Forward and Reverse Genetics in Chlamydia

描述（由申请人提供）：使用分子遗传学工具在致病菌中产生和补充突变是我们理解微生物如何引起疾病的核心。不幸的是，许多临床上重要的病原体，如沙眼衣原体——致盲性沙眼和许多性传播疾病的病原体——不适合这种分析。在此，我们建议建立一个基于化学诱变剂、基因重组和“深度”DNA测序技术的衣原体正向和反向遗传分析综合系统。我们的具体目标是：1)通过实施前向遗传筛选，包括II型分泌物的遗传分析，确定沙眼原体发病机制中的重要因素；2)开发一种系统的沙眼原体反向遗传分析方法。

DESCRIPTION (provided by applicant): The use of molecular genetic tools to generate and complement mutations in pathogenic bacteria is central to our understanding of how microorganisms cause disease. Unfortunately, many clinically important pathogens, such as Chlamydia trachomatis -the causative agent of blinding trachoma and many sexually transmitted diseases- are not amenable to such analysis. Here we propose to establish a comprehensive system for forward and reverse genetic analysis in Chlamydia based on the use of chemical mutagens, genetic recombination and "deep" DNA sequencing technologies. Our specific goals are to 1) identify factors important in C. trachomatis pathogenesis by implementing forward genetic screens, including a genetic analysis of Type II secretion and 2) develop a method for systematic reverse genetic analysis in C. trachomatis. By the completion of these experiments we will have established methods to perform routine forward and genetic screens - the lynchpin of microbial pathogenesis research. The tools and reagents generated through the proposed work will constitute an important resource for the Chlamydia research community.