Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors

泰特 生物 支原体 基因沉默 抑制因子 突变体 分泌物 细胞生物学 基因表达 遗传学 基因 结核分枝杆菌 生物化学 医学 病理 肺结核
作者
Xinzheng V. Guo,Mercedes Monteleone,Marcus Klotzsche,Annette Kamionka,Wolfgang Hillen,Miriam Braunstein,Sabine Ehrt,Dirk Schnappinger
出处
期刊:Journal of Bacteriology [American Society for Microbiology]
卷期号:189 (13): 4614-4623 被引量:107
标识
DOI:10.1128/jb.00216-07
摘要

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.
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