Organization of F Plasmid Tra Proteins in E. coli

俄克拉荷马医学研究基金会的Philip Silverman博士获得了一笔拨款，用于阐明大肠杆菌F+和F-菌株之间共轭（横向）DNA转移的结构生物学。本研究试图确定由F质粒编码的30个左右的DNA转移（Tra）蛋白是如何在细菌细胞表面组织的，并测试这些蛋白构成介导共轭DNA转移的分子机器的假设。第一个具体目标是表征Tra蛋白之间的相互作用，目的是从生物化学的角度理解Tra蛋白机器的样子。该研究的第二个具体目标是应用间接免疫荧光显微镜来确定每个细菌细胞上有多少台Tra机器，以及它们之间的空间关系、与F DNA的关系以及与细菌细胞表面的整体关系。本研究是对F质粒介导的共轭DNA转移进行详细结构/功能分析的第一步。为了扩大我们的研究对俄克拉何马州服务不足的农村学校科学教育的影响，该项目的第三个具体目标是调查俄克拉何马州牲畜肠道菌群细菌中的抗生素抗性质粒。这些R质粒将由农村高中理科学生与西尔弗曼博士创立并指导的俄克拉何马州科学项目一起分离和部分表征。几个因素使得这个项目既及时又重要。首先，夫妻DNA转移与抗生素耐药性的传播有关，这是一个日益令人担忧的公共卫生问题。其次，它是细菌细胞通过其表面屏障分泌大分子的几种机制之一；这些机制与许多细菌的致病性有关。第三，夫妻DNA转移在生物技术方面有应用；更好地理解这一过程将增加此类应用的数量和范围。最后，该项目将为俄克拉何马州的农村学生创造更多进入技术职业道路的机会，包括其规模可观的美国原住民群体。

A grant has been awarded to Dr. Philip Silverman of the Oklahoma Medical Research Foundation to elucidate the structural biology of conjugal (lateral) DNA transfer between F+ and F- strains of Escherichia coli. This research seeks to define how the 30 or so DNA transfer (Tra) proteins encoded by the F plasmid are organized at the bacterial cell surface and tests the hypothesis that these proteins constitute a molecular machine that mediates conjugal DNA transfer. The first specific aim is to characterize the interactions among the Tra proteins with the goal of understanding what the Tra protein machine looks like from a biochemical perspective. The second specific aim of the research is to apply indirect immunofluorescence microscopy to determine how many Tra machines there are on each bacterial cell and how they are related spatially to each other, to F DNA, and to the bacterial cell surface as a whole. This research is the first step towards a detailed structure/function analysis of conjugal DNA transfer mediated by the F plasmid.To broaden the impact of our research to science education in Oklahoma's underserved rural schools, a third specific aim of the project is to survey antibiotic resistance plasmids in bacteria from the intestinal flora of livestock throughout Oklahoma. These R plasmids will be isolated and partially characterized by rural high school science students in conjunction with The Oklahoma Science Project, which Dr. Silverman founded and directs. Several considerations make this project both timely and important. First, conjugal DNA transfer is connected to the dissemination of antibiotic resistance, an increasingly alarming public health problem. Second, it is one of several mechanisms by which bacterial cells secrete macromolecules across their surface barriers; these mechanisms are responsible for the pathogenicity of many bacteria. Third, conjugal DNA transfer has applications in biotechnology; a better understanding of the process will increase the number and scope of such applications. Finally, the project will create more opportunities for Oklahoma's rural students, including its sizable Native American cohort, to enter technology career paths.