Translocation of DNA across the Agrobacterium envelope

大分子在细胞间的转移是生物医学研究的一个重要领域。研究的重点是 本实验室是根癌农杆菌的IV型分泌系统。这一制度由来已久。 与细菌结合系统相关和功能相似，以及最近描述的蛋白质 细菌病原体在感染过程中使用的转位系统。这个T4S系统调停 不同底物在根癌农杆菌细胞膜上的转移。底物包括核蛋白 粒子，如病毒D2松弛酶，共价连接到单链DNA，以及蛋白质单体，如 作为病毒E2效应器蛋白。T4S系统将这些底物转移到各种系统发育上的- 不同的细菌和真核细胞靶标通过依赖于细胞间直接接触的过程。这 T4S系统由VirD4和11个VIRB亚基组装而成，对于详细的 由于根癌农杆菌的易操纵性，大分子运输的机理研究 大量的菌株、结构和手头的其他分子工具，以及丰富的可用信息 关于它和密切相关的转移系统。本实验室工作的总体目标是在 完整的分子术语：I)VIRB/D4分泌的生物发生所需的动态过程 通道，ii)分泌底物的识别，以及iii)底物跨革兰氏阴性菌的移位 信封。这些研究将结合使用最先进的生化方法和基于结构的方法 在当前的赠款周期中，该实验室开发了强大的体内技术，以定义如何 模型DNA和蛋白质底物被招募到分泌通道并通过分泌通道招募。第一，对 两种ATPase，VirD4底物受体和VirB11，对早期和晚期底物转移反应的影响 将会被确定。第二，一种新发现的DNA底物和ATP介导的现象 激活通道组装或门控所需的VirBIO亚单位的构象开关将是 完全有特点的。最后，功能相互作用的内膜亚单位对的贡献， VirB6和VirB8，以及外膜相关亚基VirB2和VirB9到底物易位 整个细胞包膜的特征将被描述。拟议的研究将发展一种机械性的 对IV型分泌物前所未有的详细了解。

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 DMA,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 DMAand 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 VirBIO 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.