Efficient Mechanism of Gene Transfer in B. fragilis

这项研究的假设是，DNA和抗生素抗性基因转移在拟杆菌属是非常有效的，涉及遗传无关的动员模块（1至3个基因），执行所有的DNA加工功能在转移启动。 此外，这些调动模块位于不同的转移因子上。 它们都与由拟杆菌属泰特元件编码的接合器（交配孔）类似地相互作用，导致它们的广泛传播。这一假设的基本原理来自于大量的初步数据，这些数据表明，当与共同的交配器共存时，拟杆菌属转移因子在拟杆菌属中具有相似的转移特性，而在大肠杆菌中具有相似的转移特性。当与广泛宿主范围质粒共存时， 每个可移动的转移因子包含一个，两个或三个基因，它们必须执行类似的DNA加工功能，尽管缺乏同源性。 然而，所有可移动的转移因子都有一个5 bp的共有nic序列。 我们现在已经捕获了拟杆菌交配器，它似乎是接合转座因子（泰特因子）的一部分。 该交配器为拟杆菌中的多种动员因子提供了交配桥梁，并在大肠杆菌中发挥作用。杆菌 此外，Tn5520和泰特元件都广泛散布在拟杆菌中。 因此，拟杆菌可能代表了DNA和抗生素耐药性传播的高效模型。本研究的具体目的是：1）确定混杂Tn5520转移因子的所有DNA加工功能均由单个动员蛋白和oriT区域提供; 2）确定pLF 9编码的交配装置是许多不同接合转移因子的共同途径，并鉴定参与起始复合物和交配孔之间相互作用的关键基因。研究包括使用交配，插入和缺失诱变，在体外和体内松弛体形成，DNA测序，蛋白质/蛋白质相互作用，和细胞定位实验。完成这些研究后，我们将确定从拟杆菌转移所涉及的DNA加工的最低要求，从而可以与其他细菌中更复杂的系统进行比较。 交配器和松弛体复合物的关键组分的鉴定和表征允许遗传多样性转移因子利用单个交配孔，这将是拟杆菌中DNA转移的综合模型的重要一步，并将为细菌抗生素耐药性如何迅速传播提供重要见解。

The hypothesis of this study is that DNA and antibiotic resistance gene transfer in Bacteroides spp is highly efficient, involving genetically unrelated mobilization modules (one to three genes) that perform all DNA processing functions during transfer initiation. Further, these mobilization modules are located on different transfer factors. They all interact similarly with a conjugation apparatus (mating pore) that is encoded by Bacteroides spp Tet elements, resulting in their widespread dissemination. The rationale for this hypothesis is derived from extensive preliminary data demonstrating that Bacteroides transfer factors have similar transfer characteristics in Bacteroides, when co- resident with a common mating apparatus, and to E. coli when co- resident with a broad host range plasmid. Each of the mobilizable transfer factors contains one, two, or three genes that must perform similar DNA processing functions, despite a lack of homology. However, all mobilizable transfer factors have a five bp consensus nic sequence. We have now captured a Bacteroides mating apparatus, which appears to be part of a conjugative transposable element (Tet element). This mating apparatus provides the mating bridge for multiple mobilization factors in Bacteroides, and also functions in E. coli. Further, both Tn5520 and the Tet element are widely disseminated in Bacteroides. Thus, Bacteroides may represent a highly efficient model for DNA and antibiotic resistance dissemination. The Specific Aims of this study are to 1) determine that all DNA processing functions of the promiscuous Tn5520 transfer factor are provided by a single mobilization protein and oriT region, and 2) to determine that pLF9 encodes a mating apparatus that serves as a common pathway for many different conjugative transfer factors, and identify key gene(s) involved in the interaction between initiation complexes and the mating pore. Studies include the use of matings, insertion and deletion mutagenesis, in vitro and in vivo relaxosome formation, DNA sequencing, protein/protein interactions, and cellular localization experiments. Upon completion of these studies, we will have determined the minimal requirements for DNA processing involved in transfer from Bacteroides, allowing comparison to more complex systems in other bacteria. The identification and characterization of the key components of the mating apparatus and relaxosome complex that allow for utilization of a single mating pore by genetically diverse transfer factors will be a major step towards a comprehensive model of DNA transfer in Bacteroides, and will provide important insights as to how bacterial antibiotic resistance is rapidly spread.