Silencing essential protein secretion in Mycobacterium smegmatis by using tetracycline repressors
Silencing essential protein secretion in Mycobacterium smegmatis by using tetracycline repressors
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DOI：
10.1128/jb.00216-07
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发表时间：
2007-07-01
影响因子：
3.2
通讯作者：
Schnappinger, Dirk
中科院分区：
文献类型：
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作者：
Guo, Xinzheng V.;Monteleone, Mercedes;Schnappinger, Dirk
人们对分枝杆菌生长所必需的许多过程知之甚少。为了促进分枝杆菌中此类过程的遗传分析，我们和其他人开发了受四环素阻遏物（TetR）抑制并用四环素诱导的调节表达系统，从而允许构建必需基因的条件突变体。这些系统的缺点是四环素起到转录诱导剂的作用，必须去除四环素才能启动基因沉默。最近，鉴定出需要四环素作为辅阻遏物的反向 TetR 突变体。在这里，我们报道了这些突变体之一，TetR r1.7，可以在无水四环素（ate）存在但不存在的情况下有效抑制耻垢分枝杆菌中的 lacZ 表达。 TetR和TetR r1.7还允许有效沉默必需的secA1基因，如通过分别去除或添加atc后条件突变体生长的抑制和SecA1蛋白的剂量依赖性消耗所证明的。 TetR 和 TetR r1.7 的 SecAl 消耗动力学相似。为了测试 secA1 的沉默是否有助于识别一般分泌途径的底物，我们分析了耻垢分枝杆菌的主要孔蛋白 MspA。这表明在 secA1 沉默后，细胞被膜相关的 MspA 量减少了 90 倍以上。因此，我们证明TetR r1.7允许构建条件分枝杆菌突变体，其中通过添加atc可以有效地沉默必需基因的表达，并且基因沉默允许鉴定分枝杆菌分泌系统的候选底物。
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 (ate). 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 SecAl protein after the removal or addition, respectively, of atc. The kinetics of SecAl 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.