Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality
Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality
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DOI：
10.1054/tube.2001.0312
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发表时间：
2001-01-01
期刊：
影响因子：
3.2
通讯作者：
Stoker, NG
中科院分区：
文献类型：
--
作者：
Parish, T;Lewis, J;Stoker, NG
背景：证明基因是必需的总是很困难，但对于结核分枝杆菌等生长缓慢的生物体来说尤其如此。目前使用的一种方法是证明导致基因失活的同源重组仅在存在第二个基因拷贝的情况下发生，但获得具有统计学意义的数据可能非常困难，基于L5的整合质粒已广泛用于分枝杆菌的遗传分析。 L5 切除酶已被用于耻垢分枝杆菌中，从染色体上切除和回收这些质粒。 目的：我们的目的是确定 L5 切除酶是否可以在结核分枝杆菌中发挥作用，去除基于 L5 的整合质粒，如果可以，则使用该技术作为改进方法的基础，以确定基因是否为必需基因。 设计：我们采用了两种携带必需基因 gInE 的结核分枝杆菌菌株，它们在基于 L5 的染色体上整合到染色体上。质粒，其中之一缺乏该基因的功能性染色体拷贝。我们用表达 L5 切除酶的载体转化它们，并寻找整合质粒的丢失。结果：我们从野生型菌株中获得了整合载体的有效切除。然而，当整合载体携带必需基因glnE的唯一功能性拷贝时，回收的菌落数量减少至背景水平。结论：L5切除酶在结核分枝杆菌中确实起作用，并且可用于确认基因的必需性。该技术还可以对必需基因进行进一步分析，而使用当前方法很难或不可能进行这些分析。 (C) 2001 年哈考特出版有限公司。
Setting: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult, L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome.Objective: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential.Design:We took two strains of M. tuberculosis carrying the essential gene gInE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid.Results: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels.Conclusion: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods. (C) 2001 Harcourt Publishers Ltd.