Translocation of DNA across the Agrobacterium envelope

描述（由申请人提供）：大分子在细胞间的易位是生物医学关注的主要领域。本实验室的研究重点是根癌农杆菌的IV型分泌（T4 S）系统。该系统与细菌接合系统以及最近描述的细菌病原体在感染过程中使用的蛋白质易位系统在祖先上相关并且在功能上相似。该T4 S系统介导多种底物跨A.根瘤菌细胞被膜。底物包括核蛋白颗粒，例如共价连接到DNA单链的VirD 2松弛酶，和蛋白质单体，例如VirE 2效应蛋白。T4 S系统通过依赖于直接细胞间接触的过程将这些底物转运至多种遗传多样的细菌和真核细胞靶标。该T4 S系统由VirD 4和11个VirB亚基组装而成，由于A.根癌农杆菌，大量的菌株，构建体和手头的其他分子工具，以及关于它和密切相关的转移系统的丰富信息。本实验室工作的总体目标是以完整的分子术语描述以下过程所需的动态过程：i）VirB/D4分泌通道的生物发生，ii）分泌底物的识别，以及iii）底物穿过革兰氏阴性包膜的易位。这些研究将使用最先进的生物化学和基于结构的方法，结合本实验室在当前资助周期内开发的强大体内技术，以确定模型DNA和蛋白质底物如何被招募到分泌通道并通过分泌通道。首先，将确定两种ATP酶，VirD 4底物受体和VirB 11，对早期和晚期底物转移反应的贡献。其次，一个新发现的现象，DNA底物和ATP介导的激活的VirB 10亚基的通道组装或门控所需的构象开关将得到充分的表征。最后，功能相互作用对内膜亚基，VirB 6和VirB 8，和外膜相关的亚基，VirB 2和VirB 9，跨细胞包膜的底物易位的贡献将被表征。拟议的研究将以前所未有的细节发展对IV型分泌的机制性理解。

DESCRIPTION (provided by applicant): Translocation of macromolecules between cells is a major area of biomedical interest. The focus of study in this laboratory is a type IV secretion (T4S) system of Agrobacterium tumefaciens. This system is ancestrally related and functionally similar to bacterial conjugation systems, as well as recently described protein translocation systems used by bacterial pathogens during the course of infection. This T4S system mediates transfer of diverse substrates across the A. tumefaciens cell envelope. The substrates include nucleoprotein particle such as the VirD2 relaxase covalently linked to a single strand of DNA, and protein monomers such as the VirE2 effector protein. The T4S system translocates these substrates to a variety of phylogenetically-diverse bacterial and eukaryotic cell targets through a process dependent on direct cell-to-cell contact. This T4S system, assembled from VirD4 and 11 VirB subunits, is an especially attractive system for detailed mechanistic studies of macromolecular transport because of the ease of manipulation of A. tumefaciens, the large numbers strains, constructs, and other molecular tools at hand, and the wealth of information available about it and closely related transfer systems. The overall goal of work in this laboratory is to describe in complete molecular terms the dynamic processes required for: i) biogenesis of the VirB/D4 secretion channel, ii) recognition of secretion substrates, and iii) translocation of substrates across the Gram-negative envelope. The studies will use state-of-the-art biochemical and structure-based approaches in combination with powerful in vivo technologies developed in this laboratory during the current grant cycle to define how model DNA and protein substrates are recruited to and through the secretion channel. First, contributions of two ATPases, the VirD4 substrate receptor and VirB11, to early- and late-stage substrate transfer reactions will be determined. Second, a newly-discovered phenomenon of DNA substrate- and ATP-mediated activation of a conformational switch of the VirB10 subunit required for channel assembly or gating will be fully characterized. Finally, the contributions of functionally interacting pairs of inner membrane subunits, VirB6 and VirB8, and outer membrane-associated subunits, VirB2 and VirB9, to substrate translocation across the cell envelope will be characterized. The proposed studies will develop a mechanistic understanding of type IV secretion in unprecedented detail.