Klotzsche Marcus, Ehrt Sabine, Schnappinger Dirk
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.
Nucleic Acids Res. 2009 Apr;37(6):1778-88. doi: 10.1093/nar/gkp015. Epub 2009 Jan 27.
Tetracycline repressor (TetR)-controlled expression systems have recently been developed for mycobacteria and proven useful for the construction of conditional knockdown mutants and their analysis in vitro and during infections. However, even though these systems allowed tight regulation of some mycobacterial genes, they only showed limited or no phenotypic regulation for others. By adapting their codon usage to that of the Mycobacterium tuberculosis genome, we created tetR genes that mediate up to approximately 50-fold better repression of reporter gene activities in Mycobacterium smegmatis and Mycobacterium bovis BCG. In addition to these repressors, for which anhydrotetracycline (atc) functions as an inducer of gene expression, we used codon-usage adaption and structure-based design to develop improved reverse TetRs, for which atc functions as a corepressor. The previously described reverse repressor TetR only functioned when expressed from a strong promoter on a multicopy plasmid. The new reverse TetRs silence target genes more efficiently and allowed complete phenotypic silencing of M. smegmatis secA1 with chromosomally integrated tetR genes.
四环素阻遏蛋白（TetR）调控的表达系统最近已被开发用于分枝杆菌，并已证明对构建条件性敲除突变体及其在体外和感染过程中的分析有用。然而，尽管这些系统能够严格调控某些分枝杆菌基因，但对其他基因仅表现出有限的或无表型调控。通过使其密码子使用适应结核分枝杆菌基因组的密码子使用，我们创建了tetR基因，其在耻垢分枝杆菌和牛分枝杆菌卡介苗中对报告基因活性的抑制作用提高了约50倍。除了这些以脱水四环素（atc）作为基因表达诱导剂的阻遏蛋白外，我们还利用密码子使用适应和基于结构的设计开发了改进的反向TetR，其中atc作为共阻遏物。先前描述的反向阻遏蛋白TetR仅在从多拷贝质粒上的强启动子表达时才起作用。新的反向TetR能更有效地沉默靶基因，并能使耻垢分枝杆菌secA1基因在染色体整合tetR基因的情况下实现完全表型沉默。
