RUI: Biochemical and Genetic Characterization of Membrane- Associated VirB-Protein COmplexes in Agrobacterium tumefaciens

;​R o o t E n t y F g 5 @ C o m p o b j b W o r d d o c u m e n t O b j e c t P O O l : 5: z 5​F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT玛西娅·斯坦伯格玛西娅·斯坦伯格@ _ 5 @ e = e {n " " " " " " " L L L L L L d n L C x | | | | | | | # # # e V T 4“ | | | | | | ” “ | x | | | | ” | " | 6 " " " " | | 4 | 9506144班塔植物病原体农杆菌在在易感植物上形成的冠瘿瘤。这种疾病是由复杂的相互作用引起的，当细菌与植物细胞表面的伤口部位结合时，这种相互作用就开始了。这种细菌有一种不寻常的能力，可以将DNA片段（T DNA）转移到寄主植物细胞中，并将DNA传递到细胞核中，在那里它成为寄主植物基因组的一部分。在过去的几年里，广泛的分子表征有助于阐明细菌内T DNA产生的过程，以及细菌起源的分子被引导到真核细胞中特定隔室的机制。这种植物病原体相互作用的一个有趣的基本方面仍然笼罩在神秘之中，即实际的运输过程，介导T DNA从细菌输出并传递到植物细胞中。有一种多蛋白通道或孔复合物被假设允许T DNA（及其相关蛋白）通过包围细菌的两层膜。已经确定了几个候选蛋白质可以组成这样的运输装置；这些蛋白质是由携带许多毒力相关基因（以及T DNA本身）的大肿瘤诱导质粒上的virB操纵子编码的。包括我们自己的实验室在内的许多实验室最近的结果揭示了许多VirB蛋白的定位和其他特征；这些观察结果支持一种模型，其中几种VirB蛋白相互作用形成T DNA运输孔，而其他许多蛋白则作为转运事件的伴侣和/或能量来源。拟议的研究旨在确定哪些VirB蛋白相互作用形成寡聚物复合物。遗传分析将利用virB10基因中导致肿瘤形成缺失的点突变；潜在的相互作用物种将通过筛选第二个virB基因的代偿性变化来确定，该基因可以抑制原始突变体的无毒表型。第二种遗传方法将利用“双杂交”策略，测试蛋白质或蛋白质片段将酵母转录激活因子（如GAL4蛋白）的两个域结合在一起的能力。这些遗传研究将辅以生化实验，旨在探索含有VirB蛋白的聚集体的组成。总之，这些研究应该提供一些关于大极性分子（如T DNA和相关蛋白质）通过包裹农杆菌细胞的两个极性膜运输的机制的见解。假设的转移装置的结构和功能的分子解剖，反过来，将有助于阐明过程和相互作用介导组装被认为是一个复杂的膜相关复合体。土壤细菌农杆菌感染易感植物，导致细胞分裂不正常和形成冠瘿瘤。这种疾病可以被认为是“植物癌”，因为与人类肿瘤一样，细胞在不适当的时间和/或地点繁殖。农杆菌的感染涉及到一段DNA （T DNA）从细菌进入寄主植物细胞的运动。这种不寻常的相互作用是唯一已知的DNA从一个王国（细菌）转移到另一个王国（植物）的自然发生的例子。我们特别感兴趣的是一个大的亲水DNA分子能够穿过包围细菌细胞的油性膜的机制。T DNA的加工和转移是由许多细菌毒力蛋白介导的。一组Vir蛋白，即VirB蛋白，被认为在T DNA离开细菌细胞的过程中起着直接作用，最有可能是通过相互作用在细菌膜上形成一个开口，T DNA通过这个开口被转移。我们计划通过研究不允许T DNA移动的有缺陷的VirB蛋白，并通过分离VirB蛋白复合物，使用遗传和生化方法来验证这一假设。*** ;@ ....()()))()() ;

; R o o t E n t r y F g 5 @ C o m p O b j b W o r d D o c u m e n t O b j e c t P o o l :z 5 :z 5 F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ _ 5 @ e = e { n " " " " " " " L L L L L d n L C x | | | | | | | # ı e V T 4 " | | | | | | " " | x | | | | " | " | 6 " " " " | | 4 | 9506144 Banta The plant pathogen Agrobacterium tumefaciens causes a disease in which crown gall tumors form on susceptible plants. The disease results from a complex interaction that is initiated when the bacterium binds to the plant cell surface at a wound site. The bacterium has the unusual ability to transfer a fragment of DNA (the T DNA) into a host plant cell and deliver the DNA to the nucleus, where it becomes part of the host plant's genome. Extensive molecular characterization over the past several years has helped elucidate the process of T DNA production within the bacterium, as well as the mechanism by which a molecule of bacterial origin is directed to a specific compartment in a eukaryotic cell. One of the fun damental aspects of this plant pathogen interaction that remains shrouded in mystery is the actual transport process mediating export of the T DNA from the bacterium and delivery into the plant cell. A multiprotein channel or pore complex has been hypothesized to allow passage of the T DNA (and its associated proteins) through the two membranes that surround the bacterium. Several candidate proteins have been identified that could make up such a transport apparatus; these proteins are encoded by the virB operon on the large tumor inducing (T) plasmid that carries many of the virulence associated genes (as well as the T DNA itself). Recent results from a number of labs including our own have shed some light on the localization and other characteristics of many of the VirB proteins; these observations support a model in which several VirB proteins interact to form a T DNA transport pore, while others many function as chaperones and/or sources of energy for the translocation event. The proposed studies are designed to determine which VirB proteins interact to form oligomeric complexes. Genetic analyses will take advantage of point mutations in the virB10 gene that result in a loss of tumor formation; potentially interacting species will be identified by screening for compensatory changes in a second virB gene that can suppress the avirulent phenotype of the original mutant. A second genetic approach will utilize the "dihybrid" strategy, in which proteins or protein fragments are tested for their ability to bring together two domains of a yeast transcriptional activator such as the GAL4 protein. These genetic studies will be complemented by biochemical experiments designed to explore the composition of VirB protein containing aggregates. Together, these studies should provide some insight into the mechanism by which a large polar molecule such as the T DNA and associated proteins is transported across the two apolar membranes that enclose the Agrobacterium cell. Molecular dissection of the structure and function of the putative transfer apparatus will, in turn, help elucidate the processes and interactions mediating assembly of what is thought to be an elaborate membrane associated complex. %%% The soil bacterium Agrobacterium tumefaciens infects susceptible plants, resulting in unregulated cell division and crown gall tumor formation. This disease can be thought of as "plant cancer" in that, as with human tumors, cells are multiplying at an inappropriate time and/or place. Infection by Agrobacterium involves the movement of a piece of DNA (the T DNA) from the bacterium into the host plant cell. This unusual interaction is the only known naturally occurring example of DNA transfer from one kingdom (bacterial) to another (plant). We are especially interested in the mechanism by which a large water loving molecule of DNA is able to cross the oily membrane that surrounds the bacterial cell. T DNA processing and transfer are mediated by a number of bacterial virulence (Vir) proteins. One group of Vir proteins, the VirB proteins, are believed to play a direct role in allowing the T DNA to exit the bacterial cell, most likely by interacting to form an opening in the bacterial membrane through which the T DNA is transferred. We plan to test this hypothesis, using both genetic and biochemical approaches, by studying defective VirB proteins that do not permit T DNA movement, and by isolating complexes of VirB proteins. *** ; @ ....()()))()() ;