NDLI: Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors▿

Content Provider	PubMed Central
Author	Guo, Xinzheng V. Monteleone, Mercedes Marcus, Klotzsche Annette, Kamionka Hillen, Wolfgang Braunstein, Miriam Ehrt, Sabine Dirk, Schnappinger
Copyright Year	2007
Abstract	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.
Related Links	http://dx.doi.org/10.1128/jb.00216-07
Ending Page	4623
Page Count	10
Starting Page	4614
File Format	PDF
ISSN	00219193
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	13
Volume Number	189
Language	English
Publisher	American Society for Microbiology
Publisher Date	2007-07-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Molecular Biology Microbiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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