Translocation of DNA across the Agrobacterium envelope

描述(由申请人提供)：大分子在细胞之间的转移是生物医学感兴趣的一个主要领域。本实验室的研究重点是根癌农杆菌的IV型分泌系统(T4S)。该系统与细菌结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转运系统在功能上相似。这个T4S系统介导了不同底物在根癌农杆菌细胞膜上的转移。底物包括核蛋白颗粒，如与单链DNA共价连接的VirD2松弛酶，以及蛋白质单体，如VirE2效应蛋白。T4S系统通过依赖于细胞间直接接触的过程，将这些底物转移到各种系统发育不同的细菌和真核细胞靶标上。该T4S系统由VirD4和11个VIRB亚基组装而成，是一个特别吸引人的系统，用于大分子运输的详细机制研究，因为根癌农杆菌的操作容易，手头有大量的菌株、结构和其他分子工具，以及关于它和密切相关的转移系统的丰富信息。该实验室的总体工作目标是用完整的分子术语描述所需的动态过程：i)VIRB/D4分泌通道的生物发生，ii)分泌底物的识别，以及iii)底物跨革兰氏阴性囊膜的转位。这些研究将使用最先进的生化和基于结构的方法，并结合本实验室在当前赠款周期中开发的强大的体内技术，来确定如何将模型DNA和蛋白质底物招募到分泌渠道并通过分泌渠道招募。首先，将确定两种ATPase，VirD4底物受体和VirB11，在早期和晚期底物转移反应中的作用。其次，新发现的DNA底物和ATP介导激活通道组装或门控所需的VirB10亚单位的构象开关的现象将得到充分表征。最后，将表征内膜亚基VirB6和VirB8以及外膜相关亚基VirB2和VirB9功能相互作用对底物跨细胞膜转运的贡献。拟议的研究将以前所未有的细节发展对IV型分泌物的机械理解。

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.