Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects
Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion in Chlamydia trachomatis and Can Reverse Cassette-Induced Polar Effects
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DOI：
10.1128/jb.00479-18
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发表时间：
2018-12-01
影响因子：
3.2
通讯作者：
Fields, K. A.
中科院分区：
文献类型：
--
作者：
Keb, G.;Hayman, R.;Fields, K. A.
作为专性细胞内细菌，衣原体属。已经进化出许多可能错综复杂的机制来创建和维持特权细胞内生态位。最近的进展，阐明和表征这些过程得到了支持的技术发展，使基本的遗传温顺。荧光报道的等位基因交换诱变（FRAEM）将染色体基因缺失与编码抗生素抗性和绿色荧光蛋白（GFP）的选择盒的插入偶联。与其他细菌类似，衣原体的许多基因存在于多顺反子操纵子中，这增加了插入盒介导的极性效应的可能性。事实上，FRAEM介导的沙眼衣原体tmeA的缺失对tine的表达产生负面影响。我们通过采用侧接loxP位点的gfp-bla盒来调整FRAEM技术。Cre重组酶在含有floxed盒的衣原体tmeA中的条件表达导致标记物的缺失和tmeB表达的恢复。沙眼感染是人类健康的重大负担。基因操纵衣原体的能力。正在克服历史上阻碍对衣原体发病机制的全面了解取得快速进展的混淆障碍。衣原体基因操作的现状。需要进一步发展，包括产生无标记基因破坏的机制。我们利用逐步Cre-lox方法从缺失的基因位点切除选择标记基因。我们发现这一过程是有效的，并且去除外来元素导致对下游基因的负极性效应逆转。该技术有助于更直接地评估基因功能，并通过促进操纵子内基因的缺失来增加衣原体分子工具箱。
As obligate intracellular bacteria, Chlamydia spp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion of Chlamydia trachomatis tmeA negatively impacts the expression of tine& We have adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in Chlamydia tmeA containing a floxed cassette resulted in deletion of the marker and restoration of tmeB expression.IMPORTANCE C. trachomatis infections represent a significant burden to human health. The ability to genetically manipulate Chlamydia spp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation in Chlamydia spp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to the Chlamydia molecular toolbox by facilitating the deletion of genes within operons.