Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis
Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis
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DOI：
10.1073/pnas.94.20.10955
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发表时间：
1997-09-30
影响因子：
11.1
通讯作者：
Guilhot, C
中科院分区：
文献类型：
--
作者：
Pelicic, V;Jackson, M;Guilhot, C
更好地了解结核分枝杆菌的毒力机制高度依赖于有效诱变系统的设计。开发了一种系统，使插入突变体失去传递载体的阳性选择。它使用ts-sacB载体，它结合了sacB基因的反选择特性和分枝杆菌的热敏复制起源，因此可以在39℃的蔗糖上有效地反选择。这种方法允许构建结核分枝杆菌转位突变文库。获得了超过10(6)个突变体，远远超过了在每个非必需基因中获得至少一个插入所需的理论数量。该系统对于基因交换诱变也很有效，正如purC基因所证明的那样：100%的选择克隆是等位基因交换突变体。因此，单一，简单的方法使我们能够开发强大的诱变系统，缺乏这种诱变系统是结核分枝杆菌遗传表征的主要障碍。
A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. A system enabling the positive selection of insertional mutants having lost the delivery vector was developed. It uses ts-sacB vectors, which combine the counterselective properties of the sacB gene and a mycobacterial thermosensitive origin of replication and can therefore be efficiently counterselected on sucrose at 39 degrees C. This methodology allowed the construction of M. tuberculosis transposition mutant libraries. Greater than 10(6) mutants were obtained, far exceeding the number theoretically required to obtain at least one insertion in every nonessential gene, This system is also efficient for gene exchange mutagenesis as demonstrated with the purC gene: 100% of the selected clones were allelic exchange mutants, Therefore, a single, simple methodology has enabled us to develop powerful mutagenesis systems, the lack of which was a major obstacle to the genetic characterization of M. tuberculosis.