal was collected over a period of (37) McFall, S. M.; Parsek, M. R.; Chakrabarty, A. M. J. Bacteriol. 1997, 179, 3655-3663. (38) McFall, S. M.; Chugani, S. A.; Chakrabarty, A. M. Gene 1998, 223, 257267.

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Table 1. Effect of the Lysis Method on the Chemiluminescence Emitted by the Sensing System after Induction of the Bacteria with 1 µM 3-Chlorocatechol for 2 ha lysis method

light intensity (counts)

RSD (%)

sonication (30 s) Polymyxin B sulfate (15 min) Polymyxin B sulfate (30 min) Triton X-100 (15 min) TritonX-100 (30min)

3.3 × 104 1.5 × 105 1.5 × 105 1.1 × 105 5.0 × 105

9.6 7.5 7.5 8.2 3.8

a The buffer used was 100 mM phosphate, pH 7.8. The chemiluminescence signal has been corrected with respect to the blank.

Figure 4. Effect of induction time on the detection limit for 3-chlorocatechol and on the total signal (light intensity) obtained with 1 µM of 3-chlorocatechol.

Figure 3. Kinetic study of the chemiluminescence emission based on the 3-chlorocatechol sensing system employing P. putida harboring the pSMM50R-B′ plasmid. The bacteria were induced with 100 mM 3-chlorocatechol for 2 h. The chemiluminescence signal has been corrected with respect to the blank. Data are the averages plus or minus one standard deviation (n ) 3).

3 s after a delay of 20 s. As shown in Figure 3, the chemiluminescence emitted by the bacteria increased with increasing incubation times with the substrate. A 45-min incubation time was chosen for the rest of the experiments. It should be noted that induction by 100 µM 3-chlorocatechol for 2 h corresponds to the maximum amount of β-galactosidase expressed under the conditions of our experiments. Moreover, the data in Figure 3 indicate that there is a linear relationship between the intensity of the light emitted and the incubation time (up to about 60 min), which demonstrates that the substrate is not a limiting reagent as long as incubation times of less than 60 min are used to determine the β-galactosidase enzymatic rates. The effect of the induction time was investigated by constructing dose-response curves after incubating the bacteria with 3-chlorocatechol for 5 min, 15 min, 30 min, 1 h, and 2 h. These curves (see Figure 4) showed that, with an increase in the induction time, the chemiluminescence signals (light intensity) increase and the detection limits decrease. Light intensity values did not change drastically for the 30-min, 1-h, and 2-h induction times, with the detection limits being on the order of 8 × 10-10 M. The detection limit is defined as the chlorocatechol concentration that corresponds to an S/N ) 3. Although the chemiluminescence signals obtained with induction times of 5 and 15 min were weaker, sufficient β-galactosidase was expressed in response 2426 Analytical Chemistry, Vol. 72, No. 11, June 1, 2000

Figure 5. Selectivity of the bacterial sensing system employing P. putida harboring the pSMM50R-B′ plasmid. The bacteria were incubated with a series of analytes for 30 min: (1) 3-chlorocatechol; (2) 4-chlorocatechol; (3) 4-chlorobiphenyl; (4) catechol; (5) biphenyl; (6) 2-chlorophenol; (7) 4-chlorophenol. The chemiluminescence signal has been corrected with respect to the blank. Data are the averages plus or minus one standard deviation (n ) 3).

to 3-chlorocatechol to allow for the sensitive detection of this compound in samples. Dose-response curves were constructed after inducing the expression of β-galactosidase with different concentrations of 3-chlorocatechol or 4-chlorocatechol for 2 h. In this system, the bacteria sense the intracellular concentration of chlorocatechols, which in turn is related to the concentration of these compounds in the sample (extracellular concentration). The detection limit for 3-chlorocatechol was 8 × 10-10 M, while 4-chlorocatechol could be detected at 2 × 10-9 M. The induction time played a key role in the sensitivity of the system to 4-chlorocatechol. When the induction time was reduced to 30 min, the detection limit for 4-chlorocatechol was on the order of 10-6 M (Figure 5). Further reduction of the induction time to 5 min worsened the detection limit for 4-chlorocatechol to 2 × 10-4 M (Figure 6). Therefore, if a sensing system for 3-chlorocatechol is desired, a 30-min induction time can be chosen to selectively discriminate against 4-chlorocatechol. Under these conditions, the detection limit for 3-chlorocatechol is 7 × 10-10 M, which is about 3 orders of magnitude better than that for

which is indicative of the high selectivity provided by the ClcR regulatory protein of the clc operon. This selectivity is higher than that observed using derivatives of the TOL plasmid, which has a broader substrate selectivity.19,40 The selectivity of the sensing system using a 5-min induction time was also evaluated by employing 4-chlorocatechol and catechol, since only these two compounds among the many structurally related organic compounds tested showed any significant response at longer induction times. The data obtained (shown in Figure 6) suggest that 4-chlorocatechol and catechol do not interfere with this system at concentrations as high as 2 × 10-4 M.

Figure 6. Dose-response curves for 3-chlorocatechol (b), 4chlorocatechol (O), and catechol (0) obtained after the bacteria were incubated with these analytes for 5 min. The x axis reflects the analyte concentration in the sample. The chemiluminescence signal has been corrected with respect to the blank. Data are the averages plus or minus one standard deviation (n ) 3).

4-chlorocatechol. The dynamic range for this sensing system was 10-8-10-5 M for 3-chlorocatechol. If a faster sensing system for 3-chlorocatechol is desired, selective detection of 3-chlorocatechol can also be accomplished by using as low as a 5-min induction time. Although the intensity of the signal produced is much weaker than that generated with a 30-min induction time, this system can still detect 3-chlorocatechol at 6 × 10-8 M, as shown in Figure 6. This detection limit is sufficient to monitor concentrations of 3-chlorocatechol in the environment.39 It should be noted that control experiments performed to evaluate whether there is any direct effect of 3-chlorocatechol on β-galactosidase activity indicated that there is no activation/inhibition of this enzyme at 3-chlorocatechol concentrations as high as 1 × 10-3 M. To study the selectivity of this system, structurally related organic compounds that could be potential interfering species, such as catechol, 2-chlorophenol, 4-chlorophenol, biphenyl, and 4-chlorobiphenyl, were evaluated. Dose-response curves for these compounds were constructed as shown in Figure 5. The data demonstrated that no appreciable levels of β-galactosidase were produced in response to any of these organic compounds tested, (39) Nagarathnamma, R.; Bajpai, P.; Bajpai, P. K. Process Biochem. 1999, 34, 939-948.

CONCLUSIONS In summary, a highly sensitive and selective chemiluminescence sensing system for chlorocatechol was developed using P. putida bacteria. When exposed to these toxic compounds, the bacteria that harbor plasmid pSMM50R-B′ express β-galactosidase in an amount related to the concentration of chlorocatechols present in the sample. By use of this sensing system, both 3-chlorocatechol and 4-chlorocatechol can be monitored at concentrations as low as 8 × 10-10 and 2 × 10-9 M, respectively. If discrimination between 3-chlorocatechol and 4-chlorocatechol is needed, the time of induction can be reduced to achieve selectivity between the two isomers. Finally, it was demonstrated that this system is extremely selective for chlorocatechols over a variety of other structurally related compounds. Because of the low detection limits and selectivity achieved, this sensing system could find applications in the determination of low concentrations of 3-chlorocatechol and 4-chlorocatechol in the environment. ACKNOWLEDGMENT We thank the National Institute of Environmental Health Sciences (Grant P42 ES 07380 to L.G.B. and S.D.) and the National Science Foundation (Grant CHE-9820808 to S.D.) for support of this research. We also thank Dr. A. M. Chakrabarty for supplying the pSMM50R-B′ plasmid and the PRS4020 strain. S.D. is a Lilly Faculty Awardee and a Cottrell Scholar. Received for review December 7, 1999. Accepted February 15, 2000. AC9913917 (40) Kobatake, E.; Niimi, T.; Haruyama, T.; Ikariyama, Y.; Aizawa, M. Biosens. Bioelectron. 1995, 10, 601-605.

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