An F-plasmid Tra Protein Linkage Map

摘要本研究的长期目标是阐明大肠杆菌F+和F-菌株之间的共轭DNA转移的结构生物学。使这项研究既及时又重要的几个考虑因素是，夫妻DNA转移与抗生素耐药性的传播密不可分，并且是细菌细胞分泌大分子越过其表面屏障的越来越多的机制之一。本研究旨在确定F质粒编码的DNA转移（Tra）蛋白如何在细菌细胞表面组织，从而产生实际介导DNA转移的细胞器。为了简化最初的项目，重点将放在15个左右的Tra蛋白（大约30个）上，这些蛋白是F+细胞精心制作F-毛所需的。f -毛是从供体细胞表面延伸出来的细丝，是DNA转移中细胞间接触的最早阶段所必需的。它们是由一个单一的亚基F-pilin构成的，尽管F-pilin的生物形成或影响F-pilin的结构或功能需要十多个其他Tra蛋白，但不会破坏纤维组装。这些Tra蛋白的组织和功能尚不清楚。本研究的第一个具体目标是通过酵母双杂交试验确定这些Tra蛋白之间的蛋白-蛋白相互作用，目的是创建Tra蛋白连锁图谱。第二个目标是确定哪些在酵母中发现的相互作用也发生在大肠杆菌中，并且是f -菌生物发生所必需的，特别关注双杂交试验表明跨越细菌细胞包膜的Tra蛋白组。条件（温度敏感）相互作用突变体、生化分析和免疫电子显微镜的结合将用于对大肠杆菌中连锁图谱预测发生的相互作用提供二次确认。该研究的第三个具体目标是表征选定的Tra蛋白，特别是那些具有嘌呤核苷酸结合基序的蛋白，因为这些蛋白具有特定的生化功能。生物化学和遗传学方法将用于确定两个具有Walker box A基序的Tra蛋白中哪一个是atp酶，如果是的话，这种活性是否对f -菌的生物发生和功能至关重要。这项研究将解决共轭的结构方面，这是一个基本的过程在细菌。这项研究的结果可能会增加我们对遗传物质（包括抗生素抗性基因）在自然界中的转移的理解，并应该提供对f -菌形成和功能至关重要的蛋白质相互作用的见解。

Silverman, Philip M.MCB- 9900553Abstract The long-term goal of this research is to elucidate the structural biology of conjugal DNA transfer between F+ and F- strains of Escherichia coli. Among several considerations that make this research both timely and important are the facts that conjugal DNA transfer is inextricably connected to the dissemination of antibiotic resistance and one of a growing number of mechanisms by which bacterial cells secrete macromolecules across their surface barriers. This research seeks to define how the DNA transfer (Tra) proteins encoded by the F plasmid are organized at the bacterial cell surface to create the organelle(s) that actually mediate DNA transfer. To simplify the project initially, the focus will be on the 15 or so Tra proteins (of about 30) that are required by F+ cells to elaborate F-pili. F-pili are filaments extending from the donor cell surface and required for the earliest, cell-cell contact stages of DNA transfer. They are built from a single subunit, F-pilin, though more than a dozen other Tra proteins are required for F-pilus biogenesis or affect F-pilus structure or function without abolishing filament assembly. The organization and functions of these Tra proteins are not at all understood. The first specific aim of the research is to identify protein-protein interactions among these Tra proteins by yeast two-hybrid assay, with the goal of creating a Tra protein linkage map. The second aim is to establish which of the interactions identified in yeast also occur in E. coli and are required for F-pilus biogenesis, with special attention paid to sets of Tra proteins suggested by two-hybrid assay to span the bacterial cell envelope. A combination of conditional (temperature-sensitive) interaction mutants, biochemical assays, and immunoelectron microscopy will be used to provide a secondary confirmation of the interactions predicted to occur by the linkage map in E. coli. The third specific aim of the research is to characterize selected Tra proteins, particularly those with purine nucleotide binding motifs, since these suggest specific biochemical functions. Biochemical and genetic approaches will be used to determine which, if either, of two Tra proteins with Walker box A motifs is an ATPase and, if so, whether or not this activity is essential for F-pilus biogenesis and function. This research will address the structural aspects of conjugation, which is a fundamental process in bacteria. The outcomes of this research may increase our understanding of the transfer of genetic material, including antibiotic resistance genes, in nature and should provide insight into the protein interactions that are essential for F-pilus formation and function.