TRANSLOCATION OF DNA ACROSS THE AGROBACTERIUM ENVELOPE

DNA 跨农杆菌包膜的易位

• 批准号: 6138462
• 负责人: PETER j. CHRISTIE
• 金额: $ 21.39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138462
• 关键词: Agrobacterium tumefaciens; adenosinetriphosphatase; bacterial proteins; binding proteins; biological signal transduction; chimeric proteins; gel filtration chromatography; gene expression; host organism interaction; intermolecular interaction; membrane permeability; membrane transport proteins; microorganism conjugation; molecular cloning; operon; phosphorylation; protein biosynthesis; protein purification; protein structure function; proteolysis; surface plasmon resonance

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.

描述（改编自研究者摘要）： 细胞之间的大分子是生物医学感兴趣的主要领域。 的 本实验室的研究重点是位于 根癌农杆菌的包膜，它输出致癌DNA作为一种 核蛋白颗粒，T-复合物，在植物细胞的感染。 的 T复合体转运体是一个特别有吸引力的模型系统， 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医学重要性的 运输机 总的来说，这些运输者负责婚姻 革兰氏阴性菌之间广泛宿主范围质粒的传递， 和至少一种多亚单位毒素的输出， 百日咳杆菌 T复合体转运蛋白本身显示了一种新的 在底物选择和靶细胞方面的多功能性 识别. 除了T复合物，IncQ质粒转移 中间体和至少两种蛋白质被认为是 易位能力的底物。 基板被运送到一个广泛的 一系列的植物物种，其他细菌，以及裂变和芽殖酵母。 传输是一个依赖于接触的过程， 用于介导供体-受体细胞接触的细胞外菌毛。 的 PI实验室工作的总体目标是了解这是如何实现的， 转运蛋白是如何组装的，它如何识别底物，以及它如何引导 底物穿过革兰氏阴性包膜到受体的运动 细胞 克里斯蒂博士已经证明，病毒B的11种产物中有10种 操纵子是底物转运所必需的， 和生化证据表明这些蛋白质组装成一个大的多聚体 在细胞膜上形成复合物。 关于运输者的定义 他将结合分子遗传学和生物化学 鉴定VirB蛋白之间的特异性接触的方法。 他将 开始时先鉴定两个细胞质膜结合的亚基相互作用 ATP酶，VirB 4和VirB 11，以及其他转运蛋白组分。 他会用一个 这些ATP酶中的大量突变来分析 的ATP水解，以运输在体外和体内的方法。 PI将尝试获得相互作用的直接生化证据 在一种有易位能力的底物和所述底物的组分之间， 运输系统。 最后，他计划测试和完善拟议的 T复合物转运系统的生物发生途径， 他在实验室里有两个重要发现，第一， 脂蛋白VirB 7与VirB 9形成分子间二硫键， 第二，VirB 7/VirB 9异二聚体对于组装一个 稳定的运输系统。