Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.

TitleControlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.
Publication TypeJournal Article
Year of Publication2005
AuthorsEhrt S, Guo XV, Hickey CM, Ryou M, Monteleone M, Riley LW, Schnappinger D
JournalNucleic Acids Res
Date Published2005 Feb 01
KeywordsAnimals, Bacterial Proteins, Cytoskeletal Proteins, Dose-Response Relationship, Drug, Gene Expression Regulation, Bacterial, Macrophages, Mice, Mycobacterium, Mycobacterium smegmatis, Mycobacterium tuberculosis, Operator Regions, Genetic, Phagosomes, Promoter Regions, Genetic, Repressor Proteins, Tetracyclines

Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.

Alternate JournalNucleic Acids Res
PubMed ID15687379
PubMed Central IDPMC548372
Grant ListR01 HL068525 / HL / NHLBI NIH HHS / United States
R01 HL68525 / HL / NHLBI NIH HHS / United States

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