First report of a tetracycline-inducible gene expression system for mollicutes
First report of a tetracycline-inducible gene expression system for mollicutes
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DOI：
10.1099/mic.0.034074-0
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发表时间：
2010-01-01
期刊：
影响因子：
2.8
通讯作者：
Renaudin, Joel
中科院分区：
文献类型：
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作者：
Breton, Marc;Sagne, Evelyne;Renaudin, Joel
可诱导启动子系统是研究原核生物基因功能的有力工具，但从未被证明在软体动物中起作用。在这项研究中，我们评价了来自枯草芽孢杆菌的四环素诱导启动子Pxyl/Teto(2)在植物病原体柑橘螺旋体和动物病原体无乳支原体中控制基因表达的效果。将携带由XYL/Teto(2)四环素可诱导启动子控制的螺林基因和由构成螺旋体启动子控制的TetR阻遏基因的柑橘链霉菌载体导入无螺林柑橘链霉菌突变体GII3-9a3。在没有四环素的情况下，TetR的表达几乎完全取消了XYL/Teto(2)启动子的螺林表达。在培养液中加入四环素(>；50ngml(-1))可诱导螺旋素的高水平表达。有趣的是，在体内也检测到了螺灵的诱导表达：在摄食含有四环素的培养基上感染柑橘链球菌的叶蝉中，以及在浇水四环素的柑橘链球菌感染的植物中。通过转座将类似的结构引入到无乳支原体的染色体中。四环素诱导螺林的表达证明了TetR-Pxyl/Teto(2)系统在反刍动物病原菌中的功能，这表明这个四环素诱导的启动子系统可能在软体动物中具有普遍用途。
Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO(2) from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO(2) tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO(2) promoter. Adding tetracycline (>50 ng ml(-1)) to the medium induced high-level expression of spiralin. interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO(2) system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.