Molecular Cloning and Functional Analysis of a Novel Tetracycline Resistance Determinant, tet(V), from Mycobacterium smegmatis |
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Authors: | Edda De Rossi Marian C. J. Blokpoel Rita Cantoni Manuela Branzoni Giovanna Riccardi Douglas B. Young Koen A. L. De Smet Orio Ciferri |
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Affiliation: | Department of Genetics and Microbiology, University of Pavia, 27100 Pavia, Italy,1. and Department of Medical Microbiology, Imperial College School of Medicine, London W2 1PG, United Kingdom2. |
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Abstract: | The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatis harboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to M. smegmatis and M. fortuitum. |
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