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Journal of Bacteriology, July 2007, p. 4614-4623, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00216-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors{triangledown}

Xinzheng V. Guo,1,2 Mercedes Monteleone,1 Marcus Klotzsche,1 Annette Kamionka,4 Wolfgang Hillen,4 Miriam Braunstein,5 Sabine Ehrt,1,2* and Dirk Schnappinger1,3*

Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10021,1 Program in Immunology and Microbial Pathogenesis,2 Program in Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, 445 East 69th Street, New York, New York 10021,3 Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany,4 Department of Microbiology and Immunology, University of North Carolina, Rm. 804, Mary Ellen Jones Building, Chapel Hill, North Carolina 275995

Received 8 February 2007/ Accepted 25 April 2007

Many processes that are essential for mycobacterial growth are poorly understood. To facilitate genetic analyses of such processes in mycobacteria, we and others have developed regulated expression systems that are repressed by a tetracycline repressor (TetR) and induced with tetracyclines, permitting the construction of conditional mutants of essential genes. A disadvantage of these systems is that tetracyclines function as transcriptional inducers and have to be removed to initiate gene silencing. Recently, reverse TetR mutants were identified that require tetracyclines as corepressors. Here, we report that one of these mutants, TetR r1.7, allows efficient repression of lacZ expression in Mycobacterium smegmatis in the presence but not the absence of anhydrotetracycline (atc). TetR and TetR r1.7 also allowed efficient silencing of the essential secA1 gene, as demonstrated by inhibition of the growth of a conditional mutant and dose-dependent depletion of the SecA1 protein after the removal or addition, respectively, of atc. The kinetics of SecA1 depletion were similar with TetR and TetR r1.7. To test whether silencing of secA1 could help identify substrates of the general secretion pathway, we analyzed the main porin of M. smegmatis, MspA. This showed that the amount of cell envelope-associated MspA decreased more than 90-fold after secA1 silencing. We thus demonstrated that TetR r1.7 allows the construction of conditional mycobacterial mutants in which the expression of an essential gene can be efficiently silenced by the addition of atc and that gene silencing permits the identification of candidate substrates of mycobacterial secretion systems.

* Corresponding author. Mailing address for Dirk Schnappinger: Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, C-546, Box 62, New York, NY 10021. Phone: (212) 746-3788. Fax: (212) 746-8587. E-mail: dis2003{at}med.cornell.edu. Mailing address for Sabine Ehrt: Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, A-275A, Box 62, New York, NY 10021. Phone: (212) 746-2994. Fax: (212) 746-8587. E-mail: sae2004{at}med.cornell.edu

{triangledown} Published ahead of print on 4 May 2007.

Journal of Bacteriology, July 2007, p. 4614-4623, Vol. 189, No. 13
0021-9193/07/$08.00+0     doi:10.1128/JB.00216-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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