Efficient Mechanism of Gene Transfer in B. fragilis

这项研究的假设是，DNA和抗生素耐药性基因在类杆菌中的转移是高效的，涉及到遗传上无关的动员模块(1到3个基因)，这些模块在转移开始时执行所有的DNA处理功能。此外，这些动员模块位于不同的转移因素上。它们都与由类杆菌Tet元件编码的接合装置(交配孔)进行类似的相互作用，导致它们广泛传播。这一假说的基本原理是根据大量的初步数据得出的，这些数据表明，当与常见的交配装置共存时，类杆菌转移因子在类杆菌中具有相似的转移特性，而当与广泛的宿主范围的质粒共存时，类杆菌转移因子在大肠杆菌中具有相似的转移特性。每个可动员的转移因子都包含一个、两个或三个基因，这些基因必须执行类似的DNA处理功能，尽管缺乏同源性。然而，所有可动员的转移因子都有一个5个碱基对的一致NIC序列。我们现在捕获了一个类杆菌交配装置，它似乎是一个共轭转座元件(Tet元件)的一部分。这种交配装置在类杆菌中为多种动员因子提供了交配桥梁，在大肠杆菌中也发挥了作用。此外，Tn5520和Tet元素都广泛分布在类杆菌中。因此，类杆菌可能是DNA和抗生素耐药性传播的一种高效模型。本研究的具体目的是：1)确定混杂Tn5520转移因子的所有DNA加工功能是由单个动员蛋白和ORIT区域提供的；2)确定pLF9编码一个交配装置，作为许多不同的接合转移因子的共同途径，并确定参与起始复合体与交配孔相互作用的关键基因(S)。研究包括配对的使用、插入和缺失突变、体外和体内松弛小体的形成、DNA测序、蛋白质/蛋白质相互作用和细胞定位实验。在这些研究完成后，我们将确定从类杆菌转移所涉及的DNA处理的最低要求，以便与其他细菌中更复杂的系统进行比较。鉴定和表征交配器和松弛小体复合体的关键成分，允许通过遗传多样性的转移因子利用单个交配孔，将是朝着类杆菌DNA转移的全面模式迈出的重要一步，并将为细菌抗生素耐药性如何迅速传播提供重要的见解。

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.