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Open Access Highly Accessed Methodology article

Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system

Zhou Yu1, Sean E Reichheld2, Leslie Cuthbertson3, Justin R Nodwell3 and Alan R Davidson12*

Author Affiliations

1 Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada

2 Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada

3 DeGroote Institute for Infectious Diseases Research, Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street W, Hamilton, ON, L8N 3Z5, Canada

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BMC Biochemistry 2010, 11:34  doi:10.1186/1471-2091-11-34

Published: 11 September 2010

Abstract

Background

Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required.

Results

We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known Km and kcat values, demonstrating the usefulness of this system for quantitative analyses.

Conclusion

Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications.