Nguyen Bidong D, Valdivia Raphael H
Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center.
J Vis Exp. 2013 Oct 23(80):e50636. doi: 10.3791/50636.
Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype-phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.
沙眼衣原体是性传播疾病和眼部感染的病原体，由于其难以用重组DNA进行实验转化，其特征仍不清楚。尽管缺乏分子遗传学工具，我们还是开发了一种在沙眼衣原体中进行遗传分析的方法。我们的方法包括：i）化学诱变，以快速生成具有不同表型的基因定义突变体的综合文库；ii）全基因组测序（WGS），以绘制潜在的基因损伤图谱，并找出突变基因与共同表型之间的关联；iii）通过将突变体和野生型细菌共同感染哺乳动物细胞来产生重组菌株。因此，我们能够建立基因型和表型之间的因果关系。化学诱导的基因变异与WGS相结合以建立相关的基因型-表型关联，应该广泛适用于目前难以进行遗传分析的大量具有医学和环境重要性的微生物。
