Analysis of the F Plasmid TraD Protein in DNA Transfer

在细菌中获得新特征的一种常见机制是通过接合在供体细胞和受体细胞之间传递DNA。本项目将对一种名为Trad的蛋白质的活性进行表征，该蛋白质对于连接特性良好的大肠杆菌F(生育)质粒是必不可少的。TRAD是一个保守的、分布广泛的细胞质膜蛋白家族的成员，它在DNA转移过程中起作用。初步研究表明，Trad是一种依赖于DNA的ATPase，可能参与通过细胞被膜上的接合专一性小孔促进DNA转移。此外，在接合过程中，Trad可能与DNA加工所必需的另外两种转运蛋白(TrA)相互作用，即细胞质TRAI(ORIT尼克酶和DNA解旋酶)和TRAM蛋白。这项工作将在各种体外和体内测试中表征野生型和突变型Trad，重点是表征Trad ATPase活性的酶参数并确定其在结合中的要求；确定Trad与TRAM/TRAI之间的相互作用；以及探测Trad胞外结构域与其他细胞被膜蛋白之间的联系，这些蛋白质是启动DNA代谢所必需的。Trad同源物由其他结合质粒表达，因此对F质粒Trad蛋白的研究可能对理解在多种细菌属之间通过接合传递遗传信息具有深远的意义。

One common mechanism for acquisition of new traits in bacteria is by the transmission of DNA between a donor and a recipient cell via conjugation. This project will characterize the activities of a protein, TraD, essential for conjugation of the well-characterized F (fertility) plasmid of Escherichia coli. TraD is a member of a conserved and broadly distributed family of cytoplasmic membrane proteins that act during the DNA transfer step of conjugation. Preliminary studies have suggested that TraD is a DNA-dependent ATPase, possibly involved in energizing DNA transfer through a conjugation-specific pore in the cell envelope. In addition, TraD probably interacts with two other transfer (Tra) proteins necessary for DNA processing during conjugation, the cytoplasmic TraI (oriT nickase and DNA helicase) and TraM proteins. This work will characterize wild type and mutant forms of TraD in a variety of in vitro and in vivo assays, focusing on characterizing the enzymatic parameters of the TraD ATPase activity and determining its requirement in conjugation; defining interactions between TraD and TraM/TraI; and probing contacts between extra-cytoplasmic domains of TraD and other cell envelope proteins required to signal the initiation of DNA metabolism. TraD homologues are expressed by other conjugal plasmids, thus the study of the F plasmid TraD protein may have profound implications for understanding the transfer of genetic information via conjugation among a wide variety of bacterial genera.