TRANSLOCATION OF DNA ACROSS THE AGROBACTERIUM ENVELOPE

描述(改编自调查员摘要)：移位 细胞间的大分子是生物医学研究的一个重要领域。这个 这个实验室的研究重点是位于 输出致癌DNA的根癌农杆菌包膜 核蛋白颗粒，T-复合体，在植物细胞感染过程中。这个 T-Complex运输机是一种特别吸引人的模型系统 大分子运输的机理研究。T-复合体转运蛋白 是一个新发现的具有重要医学价值的家族的成员 传送者。总体而言，这些转运体负责配偶 革兰氏阴性菌之间广泛宿主范围的质粒传播， 以及至少一种多亚单位毒素的出口，百日咳毒素 百日咳杆菌。T-复合体转运体本身展示了一种新的 关于底物选择和靶细胞的多功能性 承认。除T-复合体外，incq质粒转移 中间体和至少两种蛋白质被认为是 具有移位能力的底物。衬底被输送到广泛的 种类繁多的植物、其他细菌、分裂酵母和萌芽酵母。 运输是一个依赖于接触的过程，似乎需要 用于调节供受者细胞接触的胞外菌毛。这个 PI实验室工作的总体目标是了解这一点 运输器是如何组装的，它如何识别底物，以及它如何引导 底物穿过革兰氏阴性信封到达收件人 细胞。克里斯蒂博士已经证明，VIRB的11种产品中有10种 操纵子是底物转运所必需的，并提供了基因 以及这些蛋白质组装成一个大的多聚体的生化证据 在细胞膜上很复杂。关于运输者的定义 建筑，他将使用分子遗传和生化的组合 确定VIRB蛋白之间特定接触的方法。他会的 首先确定两个细胞质膜结合的亚基相互作用 ATPase、VirB4和VirB11，以及其他转运体组件。他将使用一个 这些ATPase的大量突变集合以分析其贡献 利用体外和体内两种方法转运的ATP水解物。 PI将尝试获得相互作用的直接生化证据 在一种具有转位能力的底物和 运输系统。最后，他计划测试和完善一项拟议的 T-复合体转运系统的生物发生途径研究进展 来自他实验室的两个关键发现，第一，外膜 脂蛋白VirB7与VirB9形成分子间二硫键， 其次，VirB7/VirB9杂二聚体对组装 稳定的运输综合体。

DESCRIPTION (Adapted from the investigator's abstract): Translocation of macromolecules between cells is a major area of biomedical interest. The focus of study in this laboratory is a transfer system located at the Agrobacterium tumefaciens envelope which exports oncogenic DNA as a nucleoprotein particle, the T-complex, during infection of plant cells. The T-complex transporter is an especially attractive model system for detailed mechanistic studies of macromolecular transport. The T-complex transporter is a member of a newly-recognized family of medically-important transporters. Collectively, these transporters are responsible for conjugal transmission of broad-host-range plasmids between Gram-negative bacteria, and the export of at least one multisubunit toxin, the pertussis toxin of Bordetella pertussis. The T-complex transporter itself displays a novel versatility both with respect to substrate selection and target cell recognition. In addition to T-complexes, IncQ plasmid transfer intermediates and at least two proteins are recognized as translocation-competent substrates. Substrates are delivered to a wide range of plant species, other bacteria, and fission and budding yeast. Transport is a contact-dependent process that appears to require an extracellular pilus for mediating donor-recipient cell contacts. The overall goal of work in the PI's laboratory is to understand how this transporter is assembled, how it recognizes substrates, and how it directs the movement of substrates across the Gram-negative envelope to recipient cells. Dr. Christie has shown that ten of eleven products of the virB operon are essential for substrate translocation, and has supplied genetic and biochemical evidence that these proteins assemble as a large multimeric complex at the cell envelope. Toward definition of the transporter architecture, he will use a combination of molecular genetic and biochemical approaches to identify specific contacts among the VirB proteins. He will begin by identifying subunit interactions of two cytoplasmic membrane bound ATPases, VirB4 and VirB11, with other transporter components. He will use a large collection of mutations in these ATPases to analyze the contributions of ATP hydrolysis to transport with both in vitro and in vivo approaches. The PI will attempt to gain direct biochemical evidence for interactions between one of the translocation-competent substrates and components of the transport system. Finally, he plans to test and refine steps of a proposed biogenesis pathway for the T-complex transport system that was developed from two key discoveries in his laboratory, first, that the outer membrane lipoprotein, VirB7, forms an intermolecular disulfide bridge with VirB9, and second, that the VirB7/VirB9 heterodimer is critical for assembly of a stabilized transport complex.