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E-mail: [email protected]. 2. National Chung-Hsing University. Department of Botany. Taichung, 40227, Taiwan, Republic of China. Summary.
Acta Biotechnol. 23 (2003) 2 – 3, 123 – 129

Bioluminescence Biosensor for the Detection of Organomercury Contamination

ENDO1*, G., YAMAGATA1, T., NARITA1, M., HUANG2, C.-C.

1

Tohoku Gakuin University Faculty of Engineering Laboratory of Environmental Biotechnology Miyagi, 985-8537, Japan

2

National Chung-Hsing University Department of Botany Taichung, 40227, Taiwan, Republic of China

*

Corresponding author Phone: +81 22 368 74 93 Fax: +81 22 368 70 70 E-mail: [email protected]

Summary To selectively detect organomercurial compounds in the environment, in this study a bioluminescence biosensor for organomercurials was developed using a bacterial gene expression system for the mercury resistance determinant. merB3-Luciferase (mer-lux) transcriptional fusion plasmids pHYΒ3Lux and pHY∆Β3Lux were constructed to evaluate the gene expression system with a new organomercury lyase gene merB3 from Bacillus megaterium strain MB1, which is resistant to a broad spectrum of mercury compounds, and with its 3’end-deleted defective merB3, respectively. Another plasmid, pGR1A, encoding an operator/promoter sequence, merR1, merE, merT, merP and merA from the same bacterial strain was constructed and used as a transacting gene expression vector which combines the gene expression vector of mer-lux transcriptional fusion plasmids in the same Escherichia coli cells. The transformants that carried a set of the two plasmids were used as biological sensors for the detection of organomercurials. Transformant (E. coli DH5α/pHYB3Lux, pGR1A) is available to distinguish the organomercury from inorganic mercury, since inorganic mercurials can induce the bioluminescence of both the bacterial strain lines with pHY∆B3Lux and pHYB3Lux, whereas organomercurials can only induce the bioluminescence of the lines with pHYB3Lux. These experimental results showed that the transformant with a merB 3-defective fusion plasmid, and the gene expression vector responded to only mercury chloride. On the other hand, the transformant with an intact merB3 fusion plasmid and the gene expression vector responded to mercury chloride and all organomercurials tested in the study. The sensor system responded to the existence of the phenyl mercury acetate of 50 nanomolar and was more sensitive than that of inorganic mercury (100 nanomolar). The result also indicated the capability of the system to detect bio-affecting inorganic mercury from several hundred nanomolar to several ten micromolar.

© WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2003 0138-4988/03/02-3-07-0123 $ 17.50+.50/0

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Introduction

The environmental mercury contamination, particularly by the organomercurial compounds, has caused very serious disasters such as methylmercury poisoning cases in Minamata Bay, Japan, during the 1950s [1], in Iraq from 1971 to 1972 [2, 3] and recently in the Amazon River Basin, Brazil [4, 5]. Because the mercurial compounds tend to be accumulated through biological food chain systems, detection and removal of those bio-affecting pollutants at a low level of contamination are the major goals to control the mercury poisoning problems. For the detection of the mercurial compounds, physicochemical analysis methods such as atomic absorption spectrophotometry [6] and cold vapour atomic fluorometry [7] have been developed. Besides the laborious preparation of samples, however, these procedures are not useful in detecting mercurial compounds and distinguishing those bioaffecting forms from non-bioaffecting inert forms or organomercurial from inorganic mercurial forms. In addition, the removal efficiency at lower contamination levels is yet unsatisfactory, when using a physicochemical mercury removal process through passive adsorption or immobilization treatments. As an aspect of the environmental mercury cycle, microorganisms can decompose organomercurial compounds and can detoxify the resulting inorganic mercury ions by reducing them to volatile metallic mercury [8]. One of the key enzymes in this bacterial resistance system is an organomercury lyase that mediates the protonolysis of the carbon-mercury bond as a first step of the detoxification of diverse organomercurial compounds in the environment [9]. From the preserved sediment samples of Minamata Bay, a broad-spectrum mercury resistance strain, Bacillus megaterium MB1, was isolated and a mercury resistance module, encoded in a GRAM-positive bacterial class II transposon, TnMERI1, was identified from its chromosome [10]. From upstream of the previously recognised metalloregulatory gene (merR1 gene) [11, 12], a third organomercurial lyase gene (merB3 gene) with its own operator/promoter (merB3O/P) region was newly found [13], oriented in the same direction as the merR1 gene. The complete structure of the module including two other operator/promoter regions (merR1O/P and merR2O/P) was shown as merB3 O/P-merB3-merR1O/P-merR1-merE-like-merT-merPmerA-merR2O/P-merR2-merB2-merB1 [13] (Fig. 1). Recently, the authors have demonstrated that the expression of the merB3 gene was controlled by the metalloregulator protein MerR1, that the expression was activated by an inorganic mercury ion produced by the basal expression of merB3 and that the merR1O/P-merR1-merElike-merT-merPmerA operon was not induced by organomercurials [14]. As the merB3 gene confers the broadest spectrum of organomercury resistance on the bacterial host among the three organomercurial lyase genes [13], the application of the merB3 gene in the biological remediation of environmental contamination by organomercurials is an attractive subject in environmental biotechnology. In addition, the MerR1 protein is a regulatory protein controlling the operon genes that are expressed to eliminate the mercurials. The transcriptional expression is positively induced by an inorganic mercury ion, and the expression is depressed by the elimination of an inorganic mercury ion in the environment. In this study, a biological detection method of organomercurial compounds was developed using this gene expression system.

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Fig. 1. Schematic representation of the mercury resistance module encoded in TnMERI1 from the chromosome of Bacillus megaterium MB1 (modified from HUANG et al., 1999, [13]) The ORFs and promoter/operator (O/P) regions identified from TnMERI1 are shown in the mercury resistance module with arrows. Restriction enzyme recognition sites are presented using abbreviations: B; Bgl II, E; EcoRI, H; HindIII, N; Nco I, P; Pst I, and S; Sph I. The letters B3, R1 , E, T, P, A, R2 , B2 and B1 denote merB3, merR1, merE-like, merT, merP, merA, merR2, merB2 and merB1, respectively.

Materials and Methods Bacterial Strains and Plasmids The promoterless luxAB genes from Vibrio harveyi [15] were fused downstream of the intact merB3 gene with its operator/promoter region (merB3O/P). The fusion operon was inserted at HindIII and BamHI sites of the vector plasmid pHY300PLK (TAKARA SHUZO , Kyoto, Japan) and a luciferasebased transcriptional fusion plasmid pHYB3Lux was constructed (Fig. 2A). The vector plasmid pHY300PLK is a derivative from an Inc-plasmid pACYC177 [16] and can coexist with a pUC series Inc-vector plasmid in the same Escherichia coli host cell. A host strain E. coli DH5α harbouring pGR1A [13], a pUC-based plasmid encoded with the operon genes from the merR1O/P-merR1 gene to the merA gene (Fig. 1), was co-transformed with pHYB3Lux (Fig. 2A). The transformant E. coli DH5α carrying both plasmids (pHYB3Lux and pGR1A) was used as the bacterial strain for the detection and removal of the organomercurial compounds. Another fusion plasmid, pHY∆B3Lux (Fig. 2 A), encoding the 3’ end defective merB3 gene with merB3O/P and luxAB genes, was also cotransformed with pGR1A. The transformant E. coli DH5α, carrying both plasmids (pHY∆B3Lux and pGR1A), was applied as a control strain. The defective merB3 gene construct lacked the last 44 codons of the intact merB3 gene and lost the catalytic activity for the decomposition of organomercurial compounds.

Bio-Luminescence Assays The transformant overnight cultures were diluted 100-fold with Luria-Bertani (LB) broth [17] containing 100 mg/ml sodium ampicillin and were incubated at 37 °C until the mid-log phase, when the optical density at 600 nm (OD600) reached 0.5–0.6. The cultures were diluted to a final OD600 of 0.04 in an assay medium (1.4% K2HPO4, 0.6% KH2PO4, 0.2% (NH4)2SO4, 0.1% Sodium Citrate-2 H2O, 0.5% Glucose, 0.02% MgSO4 × 7H2O, 0.02% Arginine, 0.02% Leucine, 0.02% Threonine, 0.02%

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Tryptophan, 0.1% Casamino acids) containing mercurial compounds of 10 mM, 1 mM, 0.1 mM of mercury chloride (MC) or 5 mM, 0.5 mM, 0.05 mM of phenylmercury acetate (PMA) and were incubated at 37 °C for some hours. For the luciferase assay, an aliquot (290 µl) of culture was transferred to a luminometric cuvette and the biological luminescence reaction was started by adding an aliquot (10 µl) of 10 % 1-decanal (v/v, dissolved in ethanol). Light produced by the cloned bacterial strains harbouring luxAB gene fusion plasmids was monitored using a Lumat LB 9507 luminometer (EG&G BERTHOLD, Bad Wildbad, Germany) and the total light emitted for the period of six seconds subsequent to adding the decanal solution was recorded as Luminescence Units (LU). Total LU per culture OD600 unit of bacterial culture was calculated, and the relative luminescence activities (RLA) induced by the mercurial compounds were determined by the ratio between LUs per culture OD600 under induced and uninduced conditions. The mean values of RLA were obtained from three independent measurements with those standard deviations.

Fig. 2. Constitution and physical maps of plasmids constructed in this study A. pHY∆B3Lux and pHYB3Lux; B. pGR1A. The letters R, E, T, P, A and B are the same as in Fig.1.

Results and Discussion Detection of the Mercury Ion

Given that the MerR1 protein is induced as an activator only by inorganic mercuric ions [18], mercury chloride was added as the inducer to characterise the system. Light emissions of 0.1 µM, 1 µM, 10 µM of MC were examined. The intensities of light emission were illustrated dependent on the concentration of MC (Fig. 3 A, 3 B). Since the merR1 gene with other operon genes was constructed separately from merB3O/P, the result demonstrated that the MerR1 protein is a trans-acting element in the gene expression. The result also indicates the possibility that the system may be able to detect bioaffecting inorganic mercury from 100 nanomolar to 10 micromolar (Fig. 3 A, 3 B). Detection of Phenylmercury

For the phenylmercury detection, light emissions from the media containing 5 µM, 0.5 µM, and 0.05 µM of PMA were tested. In those cases, the intensities of the light

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emission were also dependent on the concentrations of PMA (Fig. 3 D). The result illustrates that the sensor system responded to the existing concentration of the PMA from 50 nanomolar to 5 micromolar and was more sensitive than that of inorganic mercury. On the other hand, there was no response for the existence of PMA when the defected merB3 gene with the luxAB fusion plasmid (pHY∆B3Lux) was used instead of the pHYB3Lux (Fig. 3 C). This result also provides a hint that the transformant (E. coli DH5α/pHYB3Lux, pGR1A) is able to distinguish the organomercury from inorganic mercury, because inorganic mercurials can induce the bioluminescence of both bacterial strain lines with pHY∆B3Lux and pHYB3Lux, but organomercurials can only induce the bioluminescence of the lines with pHYB3Lux. The result also reconfirms that the MerB3 enzyme is needed to yield the inorganic mercury ion for triggering the gene expression regulated by the MerR1 metalloregulatory protein, and that other organomercurials can also only induce the bioluminescence by using all the lines with pHYB3Lux (data not shown).

Fig. 3. Relative luminescence activities induced with mercury compounds A. E. coli DH5α/pHY∆B3Lux+pGR1A was induced with mercury chloride (MC). Symbols: H – induced with 0.1 µM MC, Ì – induced with 1.0 µM MC, ∆ – induced with 10.0 µM MC. B. E. coli DH5α/pHYB3Lux +p GR1A was induced with MC. Symbols: G – induced with 0.1 µM MC, I – induced with 1.0 µM MC, L – induced with 10.0 µM MC. C. E. coli DH5α/pHY∆B3Lux+pGR1A was induced with phenylmercury acetate (PMA). Symbols: Ì – induced with 0.5 µM PMA. D. E. coli DH5α/pHYB3Lux+pGR1A was induced with PMA. Symbols: G – induced with 0.05 µM PMA, I – induced with 0.5 µM PMA, L – induced with 5.0 µM PMA.

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The reason why the PMA increased the sensitivity of the system compared to the sensitivity for the inorganic mercury ion is still unknown. Since organomercurial compounds benefit from the membrane permeability, the difference of uptake routes between organomercurials and the inorganic mercury ion [19] is considered an important factor that affects the sensitivity of detection by the bioluminescence mercury sensor systems. While many mercury biosensors were developed based on the regulatory gene (merR gene) and its expression system [20–22], a system based on an organomercury lyase gene (merB3 gene) and its expression system has not been developed until now. Our study presented in this paper stands for a prototype of a biological detection system for the estimation of the bio-availability of those toxic organomercurial compounds.

Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (JSPS) and a Grant-in-Aid for Scientific Research from the Japanese Ministry of Health, Labour and Welfare.

Received 9 December 2002 Received in revised form 11 June 2003 Accepted 20 June 2003

References [1] HARADA, M.: Minamata disease: Methylmercury poisoning in Japan caused by environmental pollution. Crit. Rev. Toxicol. 25 (1995), 1−24. [2] GREENWOOD, M. R.: Methylmercury poisoning in Iraq. An epidemiological study of the 1971−1972 outbreak. J. Appl. Toxicol. 5 (1985), 148−159. [3] COX, C., MARSH, D., MYERS, G., CLARKSON, T.: Analysis of data on delayed development from the 1971−72 outbreak of methylmercury poisoning in Iraq. Assessment of influential points. Neurotoxicology 16 (1995), 727−730. [4] LODENIUS, M., MALM, O.: Mercury in the Amazon. Rev. Environ. Contam. T. 157 (1998), 25−52. [5] GRANDJEAN, P., WHITE, R. F., NIELSEN, A., CLEARY, D., DE OLIVEIRA SANTOS, E. C.: Methylmercury neurotoxicity in Amazonian children downstream from gold mining. Environ. Health Persp. 107 (1999), 587−591. [6] OMANG, S . H.: Determination of mercury in natural waters and effluents by flameless atomic absorption spectrophotometry. Anal. Chim. Acta 53 (1971), 415−419. [7] BLOOM, N., FITZGERALD, W. F.: Determination of volatile mercury species at the pico-gram level by low-temperature gas chromatography with cold-vapour atomic fluorescence detection. Anal. Chim. Acta 208 (1988), 151−161. [8] SILVER, S., ENDO, G., NAKAMURA, K.: Mercury in the environment and laboratory. J. Japan. Soc. Water Environ. 17 (1994), 235−243. [9] HOBMAN, J. L., BROWN , N. L.: Bacterial mercury-resistance genes. In: SIGEL, H., SIGEL, A. (eds.). Metal Ions in Biological Systems, Vol. 34, New York: Marcel Dekker Inc., 1997, 527−568. [10] HUANG, C. C., NARITA, M., YAMAGATA, T., ITOH, Y., ENDO, G.: Structure analysis of a class II transposon encoding the mercury resistance of the GRAM-positive bacterium, Bacillus megaterium MB1, a strain isolated from Minamata Bay, Japan. Gene 234 (1999), 361−369.

ENDO, G., YAMAGATA, T. et al., Bioluminescence Organomercury Biosensor

129

[11] HELMANN, J. D., WANG, Y., MAHLER, I., WALSH, C. T.: Homologous metalloregulatory proteins from both GRAM-positive and GRAM-negative bacteria control transcription of mercury resistance operons. J. Bacteriol. 171 (1989), 222−229. [12] GUPTA, A., PHUNG, L. T., CHAKRAVARTY, L., SILVER, S.: Mercury resistance in Bacillus cereus RC607: transcriptional organization and two new open reading frames. J. Bacteriol. 181 (1999), 7080−7086. [13] HUANG, C. C., NARITA, M., YAMAGATA, T., ENDO, G.: Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1. Gene 239 (1999b), 361−366. [14] HUANG, C. C., NARITA, M ., YAMAGATA, T., ENDO, G., SILVER, S.: Characterization of two regulatory genes of the mercury resistance determinants from TnMERII by luciferase-base examination. Gene 301 (2002), 13−20. [15] BELAS, R., MILEHAM, A., COHN, D., HILMAN, M., SIMON, M., SILVERMAN, M.: Bacterial bioluminescence: Isolation and expression of the luciferase genes from Vibrio harveyi. Science 218 (1982), 791−793. [16] ISHIWA, H., TSUCHIDA, N.: New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Construction and characterization of plasmid pHY460 with twelve unique cloning sites. Gene 32 (1984), 129−134. [17] SAMBROOK, J., FRITSCH, E. F., MANIATIS, T.: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor. New York: Cold Spring Harbor Laboratory, 1989. [18] YAMAGATA, T., ISHII, M., NARITA , M., HUANG, C. C., ENDO, G.: Bio-affecting mercury detection using mercury resistance gene module fused with bioluminescence reporter genes. Water Sci. Technol. 46 (2002) 11−12, 253−256. [19] KIYONO, M., OMURA, T., FUJIMORI, H., PAN-HOU, H.: Lack of involvement of merT and merP in methylmercury transport in mercury resistant Pseudomonas K-62. FEMS Microbiol. Lett. 128 (1995), 301−306. [20] KLEIN, J., ALTENBUCHNER, J., MATTES, R.: Genetically modified Escherichia coli for colorimetric detection of inorganic and organic Hg compounds. EXS 80 (1997), 133−151. [21] RASMUSSEN, L. D., TURNER, R. R., BARKEY, T.: Cell-density-dependent sensitivity of a mer-lux bioassay. Appl. Environ. Microbiol. 63 (1997), 3291−3293. [22] SELFONOVA, O., BURLAGE, R., BARKEY, T.: Bioluminescent sensors for detection of bioavailable Hg (II) in the environment. Appl. Environ. Microbiol. 59 (1993), 3083−3090.

Book Review MICHELS, Corinne A. Genetic Techniques for Biological Research A Case Study Approach

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