Efficient Mechanism of Gene Transfer in B. fragilis

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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.