GENETIC SYSTEMS FOR FUSOBACTERIUM NUCLEATUM

描述（改编自研究者摘要）：具核梭杆菌 是牙周病中常见的重要病原体 和其他常见的人类感染。 人们对它的毒性知之甚少 这种病原体的机制和调查一直受到缺乏 基因操作系统。 F的重要性。口腔核仁 和系统性疾病引起了人们对基因组测序的兴趣， 未来几年 分子分析系统将被要求 有效利用序列数据研究毒力特性。 的 首席研究员的实验室最近分离并鉴定了 a F.有核质粒pFN1。 在这个提议中，要检验的假设 pFN1可用于开发基因转移系统， 宿主载体系统，为分子生物学技术在F. 核质 在Specific Aim 1中，建立了F.核质将 使用基于pFN1的质粒进行电穿孔， 四环素选择标记 转型研究将确定一个 “第一代”穿梭质粒，F.具核菌株 转化效率和其他抗生素耐药性决定因素 在F.核质 基因转移 系统将在具体目标2中优化， 穿梭质粒和转化程序。 表征 基于pFN1的质粒将包括对以下theta机制的确认： 复制，以及分离和结构稳定性的测定， pFN 1最小复制子和质粒拷贝数。 一个“秒 第二代”质粒将通过消除非必需区域来开发。 pFN1 DNA 转换效率将通过 第二代穿梭质粒，并在代表性的F. 具核菌株 F.核质， 缺少一个F核复制子，但含有F.核质基因组DNA 序列和tetM决定簇，将在具体目标3中开发。 的 转化体的基因组结构将通过Southern和PCR进行评估 分析以确认染色体整合，并表征 整合的场所和机制。 这些研究的结果将创造 为F.显核分子 诱变和互补技术。 因为基因系统 目前，F不具备转让条件。nucleatum，这些结果将 对我们理解F.核毒力和 潜在地用于预防人类传染病。

DESCRIPTION (Adapted from investigator's Abstract): Fusobacterium nucleatum is an important pathogen that is commonly involved in periodontal disease and other common human infections. Little is known about the virulence mechanisms of this pathogen and investigation has been hampered by a lack of systems for genetic manipulation. The importance of F. nucleatum in oral and systemic diseases has led to interest in genomic sequencing within the next few years. Systems for molecular analysis will be required to effectively utilize sequence data in the study of virulence properties. The Principal Investigator's laboratory has recently isolated and characterized a F. nucleatum plasmid, pFN1. In this proposal, the hypothesis to be tested is that pFN1 can be used to develop a system of gene transfer as well as a host vector system for the application of molecular techniques in F. nucleatum. In Specific Aim 1 a gene transfer system for F. nucleatum will be developed using electroporation with pFN1-based plasmids that encode a tetracycline selectable marker. Transformation studies will identify a "first generation" shuttle plasmid, a F. nucleatum strain with high transformation efficiency, and additional antibiotic resistance determinants that confer a selectable phenotype in F. nucleatum. The gene transfer system will be optimized in Specific Aim 2 through refinement of both the shuttle plasmid and the transformation procedures. Characterization of the pFN1-based plasmids will include confirmation of the theta mechanisms of replication, and determination of segregational and structural stability, the pFN1 minimal replicon, and the plasmid copy number. A "second generation" plasmid will be developed by eliminating nonessential regions of the pFN1 DNA. The efficiency of transformation will be optimized with the second generation shuttle plasmid and evaluated in representative F. nucleatum strains. A chromosomal integration plasmid for F. nucleatum, which lacks a F. nucleatum replicon but contains F. nucleatum genomic DNA sequences and a tetM determinant, will be developed in Specific Aim 3. The genomic organization of transformants will be evaluated by Southern and PCR analyses to confirm the chromosomal integration, and to characterize the site and mechanism of integration. The results of these studies will create the foundation for genetic analysis in F. nucleatum by developing molecular techniques of mutagenesis and complementation. Because systems of gene transfer are not currently available for F. nucleatum, these results will have significant impact on our understanding of F. nucleatum virulence and potentially in the prevention of human infectious diseases.