Published online 1 February 2005
Methods Online |
Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor
1 Department of Microbiology and Immunology, Weill Medical College of Cornell University New York, NY 10021, USA 2 Division of Infectious Diseases, School of Public Health, University of California Berkeley, CA 94720, USA 3 Graduate Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University New York, NY 10021, USA 4 Graduate Program in Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University New York, NY 10021, USA
*To whom correspondence should be addressed at Sabine Ehrt, WMC of Cornell University, Department of Microbiology and Immunology, 1300 York Avenue, New York, NY, 10021, USA. Tel: +1 212 746 2994; Fax: +1 212 746 8587; Email: sae2004{at}med.cornell.edu
Received November 19, 2004. Revised December 21, 2004. Accepted December 21, 2004.
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.
Correspondence may also be addressed to Dirk Schnappinger, WMC of Cornell University, Department of Microbiology and Immunology, 1300 York Avenue, New York, NY, 10021, USA. Tel: +1 212 746 3788; Fax: +1 212 746 8587; Email: dis2003{at}med.cornell.edu
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