A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae
A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae
复制标题
DOI：
10.1073/pnas.0803281105
复制
发表时间：
2008-06-24
影响因子：
11.1
通讯作者：
Mekalanos, John J.
中科院分区：
文献类型：
--
作者：
Cameron, D. Ewen;Urbach, Jonathan M.;Mekalanos, John J.
定义的突变体库允许有效的基因组规模的筛选，并提供了一个方便的收集突变的几乎任何非必需的基因的兴趣。在这里，我们提出了一个近饱和的转座子插入库霍乱弧菌菌株C6706，临床分离属于01埃尔托生物型负责目前的霍乱大流行。对23，312个突变体的自动测序分析使我们能够构建一个3，156个成员的子集文库，其中包含每个破坏的ORF中的代表性插入。由于影响运动性的未表征突变已显示出在减毒霍乱弧菌活疫苗中的效用，因此我们使用该全基因组亚组文库来定义运动性所需的所有基因，并进一步评估文库的准确性和纯度。在这个屏幕上，我们确定了假设的基因VC 2208（flgT）的运动性是必不可少的。flgT突变体中的鞭毛细胞非常罕见，转录分析表明它特别停滞在霍乱弧菌鞭毛调节系统的III/IV类装配检查点。因为预测FlgT与TolB（一种参与决定外膜结构的蛋白质）具有结构同源性，并且霍乱弧菌鞭毛的鞘似乎来源于细胞的外膜，所以FlgT可能在鞭毛鞘的形成中起直接作用。
Defined mutant libraries allow for efficient genome-scale screening and provide a convenient collection of mutations in almost any nonessential gene of interest. Here, we present a near-saturating transposon insertion library in Vibrio cholerae strain C6706, a clinical isolate belonging to the 01 El Tor biotype responsible for the current cholera pandemic. Automated sequencing analysis of 23,312 mutants allowed us to build a 3,156-member subset library containing a representative insertion in every disrupted ORF. Because uncharacterized mutations that affect motility have shown utility in attenuating V. cholerae live vaccines, we used this genome-wide subset library to define all genes required for motility and to further assess the accuracy and purity of the library. In this screen, we identified the hypothetical gene VC2208 (flgT) as essential for motility. Flagellated cells were very rare in a flgT mutant, and transcriptional analysis showed it was specifically stalled at the class III/IV assembly checkpoint of the V. cholerae flagellar regulatory system. Because FlgT is predicted to have structural homology to TolB, a protein involved in determining outer membrane architecture, and the sheath of the V. cholerae flagellum appears to be derived from the cell's outer membrane, FlgT may play a direct role in flagellar sheath formation.