Treponomics: enhanced tools for genetic manipulation in spirochetes

描述（由申请人提供）：微生物基因组学时代的主要挑战之一是通过实验建立基因组数据与基因功能、生理、行为和致病性之间的联系。齿龈密螺旋体（Treponema denticola）是一种牙龈粘膜组织的机会致病菌，为微生物-宿主相互作用、微生物生理学和分子进化等研究提供了一系列靶点。虽然口腔螺旋体的致病行为和疾病关联与苍白螺旋体明显不同，但齿状螺旋体的致病行为和疾病关联与苍白螺旋体不同

DESCRIPTION (provided by applicant): One of the major challenges in the era of microbial genomics is to experimentally establish connections between genome data and gene function, physiology, behavior and pathogenicity. Treponema denticola, an opportunistic pathogen of gingival mucosal tissue, offers a range of targets for research into microbe-host interactions, microbial physiology and molecular evolution. While pathogenic behaviors and disease associations of oral spirochetes are clearly distinct from those of T. pallidum, T. denticola is of high interest as a model for studying physiology, biosynthetic pathways and microbe-host interactions in spirochetal diseases including syphilis. This is due both to physiological similarities between Td and T. pallidum and to the fact that T. pallidum genetic studies remain impracticable without an in vitro culture system. This project will develop and demonstrate significant advances in methods for genetic manipulation of cultivable spirochetes of the genus Treponema that will "jump-start" Treponema genetic research, thus providing opportunities to rationally address novel biological questions, particularly in uncultivable spirochetes. We propose a T. denticola-specific adaptation of counter selection mutagenesis coupled with knockouts of restriction-modification systems as key features to facilitate construction of T. denticola strains with (1) greatly increased ability to take up and incorporate heterologous DNA's, and (2) an expanded range of possible mutant types. Building on "proof of concept" studies primarily conducted by our group, results of this work will both facilitate in-depth molecular analysis of T. denticola and establish the utility of T. denticola as a model organism for molecular analysis of agents of other treponemal infections, particularly T. pallidum.