Lecointe François, Coste Geneviève, Sommer Suzanne, Bailone Adriana
Institut de Génétique et Microbiologie, UMR 8621, Bât. 409, Université Paris-Sud, F-91405 Orsay, France.
Gene. 2004 Jul 7;336(1):25-35. doi: 10.1016/j.gene.2004.04.006.
Deinococcus radiodurans possesses an exceptional capacity to withstand the lethal and mutagenic effects of most form of DNA damage and has received considerable interest for use in both fundamental and applied research. Here we describe vectors that allow regulated expression of Deinococcal genes for functional analysis. The vectors contain the IPTG-regulated Spac system (Pspac promoter and lacI repressor gene), originally designed for Bacillus subtilis, that we have adapted to be functional in D. radiodurans. We show that the Spac system can control the expression of a lacZ reporter gene over two orders of magnitude depending on the inducer concentration and the copy number of the lacI regulatory gene. Furthermore, we demonstrate that the Spac system can be used to regulate the synthesis of a critical repair protein, such as RecA, resulting in a conditional mitomycin-resistant cell phenotype. We have also developed tools for the construction of conditional mutants where the expression of the target gene is regulated by an inducible promoter. The utility of these conditional gene inactivation systems is exemplified by the conditional lethal phenotype of a mutant expressing gyrA from the Pspac promoter.
耐辐射球菌具有非凡的能力，能够承受大多数形式的DNA损伤所带来的致死和诱变效应，并且在基础研究和应用研究中都引起了广泛关注。在此，我们描述了用于功能分析的、可调控耐辐射球菌基因表达的载体。这些载体包含IPTG调控的Spac系统（Pspac启动子和lacI阻遏基因），该系统最初是为枯草芽孢杆菌设计的，我们已对其进行改造使其在耐辐射球菌中发挥功能。我们表明，Spac系统可根据诱导剂浓度和lacI调控基因的拷贝数，将lacZ报告基因的表达控制在两个数量级以上。此外，我们证明Spac系统可用于调控关键修复蛋白（如RecA）的合成，从而产生条件性丝裂霉素抗性细胞表型。我们还开发了用于构建条件突变体的工具，其中靶基因的表达由可诱导启动子调控。表达来自Pspac启动子的gyrA的突变体的条件致死表型例证了这些条件基因失活系统的实用性。
