GENETIC SYSTEMS FOR FUSOBACTERIUM NUCLEATUM

描述(改编自调查人员摘要)：核梭杆菌 是一种重要的病原体，通常与牙周病有关。 以及其他常见的人类感染。人们对这种病毒的毒性知之甚少 这种病原菌的发病机制和研究一直受到缺乏 用于基因操作的系统。核盘藻在口腔中的重要性 和系统性疾病导致了人们对基因组测序的兴趣 接下来的几年。分子分析系统将被要求 在毒力特性研究中有效利用序列数据。这个 首席调查员的实验室最近分离并确定了 核盘菌表达载体pFN1。在这个提案中，要检验的假设 是PFN1可以用来开发一种基因转移系统以及 寄主载体系统为分子技术在F. 核糖体。在特定目标1中，核盘藻的基因转移系统将 使用基于PFN1的质粒进行电穿孔，以编码 四环素可选标记。转型研究将确定 “第一代”穿梭质粒，一株高表达的核盘菌 转化效率和其他抗生素耐药性决定因素 这赋予了核盘藻一种可选择的表型。基因转移 系统将在具体目标2中进行优化，通过改进 穿梭质粒及转化程序。刻画人物形象 基于PFN1的质粒将包括确认Theta机制 复制，以及分离和结构稳定性的确定， PFN1最小复制子，以及质粒拷贝数。“第二” 通过消除不必要的区域，将产生“代”质粒。 PFN1的DNA。转型的效率将通过 第二代穿梭质粒，在代表性的F. 核酸菌菌株。核盘藻的染色体整合质粒， 它缺乏核盘藻复制子，但含有核盘藻基因组DNA。 序列和TETM决定簇，将在具体目标3中开发。 转化子的基因组组织将通过Southern和PCR进行鉴定 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 利用分子生物学技术进行核针茅遗传分析的基础 诱变和互补技术。因为基因系统 转移目前不适用于核盘藻，这些结果将 对我们理解核盘藻的毒力和 有可能在预防人类传染病方面发挥作用。

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.