Organization of F Plasmid Tra Proteins

智力优势：这项研究的长期目标是阐明由细菌IV型分泌系统(TFSS)介导的水平DNA转移的结构生物学和机制。这些系统包括一组广泛分布的分子纳米机器，它们的功能是将大分子从革兰氏阴性细菌分泌到其他细菌或真核细胞。通过序列比较可以区分几个TFSS类别，这些系统可能在细菌进化过程中出现过不止一次。IV型分泌系统在细菌群落内以及细菌与包括植物和动物在内的其他有机体之间的相互作用网络中发挥着至关重要的作用。作为一个整体，IV型系统具有转移DNA和蛋白质的能力，除了抗生素耐药性的传播外，还直接参与了动植物细菌的致病和生物膜的形成等现象。鉴于它们在细菌群落自然历史中的普遍存在和重要性，对这些系统的详细分析显然是重要的。这个更新项目解决了一个典型的TFSS，它是由大肠杆菌的F质粒编码的。TFSS-F包括大约14个trA基因，它们的同源TrA(DNA转移)蛋白需要形成接合(在这种情况下是F-)菌毛。结合菌毛是表面细丝，外径约9纳米，长几微米，介导革兰氏阴性细菌之间水平DNA转移所需的早期事件。Silverman博士之前由NSF支持的工作已经定义了在F-菌毛组装中起作用的反式蛋白相互作用基团。这个项目扩展了这项工作，专注于一个稳定的TrA蛋白亚组分，据信它横跨细胞被膜，从内膜(TRAb)到周质(TRAb/Trak)再到外膜(Trak/Trav)。其他TFSS中的类似蛋白质被描述为核心复合体的组成部分，该复合体具有易位通道或易位子的功能。基于TRAB/K/V亚组件同样是跨包膜转运子的一部分这一工作假设，特定的目标结合了生化和遗传策略来解决三个问题：核心复合体由哪些TrA蛋白组成？反式蛋白对体内复合体的形成或稳定性的要求是什么？单个复杂组件如何与其他组件交互以产生功能组装？其具体目的是：1，建立TRAb在Tra细胞中的定位和定位及其与Trav脂蛋白的关系，分离和鉴定TRAb/K/V复合体，并通过蛋白质印迹和质谱学测定纯化的复合体的Tra蛋白组成；2，确定TRAb/K/V复合体是否需要其他Tra蛋白来稳定或在体内组装，如果需要，鉴定它们；以及3，鉴定复杂成分之间的蛋白质相互作用结构域，绘制相互作用结构域，并分离在TrA片段和体内完整的Tra蛋白之间形成的复合体。通过专注于F-Pilin的分泌，该项目补充了许多实验室为建立由IV型分泌系统介导的DNA转移机制所做的努力。预计这项工作将阐明结合菌毛素转位和DNA-蛋白质复合体的转位如何在力学上相关，并指导体外测试的发展，以了解影响结合菌毛的反式蛋白质和蛋白质组件的功能。广泛影响：该项目包括与俄克拉何马大学生物学教授吉姆·比德拉克博士的合作，该大学是一所以本科为主的机构，并将为该机构的本科生提供研究机会。西尔弗曼博士最近被任命为中俄克拉荷马大学的“驻校领导”，在06-07学年期间，他将成为教职员工的榜样和导师。

Intellectual merit: The long-term goal of this research is to elucidate the structural biology and mechanism of horizontal DNA transfer mediated by bacterial type IV secretion systems (TFSS's). These systems comprise a broadly distributed group of molecular nano-machines that function to secrete macromolecules from gram-negative bacteria to other bacterial or eukaryotic cells. Several TFSS classes are distinguishable by sequence comparisons, and these systems may have arisen more than once during bacterial evolution. Type IV secretion systems play a crucial role in interaction networks within bacterial communities and between bacteria and other organisms, including plants and animals. With their capacity to transfer DNA as well as proteins, type IV systems as a group are directly involved in such phenomena as bacterial pathogenesis in plants and animals and biofilm formation, in addition to the dissemination of antibiotic resistance. Given their ubiquity and importance in the natural history of bacterial communities, detailed analysis of these systems is clearly important.This renewal project addresses an archetypical TFSS, that which is encoded by the F plasmid of Escherichia coli. TFSS-F includes about 14 tra genes whose cognate Tra (DNA transfer) proteins are required to form conjugative (in this case F-) pili. Conjugative pili are surface filaments, approximately 9 nanometers in outer diameter and several micrometers long, that mediate early events required for horizontal DNA transfer among gram-negative bacteria. Dr. Silverman's previous NSF-supported work has defined Tra protein interaction groups that function in the assembly of F-pili. This project extends that work, focusing on a stable subassembly of Tra proteins that is believed to span the cell envelope, from the inner membrane (TraB) through the periplasm (TraB/TraK) to the outer membrane (TraK/TraV). Similar proteins in other TFSS's have been described as components of a core complex that functions as a translocation channel, or translocon. Based on the working hypothesis that the TraB/K/V subassembly is likewise part of a trans-envelope translocon, the specific aims incorporate biochemical as well as genetic strategies to address three issues: what Tra proteins comprise the core complex? what are the Tra protein requirements for complex formation or stability in vivo? and how do individual complex components interact with the others to yield the functional assembly? The specific aims are: 1, to establish the localization and orientation of TraB in Tra+ cells and its relation to the TraV lipoprotein, isolate and characterize the TraB/K/V complex, and determine the Tra protein composition of purified complexes by Western blot and mass spectometry; 2, to determine if the TraB/K/V complex requires other Tra proteins for stability or assembly in vivo and, if so, identify them, and 3, to identify protein-protein interaction domains among complex components, map interaction domains, and isolate complexes that form between the Tra segments and intact Tra proteins in vivo. By focusing on F-pilin secretion, this project complements efforts in numerous laboratories to establish the mechanism of DNA transfer mediated by type IV secretion systems. It is anticipated that the work will elucidate how conjugative pilin translocation and translocation of DNA-protein complexes are mechanistically related and guide development of in vitro assays necessary to understand the function of Tra proteins and protein assemblies that affect conjugative pili.Broader impacts: The project includes a collaboration with Dr. Jim Bidlack, Professor of Biology at the University of Central Oklahoma, a predominantly undergraduate institution, and will provide research opportunities for undergraduate students from that institution. Dr. Silverman has recently been appointed "Leader in Residence" at the University of Central Oklahoma, whereby he will serve as a role model and mentor for the faculty during the 06-07 academic year.