Analysis of DNA Transfer During Conjugation

接合是在不同细菌物种之间传递遗传信息的有效手段，并导致多种性状的传播，包括抗生素抗性决定簇和分解代谢途径。缀合依赖于供体细胞与受体细胞接合以形成配对，在细胞包膜中组装DNA可以通过其转移的装置，并处理DNA以产生转移的底物。在更广泛的背景下，接合过程与病原体用于将毒力决定簇输出到真核细胞中的机制有关。该项目的重点是表征的DNA转移阶段的细菌接合的大肠杆菌F质粒，使用遗传和生物化学的方法。实验将探索介导转移的DNA分子通过膜的装置的性质，以及该装置如何接触DNA加工机器。 这些研究将定义参与这些过程的不同转移（Tra）蛋白（特别是TraI，TraD和TraM）的重要结构域，并测试这些蛋白质之间的相互作用。TraD和TraI突变体的集合将使相关蛋白质结构和蛋白质-蛋白质相互作用的分析成为可能。野生型和突变蛋白的行为的比较将测试所观察到的相互作用对缀合过程的重要性。

Conjugation is an efficient means for transfer of genetic information among diverse bacterial species and leads to the dissemination of a variety of traits, including antibiotic resistance determinants and catabolic pathways. Conjugation relies on a donor cell engaging a recipient cell to form a mating pair, assembling an apparatus in the cell envelope through which DNA can be transferred, and processing DNA to produce the transferred substrate. In a broader context, the process of conjugation is related to a mechanism that pathogens use to export virulence determinants into eukaryotic cells. This project focuses on the characterization of the DNA transfer stage of bacterial conjugation for the F plasmid of Escherichia coli, using both genetic and biochemical approaches. Experiments will explore the nature of the apparatus that mediates the passage of the transferred DNA molecule through the membrane and how this apparatus contacts the DNA processing machinery. The studies will define important domains for different transfer (Tra) proteins involved in these processes (specifically, TraI, TraD, and TraM) and test interactions between those proteins. A collection of TraD and TraI mutants will enable analysis of relevant protein structure and protein-protein interactions. Comparisons of the behaviors of wildtype and mutant proteins will test for the importance of observed interactions to the conjugation process.