Targeted inactivation of Francisella tularensis genes by group II introns
Targeted inactivation of Francisella tularensis genes by group II introns
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DOI：
10.1128/aem.02905-07
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发表时间：
2008-05-01
影响因子：
4.4
通讯作者：
Klose, Karl E.
中科院分区：
文献类型：
--
作者：
Rodriguez, Stephen A.;Yu, Jieh-Juen;Klose, Karl E.
图拉氏方济氏菌是图拉热症的病原体，由于缺乏在最具毒性的亚种中快速靶向基因中断的遗传技术，对其发病机制的分子机制的研究一直受到阻碍。在这里，我们描述了利用针对图拉氏杆菌而优化的移动第二组内含子(目标启动子)在图拉氏杆菌中进行有效的靶向基因破坏。利用编码氨苄西林耐药性的靶向基因blab，我们证明了该系统在三个不同亚种中高效工作：图拉氏丝孢子虫亚种。Tularensis、图拉氏亚种F.tularensis subsp.Holarctica和“F·tularensis subsp.novicida”。还利用靶标灭活了图拉氏丝孢子菌亚种。Holarctica iglC，一种毒力所需的基因。IglC基因位于方济氏菌致病岛(Fpi)内，该基因已在最强毒力亚种中复制。重要的是，iglC靶子同时针对两个拷贝，导致一个菌株在一个步骤中同时突变了两个iglC基因。这个系统将有助于阐明特定的基因，特别是那些在FPI内的基因，对这种研究较少的生物体的发病机制做出了贡献。
Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.