A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis.
A broad-spectrum cloning vector that exists as both an integrated element and a free plasmid in Chlamydia trachomatis.
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DOI：
10.1371/journal.pone.0261088
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Rockey D
中科院分区：
文献类型：
--
作者：
Garvin L;Vande Voorde R;Dickinson M;Carrell S;Hybiske K;Rockey D
衣原体的质粒转化为研究衣原体感染过程中宿主-微生物相互作用创造了新的机会;然而，仍然存在局限性。质粒转化需要来自天然衣原体质粒的复制子，并且这些转化是物种特异性的。我们探索了广泛的宿主范围质粒pBBR 1 MCS-4转化衣原体的效用，目的是简化转化过程。将质粒修饰为含有来自C.以促进同源重组。将印加侧翼序列与来自pSW 2-GFP衣原体穿梭载体的GFP：CAT盒一起沿着克隆到pBBR 1 MCS-4载体中。将最终的质粒构建体pBVR 2成功地转化到C.沙眼衣原体菌株L2-434。通过免疫荧光显微镜分析衣原体转化体，并使用有限稀释法依次纯化阳性克隆。使用PCR和基于PacBio-based的全基因组测序来确定质粒是否保持在染色体内或作为附加体。对克隆转化体的PacBio测序揭示了染色体和质粒pBVR 2之间的等位基因交换事件，该事件用质粒GFP：CAT盒取代了染色体印加。数据还显示了质粒完全整合到细菌染色体中的证据。虽然有些质粒完全整合，但有些质粒仍以附加体形式存在，可以纯化并重新转化到E.杆菌因此，质粒可以成功地转化到没有衣原体复制起点的衣原体中，并且可以在转化的群体中以多种状态存在。
Plasmid transformation of chlamydiae has created new opportunities to investigate host–microbe interactions during chlamydial infections; however, there are still limitations. Plasmid transformation requires a replicon derived from the native Chlamydia plasmid, and these transformations are species-specific. We explored the utility of a broad host-range plasmid, pBBR1MCS-4, to transform chlamydiae, with a goal of simplifying the transformation process. The plasmid was modified to contain chromosomal DNA from C. trachomatis to facilitate homologous recombination. Sequences flanking incA were cloned into the pBBR1MCS-4 vector along with the GFP:CAT cassette from the pSW2-GFP chlamydial shuttle vector. The final plasmid construct, pBVR2, was successfully transformed into C. trachomatis strain L2-434. Chlamydial transformants were analyzed by immunofluorescence microscopy and positive clones were sequentially purified using limiting dilution. PCR and PacBio-based whole genome sequencing were used to determine if the plasmid was maintained within the chromosome or as an episome. PacBio sequencing of the cloned transformants revealed allelic exchange events between the chromosome and plasmid pBVR2 that replaced chromosomal incA with the plasmid GFP:CAT cassette. The data also showed evidence of full integration of the plasmid into the bacterial chromosome. While some plasmids were fully integrated, some were maintained as episomes and could be purified and retransformed into E. coli. Thus, the plasmid can be successfully transformed into chlamydia without a chlamydial origin of replication and can exist in multiple states within a transformed population.
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