Quantifying promoter activity during the developmental cycle of Chlamydia trachomatis.
Quantifying promoter activity during the developmental cycle of Chlamydia trachomatis.
复制标题
沙眼衣原体发育周期中启动子活性的定量
DOI：
10.1038/srep27244
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发表时间：
2016-06-06
影响因子：
4.6
通讯作者：
Shen L
中科院分区：
文献类型：
--
作者：
Cong Y;Gao L;Zhang Y;Xian Y;Hua Z;Elaasar H;Shen L
沙眼衣原体是一种重要的人类病原体，其经历与阶段特异性基因表达谱相关的特征性发育周期。利用C.本研究构建了一个多功能的绿色荧光蛋白（GFP）报告系统，用于研究沙眼衣原体的发育依赖功能。沙眼衣原体启动子，试图阐明控制沙眼衣原体的机制。沙眼适应性我们通过观察早期euo基因启动子的活性来验证GFP报告系统的使用。此外，我们利用GFP报告系统结合5′端cDNA末端快速扩增（RACE）、体外转录检测、实时定量RT-PCR（RT-qPCR）和流式细胞术，发现了一个新的ompA启动子，命名为P3。P3区的突变证实P3是一个新的C.沙眼衣原体σ66依赖性启动子，其需要延伸的-10 TGn基序进行转录。这些结果证实了C.沙眼利用遗传标记的C.使用P3驱动的GFP的沙眼衣原体生物体允许观察响应于发育信号的ompA表达的变化。本研究的结果可用于补充以前的研究结果，并促进对C。沙眼衣原体基因表达
Chlamydia trachomatis is an important human pathogen that undergoes a characteristic development cycle correlating with stage-specific gene expression profiles. Taking advantage of recent developments in the genetic transformation in C. trachomatis, we constructed a versatile green fluorescent protein (GFP) reporter system to study the development-dependent function of C. trachomatis promoters in an attempt to elucidate the mechanism that controls C. trachomatis adaptability. We validated the use of the GFP reporter system by visualizing the activity of an early euo gene promoter. Additionally, we uncovered a new ompA promoter, which we named P3, utilizing the GFP reporter system combined with 5′ rapid amplification of cDNA ends (RACE), in vitro transcription assays, real-time quantitative RT-PCR (RT-qPCR) and flow cytometry. Mutagenesis of the P3 region verifies that P3 is a new class of C. trachomatis σ66-dependent promoter, which requires an extended −10 TGn motif for transcription. These results corroborate complex developmentally controlled ompA expression in C. trachomatis. The exploitation of genetically labeled C. trachomatis organisms with P3-driven GFP allows for the observation of changes in ompA expression in response to developmental signals. The results of this study could be used to complement previous findings and to advance understanding of C. trachomatis genetic expression.