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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...

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Autores principales: Yu, Zhou, Reichheld, Sean E, Cuthbertson, Leslie, Nodwell, Justin R, Davidson, Alan R
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949611/
https://www.ncbi.nlm.nih.gov/pubmed/20831817
http://dx.doi.org/10.1186/1471-2091-11-34
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author Yu, Zhou
Reichheld, Sean E
Cuthbertson, Leslie
Nodwell, Justin R
Davidson, Alan R
author_facet Yu, Zhou
Reichheld, Sean E
Cuthbertson, Leslie
Nodwell, Justin R
Davidson, Alan R
author_sort Yu, Zhou
collection PubMed
description 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 K(m )and k(cat )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.
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spelling pubmed-29496112010-11-03 Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system Yu, Zhou Reichheld, Sean E Cuthbertson, Leslie Nodwell, Justin R Davidson, Alan R BMC Biochem Methodology Article 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 K(m )and k(cat )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. BioMed Central 2010-09-11 /pmc/articles/PMC2949611/ /pubmed/20831817 http://dx.doi.org/10.1186/1471-2091-11-34 Text en Copyright ©2010 Yu et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Yu, Zhou
Reichheld, Sean E
Cuthbertson, Leslie
Nodwell, Justin R
Davidson, Alan R
Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title_full Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title_fullStr Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title_full_unstemmed Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title_short Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
title_sort characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2949611/
https://www.ncbi.nlm.nih.gov/pubmed/20831817
http://dx.doi.org/10.1186/1471-2091-11-34
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