Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
Microbiology (Reading). 2011 Dec;157(Pt 12):3314-3323. doi: 10.1099/mic.0.052548-0. Epub 2011 Sep 15.
A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P(xyl/tet). Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR-P(xyl/tet) regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P(xyl/tet)-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.
本文介绍了一套用于优化金黄色葡萄球菌中四环素依赖型基因调控的载体。质粒 pRAB11 是通过在 TetR 调控的启动子 P(xyl/tet) 内添加第二个 tet 操纵子，从 pRMC2 衍生而来的。在没有脱水四环素（ATc）的情况下观察到明显的抑制作用，同时在存在药物的情况下观察到强烈的诱导作用，这在携带金黄色葡萄球菌核酸酶 nuc1、lacZ 或 gfp 的 pRAB11 中得到了证明。此外，在质粒 pCG261 中，pRAB11 tetR-P(xyl/tet) 调控结构允许紧密抑制和靶基因 rny（假定的核糖核酸酶）的转录物数量逐步增加，与 ATc 浓度的升高相关。此外，构建了携带半理性设计的 P(xyl/tet)-样片段的 pRAB11 衍生载体，这些片段在多达六个定义的位置发生突变。分析了 16 个具有单到四交换的突变序列的转录强度和 ATc 依赖性诱导能力。提出了一组具有逐渐降低活性和改善抑制作用的启动子。最后，实施了反向 TetR revtetR-r2，它在存在 ATc 的情况下表现出三个氨基酸交换并与 tetO 结合，在 pRAB11 系统内产生了一种有效共抑制的载体。有趣的是，仅在金黄色葡萄球菌中繁殖后才发现 revtetR 含有第四个突变。我们预测，所描述的载体构成了葡萄球菌遗传学的有价值工具。
