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


CONCLUSIONS
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.
Yu
Zhou
Z
Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
Reichheld
Sean E
SE
Cuthbertson
Leslie
L
Nodwell
Justin R
JR
Davidson
Alan R
AR
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013752:Tetracycline / Q000378:metabolism*; D013753:Tetracycline Resistance
D000900:Anti-Bacterial Agents; D004798:Enzymes; D029968:Escherichia coli Proteins; C470187:TetR(B) protein, E coli; D013752:Tetracycline