Improved tetracycline repressors for gene silencing in mycobacteria

被引:74
作者
Klotzsche, Marcus [1 ]
Ehrt, Sabine [1 ]
Schnappinger, Dirk [1 ]
机构
[1] Weill Cornell Med Coll, Dept Microbiol & Immunol, New York, NY 10065 USA
基金
美国国家卫生研究院; 英国惠康基金;
关键词
REGULATORY ELEMENTS; TET REPRESSOR; BACILLUS-SUBTILIS; ESCHERICHIA-COLI; IN-VIVO; EXPRESSION; TUBERCULOSIS; SMEGMATIS; RESISTANCE; BACTERIA;
D O I
10.1093/nar/gkp015
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 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.
引用
收藏
页码:1778 / 1788
页数:11
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