RUI: Protein-Protein Interactions Mediating Substrate Recognition by the VirB Complex of Agrobacterium Tumefaciens

RUI：介导根癌农杆菌 VirB 复合物识别底物的蛋白质-蛋白质相互作用

• 批准号: 9905126
• 负责人: Lois Banta
• 金额: $ 18万
• 依托单位: Haverford College
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9905126&HistoricalAwards=false
• 关键词: RUI; Protein; Interactions; Mediating; Substrate

Banta99051261. Technical Agrobacterium tumefaciens causes crown gall tumors to form on susceptible plants by stably transforming the host organism with a 20-kb fragment of single-stranded DNA (the T-DNA) encoding plant growth hormone biosynthetic enzymes. The VirB proteins mediate export of this oncogenic DNA from the bacterium. The focus of this study is the protein-protein interactions mediating recognition by the transport machinery of the various substrates that utilize the VirB pore. These substrates include not only the T-DNA, but also a single-stranded DNA-binding protein (VirE2) involved in targeting the T-DNA to the nucleus of the host cell, and a DNA-protein complex derived from the mobilizable plasmid RSF1010, which appears to compete for access to the transport apparatus, inhibiting delivery of the other substrates. A novel chromosomally-encoded, 40 kD membrane protein, which the PI discovered earlier, interacts with VirE2. This protein will be purified, taking advantage of its affinity for VirE2, and its partial amino acid sequence determined. Degenerate primers will be designed accordingly and used to isolate a genomic clone for quantitative production and further characterization of this protein. Generation of alkaline phosphatase fusions and antisera will facilitate studies on the membrane topology and probing potential interactions with other components of the VirB transport machinery. Gene disruption will be used to assess directly the role of the 40 kD protein in virulence, specifically in the delivery of VirE2 to host plant cells. Strikingly, this protein appears to be destabilized in cells carrying RSF1010 or by over-production of one component of the VirB pore, VirB10; both of these conditions also result in an inhibition of VirE2 export. To pursue these observations, turnover of the 40 kD protein in response to production of an RSF1010-derived transfer intermediate will be measured. In a second set of experiments, a mutant screen will be initiated in the hopes of identifying one or more VirB proteins responsible for activating release of a substrate through the VirB pore in response to host-cell contact. Screening for plant cell-independent secretion of VirE2 will identify such mutants. Together, these studies should provide some insight into the process by which DNA and/or associated proteins are recognized and transported across the two apolar membranes that enclose the Agrobacterium cell. Molecular dissection of the mechanism mediating export of these virulence determinants will, in turn, enhance our understanding of pathogenesis in other organisms, including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila, that utilize homologous transport systems to deliver their pathogenic substrates.2. Non-technical The bacterium Agrobacterium tumefaciens causes a disease in which tumors form on certain plants. The symptoms result from the transfer of a piece of DNA from the bacterium to the susceptible plant cell. Movement of a protein, VirE2, is also required for efficient tumor formation. The goal of this study is to elucidate the mechanism by which the disease-causing DNA and VirE2 are recognized and delivered through the membrane surrounding the bacterium into the host plant cell. The VirB proteins are known to mediate this transport; we have identified an additional novel, non-VirB, component that appears to interact with VirE2. The hypothesis to be tested is that this protein may allow the VirB transport apparatus to engage with VirE2, since the protein is no longer detectable under conditions that are known to block VirE2 movement from the bacterium. The gene encoding this protein will be cloned to generate this protein in quantity so that if and how the protein interacts with the VirB proteins can be determined. A second line of experimentation will also be initiated to investigate how contact with the plant cell triggers transport of the VirE2 from the bacterium. This will be done by searching for mutants that release VirE2 even in the absence of a host plant cell.

班塔99051261。技术性根癌农杆菌通过用编码植物生长激素生物合成酶的 20 kb 单链 DNA（T-DNA）片段稳定转化宿主生物体，导致易感植物上形成冠瘿瘤。 VirB 蛋白介导细菌中致癌 DNA 的输出。 本研究的重点是蛋白质-蛋白质相互作用介导利用 VirB 孔的各种底物的运输机制的识别。 这些底物不仅包括 T-DNA，还包括参与将 T-DNA 靶向宿主细胞核的单链 DNA 结合蛋白 (VirE2)，以及源自可移动质粒 RSF1010 的 DNA-蛋白质复合物，该复合物似乎竞争进入运输装置，抑制其他底物的递送。 PI 之前发现的一种新型染色体编码的 40 kD 膜蛋白与 VirE2 相互作用。该蛋白质将利用其对 VirE2 的亲和力进行纯化，并确定其部分氨基酸序列。将相应地设计简并引物并用于分离基因组克隆，以定量生产并进一步表征该蛋白质。碱性磷酸酶融合物和抗血清的产生将有助于对膜拓扑结构的研究以及探索与 VirB 运输机制其他组件的潜在相互作用。 基因破坏将用于直接评估 40 kD 蛋白在毒力中的作用，特别是在将 VirE2 递送至宿主植物细胞中的作用。 引人注目的是，这种蛋白质似乎在携带 RSF1010 的细胞中不稳定，或者由于 VirB 孔的一种成分 VirB10 的过量产生而不稳定。这两种情况也会导致 VirE2 输出受到抑制。 为了进行这些观察，将测量 40 kD 蛋白质响应 RSF1010 衍生的转移中间体的产生的周转率。 在第二组实验中，将启动突变体筛选，希望能够鉴定出一种或多种 VirB 蛋白，该蛋白负责响应宿主细胞接触而通过 VirB 孔激活底物的释放。筛选植物细胞独立分泌的 VirE2 将鉴定出此类突变体。 总之，这些研究应该为 DNA 和/或相关蛋白质被识别并穿过包围农杆菌细胞的两个非极性膜的过程提供一些见解。 对介导这些毒力决定子输出的机制进行分子剖析反过来将增强我们对其他生物体发病机制的理解，包括百日咳博德特氏菌、幽门螺杆菌和嗜肺军团菌，它们利用同源运输系统来传递其致病底物。2。非技术性 根癌农杆菌引起一种在某些植物上形成肿瘤的疾病。 这些症状是由细菌的 DNA 片段转移到易受影响的植物细胞引起的。 蛋白质 VirE2 的运动也是有效肿瘤形成所必需的。本研究的目的是阐明致病 DNA 和 VirE2 被识别并通过细菌周围的膜传递到宿主植物细胞中的机制。 VirB 蛋白已知可介导这种运输；我们还发现了一种新的非 VirB 成分，它似乎与 VirE2 相互作用。 待测试的假设是，该蛋白质可能允许 VirB 转运装置与 VirE2 接合，因为在已知阻止 VirE2 从细菌中移动的条件下，不再检测到该蛋白质。编码该蛋白质的基因将被克隆以大量产生该蛋白质，从而可以确定该蛋白质是否以及如何与 VirB 蛋白质相互作用。 第二线实验也将启动，以研究与植物细胞的接触如何触发细菌中 VirE2 的转运。 这将通过寻找即使在没有宿主植物细胞的情况下也能释放 VirE2 的突变体来完成。