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

Banta99051261。农杆菌通过编码植物生长激素生物合成酶的20 kb单链DNA片段（T-DNA）稳定地转化寄主，在易感植物上形成冠瘿瘤。VirB蛋白介导这种致癌DNA从细菌中输出。本研究的重点是蛋白质-蛋白质相互作用，通过利用VirB孔的各种底物的运输机制介导识别。这些底物不仅包括T-DNA，还包括将T-DNA靶向到宿主细胞核的单链dna结合蛋白（VirE2），以及来自可移动质粒RSF1010的dna -蛋白复合物，后者似乎竞争进入运输装置，抑制其他底物的递送。PI先前发现的一种新的染色体编码的40 kD膜蛋白与VirE2相互作用。该蛋白将被纯化，利用其对VirE2的亲和力，并确定其部分氨基酸序列。简并引物将被设计并用于分离基因组克隆，用于定量生产和进一步表征该蛋白。碱性磷酸酶融合物和抗血清的产生将有助于研究膜拓扑结构和探测与VirB运输机制的其他成分的潜在相互作用。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传递到宿主植物细胞中的作用。引人注目的是，这种蛋白在携带RSF1010的细胞中或由于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抑制VirE2的输出。为了实现这些观察结果，将测量40kd蛋白在rsf1010衍生的转移中间体产生后的周转率。在第二组实验中，将启动突变筛选，希望鉴定一个或多个VirB蛋白，这些蛋白负责激活宿主细胞接触时通过VirB孔释放底物。筛选植物细胞独立分泌的VirE2将鉴定出这样的突变体。总之，这些研究应该对DNA和/或相关蛋白质被识别并通过包围农杆菌细胞的两极膜运输的过程提供一些见解。分子解剖介导这些毒力决定因子输出的机制，反过来，将增强我们对其他生物发病机制的理解，包括百日杆菌、幽门螺杆菌和嗜肺军团菌，它们利用同源转运系统递送致病性底物。农杆菌：农杆菌在某些植物上引起肿瘤形成的一种疾病这些症状是由细菌的一段DNA转移到易感植物细胞引起的。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寄主植物细胞的机制。已知VirB蛋白介导这种转运；我们已经确定了一个新的，非virb，似乎与VirE2相互作用的组件。需要测试的假设是，这种蛋白质可能允许VirB转运装置与VirE2结合，因为在已知阻止VirE2从细菌中移动的条件下，这种蛋白质不再被检测到。编码这种蛋白质的基因将被克隆，以产生大量这种蛋白质，以便确定这种蛋白质是否以及如何与VirB蛋白相互作用。第二项实验也将启动，以研究与植物细胞的接触是如何触发从细菌中运输VirE2的。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的突变体来实现。

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.