Development of Transposon Mutagenesis for Chlamydia muridarum.
Development of Transposon Mutagenesis for Chlamydia muridarum.
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鼠衣原体转座子诱变的发展。
DOI：
10.1128/jb.00366-19
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发表时间：
2019
影响因子：
3.2
通讯作者：
Hybiske,Kevin
中科院分区：
文献类型：
--
作者：
Wang,Yibing;LaBrie,ScottD;Carrell,StevenJ;Suchland,RobertJ;Dimond,ZoeE;Kwong,Forrest;Rockey,DanielD;Hefty,PScott;Hybiske,Kevin
衣原体的功能遗传学分析一直是一个挑战，由于历史的遗传棘手性衣原体，虽然最近的进展衣原体基因操作已经开始消除这些障碍。在这里，我们报告了鼠衣原体的Himar C9转座子系统的发展，鼠衣原体是一种小鼠适应的衣原体，广泛用于衣原体感染模型。我们展示了33个氯霉素（Cam）抗性，绿色荧光蛋白（GFP）表达的C.鼠伤寒沙门氏菌转座子突变体大多数突变体含有遍布整个C.海蛞蝓染色体结果表明，该文库在编码开放阅读框（ORF）中含有31个转座子插入，在基因间区含有7个转座子插入。17个突变克隆的全基因组测序分析证实了插入的染色体位置。进一步研究了lgB、pmpI、pmpA和pmpD四种转座子插入突变体的体外和体内表型，包括生长、包涵体形态和与宿主细胞的粘附。glgB突变体不能完成糖原的生物合成并在突变体包涵体的内腔中积累。在3 pmp突变体中，pmpI表现出最明显的生长衰减缺陷。这个初始文库证明了稳定的、同基因的转座子突变体对C. muridarum。一个完整的C.重要提示：衣原体基因操作的历史问题已经阻碍了衣原体基因组中1,000多个基因的严格功能遗传学表征。在这里，我们报告了一个转座子诱变系统的C。muridarum是一种小鼠适应的衣原体种，广泛用于衣原体发病机制的体内研究。这一进展建立在该系统的开拓性发展的C。沙眼我们证明了一个初始库的33个突变体含有稳定的单或双转座子插入的产生。使用这些突变体克隆，我们表征了与糖原生物合成和三种多态性外膜蛋白的遗传破坏相关的体外表型。
Functional genetic analysis ofChlamydiahas been a challenge due to the historical genetic intractability ofChlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adaptedChlamydiaspecies that is widely used inChlamydiainfection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions inglgB,pmpI,pmpA, andpmpDwere investigated further forin vitroandin vivophenotypes, including growth, inclusion morphology, and attachment to host cells. TheglgBmutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3pmpmutants,pmpIwas shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements forChlamydiainfectionin vivo.IMPORTANCEHistorical issues with genetic manipulation ofChlamydiahave prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adaptedChlamydiaspecies that is widely used forin vivoinvestigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterizedin vitrophenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.