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Appl Environ Microbiol, March 1998, p. 1006-1012, Vol. 64, No. 3
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Development and Testing of a Bacterial Biosensor for Toluene-Based Environmental Contaminants

Barry M. Willardson,1,* Jon F. Wilkins,2,dagger Timothy A. Rand,1 James M. Schupp,3 Karen K. Hill,2 Paul Keim,3 and Paul J. Jackson2

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 846021; Environmental Molecular Biology Group, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 875452; and Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 86011-56403

Received 2 September 1997/Accepted 2 January 1998

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, Pu. A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and Pu. When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K1/2 values ranging from 39.0 ± 3.8 µM for 3-xylene to 2,690 ± 160 µM for 3-methylbenzylalcohol (means ± standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.

* Corresponding author. Mailing address: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602. Phone: (801) 378-2785. Fax: (801) 378-5474. E-mail: barry_willardson{at}byu.edu.

dagger Present address: Department of Biochemistry, University of Wisconsin---Madison, Madison, WI 53706.




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