Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.
Site-specific, insertional inactivation of incA in Chlamydia trachomatis using a group II intron.
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DOI：
10.1371/journal.pone.0083989
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Fisher DJ
中科院分区：
文献类型：
--
作者：
Johnson CM;Fisher DJ
沙眼衣原体是一种专性的细胞内细菌病原体，直到最近仍然难以进行基因操作。然而，由于缺乏创建可选择的、有针对性的染色体突变的工具，该领域仍然受到阻碍。先前对移动组 II 内含子的研究表明，可以通过改变内含子底物识别区域内的 DNA 序列来重新定位它们，以创建位点特异性基因插入。该平台（商品名为 TargeTron™，Sigma）已成功应用于多种细菌。随后，我们修改了 TargeTron™ 以用于沙眼衣原体，并作为原理证明，使用我们的系统插入灭活 incA，这是一种编码衣原体包涵体同型融合所需蛋白质的染色体基因。用氨苄青霉素选择沙眼衣原体 incA::GII(bla) 突变体，然后分离噬菌斑纯化的克隆用于基因型和表型分析。 PCR、Southern blotting 和 DNA 测序验证了 GII(bla) 的正确插入，而在没有选择的情况下连续传代则表明插入是稳定的。正如天然存在的 IncA− 突变体所见，光学和免疫荧光显微镜证实，在感染复数大于 1 的 incA::GII(bla) 突变体感染的细胞中存在非融合内含物。通过蛋白质印迹进一步证实突变克隆不产生 IncA。最终，重新定位内含子的容易性、选择突变体的能力以及在没有选择的情况下内含子的稳定性使得该方法成为不断增长的衣原体分子工具箱的有力补充。
Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that has until more recently remained recalcitrant to genetic manipulation. However, the field still remains hindered by the absence of tools to create selectable, targeted chromosomal mutations. Previous work with mobile group II introns demonstrated that they can be retargeted by altering DNA sequences within the intron’s substrate recognition region to create site-specific gene insertions. This platform (marketed as TargeTron™, Sigma) has been successfully employed in a variety of bacteria. We subsequently modified TargeTron™ for use in C. trachomatis and as proof of principle used our system to insertionally inactivate incA, a chromosomal gene encoding a protein required for homotypic fusion of chlamydial inclusions. C. trachomatis incA::GII(bla) mutants were selected with ampicillin and plaque purified clones were then isolated for genotypic and phenotypic analysis. PCR, Southern blotting, and DNA sequencing verified proper GII(bla) insertion, while continuous passaging in the absence of selection demonstrated that the insertion was stable. As seen with naturally occurring IncA− mutants, light and immunofluorescence microscopy confirmed the presence of non-fusogenic inclusions in cells infected with the incA::GII(bla) mutants at a multiplicity of infection greater than one. Lack of IncA production by mutant clones was further confirmed by Western blotting. Ultimately, the ease of retargeting the intron, ability to select for mutants, and intron stability in the absence of selection makes this method a powerful addition to the growing chlamydial molecular toolbox.
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影响因子：
3.7
作者：
Mital J;Miller NJ;Dorward DW;Dooley CA;Hackstadt T
通讯作者：
Hackstadt T
影响因子：
3.7
作者：
Fisher DJ;Fernández RE;Adams NE;Maurelli AT
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Maurelli AT
影响因子：
2.6
作者：
Akhtar P;Khan SA
通讯作者：
Khan SA
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作者：
Pannekoek, Y;Spaargaren, J;van der Ende, A
通讯作者：
van der Ende, A
影响因子：
3.4
作者：
Hackstadt, T;Scidmore-Carlson, MA;Fischer, ER
通讯作者：
Fischer, ER