DNA Translocation Across the Agrobacterium Envelope

描述(申请人提供)：大分子跨 原核膜和真核膜是生物医学研究的重要领域。在……里面 近年来，与细菌鞭毛和细菌鞭毛有关的分泌系统 结合系统已被证明在致病机制中发挥重要作用 将效应分子转移到真核细胞胞浆中 感染病程。这些系统现在被指定为类型III和类型IV 分别是分泌途径。这个实验室的工作重点是 根癌农杆菌使用的IV型转移系统将致癌的T-DNA运送到 敏感的植物细胞。T-DNA转移系统是一个很好的模型 IV型分泌物的详细机制研究。最近的研究表明， T-DNA转移系统在底物方面具有特殊的多功能性 选择和目标小区识别。除了T-DNA的分泌 底物，该系统可以输出其他DNA底物以及效应器 与DNA无关的蛋白质。此外，该系统还可以移位 其他细菌和各种各样的真核细胞类型的底物， 包括植物、真菌和人类。这方面工作的总体目标是 实验室的任务是：i)确定这种IV型转移的组装途径 系统，二)描述其体系结构安排，三)阐明 衬底加工和传送到 分泌通道，以及iv)定义了跨 革兰氏阴性细菌包膜。现在已经确定，底物转移 需要一个用于调节细胞-细胞接触的管状物和一个用于 在细菌细胞膜上的移位。我们将结合使用 分子、遗传和生物化学方法在以下特定目的。 首先，我们将探索内膜的结构-功能关系 VirB11 ATPase确定其在转运体生物发生和底物中的作用 移位；对于这些研究，我们将利用我们的 功能改变突变体的大量集合和一种 VirB11同源基因。第二，我们将确定衬底输送中的步骤 途径，重点是模型的VirE2底物和VirE1的分泌 监护人。最后，我们将描述组装过程中的关键步骤 ；这些研究将确定具体的联系人 涉及转运蛋白组装和结构所必需的三种蛋白质 整体性，多角体内膜蛋白VirB6和两个外膜 蛋白质、病毒B7脂蛋白和病毒B9。

DESCRIPTION (provided by applicant):Macromolecular translocation across prokaryotic and eukaryotic membranes is a major area of biomedical interest. In recent years, secretion systems ancestrally-related to bacterial flagellar and conjugation systems have been shown to play important roles in pathogenesis by translocating effector molecules to the eukaryotic cell cytosol during the course of infection. These systems are now designated as types III and IV secretion pathways, respectively. The focus of work in this laboratory is the type IV transfer system used by A. tumefaciens to deliver oncogenic T-DNA to susceptible plant cells. The T-DNA transfer system is an excellent model for detailed mechanistic studies of type IV secretion. Recent work has shown that the T-DNA transfer system is exceptionally versatile both in terms of substrate selection and target cell recognition. In addition to the T-DNA secretion substrate, this system can export other DNA substrates as well as effector proteins independently of DNA. Furthermore, this system can translocate substrates to other bacteria and to a wide variety of eukaryotic cell types, including those of plants, fungi, and humans. The overall goals of work in this laboratory are to: i) define the assembly pathway for this type IV transfer system, ii) characterize its architectural arrangement, iii) elucidate the reaction mechanisms underlying substrate processing and delivery to the secretion channel, and iv) define the route of translocation across the Gram-negative bacterial envelope. It is now established that substrate transfer requires a pilus for mediating cell-cell contacts and a channel for translocation across the bacterial cell envelope. We will use a combination of molecular, genetic, and biochemical approaches in the following specific aims. First, we will explore structure - function relationships of the inner membrane VirB11 ATPase to define its role in transporter biogenesis and substrate translocation; for these studies we will capitalize on our assemblage of a large collection of altered-function mutants and a crystal structure of a VirB11 homolog. Second, we will identify steps in the substrate delivery pathway, focusing on the model VirE2 substrate and the VirE1 secretion chaperone. Finally, we will characterize critical steps in the assembly pathway of this transfer system; these studies will identify specific contacts involving three proteins essential for transporter assembly and structural integrity, the polytopic inner membrane protein VirB6 and two outer membrane proteins, VirB7 lipoprotein and VirB9.