Analysis of the F Plasmid TraD Protein in DNA Transfer

在细菌中获得新特性的一种常见机制是通过结合在供体细胞和受体细胞之间传递DNA。本项目将对一种蛋白质TraD的活性进行表征，TraD对大肠杆菌F（育性）质粒的结合至关重要。TraD是一个保守的、广泛分布的细胞质膜蛋白家族的成员，在DNA结合的转移步骤中起作用。初步研究表明，TraD是一种DNA依赖的atp酶，可能参与激活DNA通过细胞包膜中结合特异性孔的转移。此外，TraD可能与接合过程中DNA加工所必需的另外两种转移（Tra）蛋白，细胞质TraI （oriT nickase和DNA解旋酶）和TraM蛋白相互作用。这项工作将在各种体外和体内试验中表征野生型和突变型TraD，重点是表征TraD atp酶活性的酶参数，并确定其在偶联中的要求；界定TraD与TraM/TraI之间的相互作用；以及探测TraD胞质外结构域与其他细胞包膜蛋白之间的接触，这些包膜蛋白是DNA代谢启动的必要信号。TraD同源物可以通过其他偶联质粒表达，因此对F质粒TraD蛋白的研究可能对理解多种细菌属间通过偶联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.