Lehrbereich Mikrobielle Genetik, Eberhard Karls Universität Tübingen, Tübingen, Germany.
Appl Environ Microbiol. 2010 Feb;76(3):680-7. doi: 10.1128/AEM.02416-09. Epub 2009 Dec 4.
Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (tet) system for Staphylococcus aureus. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of tetR or a variant allele, rev-tetR(r2). The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev-TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)tetR constructs, expression from a chromosomal P(xyl/tet)-gfpmut2 fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native S. aureus gene (zwf) was put under tet control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev-TetR control of P(xyl/tet)-gfpmut2. Additionally, TetR was used in trans to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the fabI gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new tet systems and their flexible use for different purposes.
可诱导表达是阐明基因功能的一种有价值的方法。在这里，我们提出了新的金黄色葡萄球菌四环素依赖性基因调控（tet）系统的配置。为了提供改进和扩展的控制模式，构建了用于tetR 或变体等位基因 rev-tetR(r2) 组成型表达的菌株和质粒。编码的调节剂对效应物脱水四环素 (ATc) 的反应不同，ATc 导致靶基因表达被 TetR 诱导或被 rev-TetR 抑制。为了定量和比较质体或染色体（rev-）tetR 构建体介导的调控，测量了来自染色体 P(xyl/tet)-gfpmut2 融合的表达。染色体编码的 TetR 表现出紧密的抑制作用，并允许在 ATc 存在下以剂量依赖的方式进行高水平的基因表达。使用一种在其天然染色体位置下将天然金黄色葡萄球菌基因（zwf）置于 tet 控制下的菌株进一步验证了调节能力。在抑制条件下几乎检测不到转录物量，而在 ATc 处理的细胞中则很高，这反映了紧密的抑制作用。在报告基因测定中，这种类型的控制，称为 Tet-on，比 Tet-off 调节更有效，后者通过 ATc 的添加导致靶基因的下调。通过直接 rev-TetR 对 P(xyl/tet)-gfpmut2 的控制实现并量化了后者。此外，TetR 被用于反式控制用于转录后基因沉默的反义 RNA 的表达。fabI 基因的反义 RNA 表达的诱导导致持续数小时的明显生长迟缓。这些结果证明了新的 tet 系统的效率及其在不同目的下的灵活用途。
