Genetic Systems for Fusobacterium nucleatum

产品说明：革兰氏阴性菌种具核梭杆菌（Fn）是在牙菌斑生物膜和具有显著社会影响的感染（包括牙周炎）中重要的厌氧菌的异质菌种。基因组DNA序列可用于Fn的三个菌株，代表具有不同表型的主要亚种。我们的实验室首次报道了Fn的转化。最近的发展包括可靠的两个Fn菌株与第二代穿梭质粒的转化，并在两个Fn染色体基因的定点突变的示范。微生物特性研究的最大希望在于在具有确定基因组的宿主菌株中进行的分子操作。我们建议进一步开发Fn的遗传转移系统，重点是具有可用基因组序列的菌株。在具体目标1中，我们将使用选择的Fn限制性内切酶的插入失活或在E.螺旋用于预甲基化DNA作为克服限制性障碍的策略。在具体目标2中，我们将测试假设，即天然梭菌质粒pFN 3的新复制子将为具有不同宿主范围的替代稳定载体系统提供基础。将表征pFN 3复制子，并与先前描述的Fn复制子相比测试其宿主范围。最后，序列分析表明，原生梭菌质粒可能是可移动的接合。在具体目标3中，我们将检验接合基因转移可用于Fn的假设。这在绕过宿主限制性屏障方面可能特别有益;因为通常对限制性内切酶具有抗性的单链DNA被转移到受体菌株中。这些研究将使一个多样化的和重要的土著病原体的代表性菌株的分子分析，从而为未来的生态学和发病机制的研究提供了一个重要的基础。

DESCRIPTION: The Gram-negative species Fusobacterium nucleatum (Fn) is a heterogeneous species of anaerobic bacteria important in Dental plaque biofilms and infections with significant societal impact, including periodontitis. Genomic DNA sequence is available for three strains of Fn, representative of the major subspecies with distinct phenotypes. Our laboratory first reported transformation of Fn. Recent developments include reliable transformation of two Fn strains with second generation shuttle plasmids, and the demonstration of site-directed mutagenesis in two Fn chromosomal genes. The greatest promise for investigations of microbial properties lies in molecular manipulations conducted in host strains with defined genomes. We propose to further develop systems of genetic transfer for Fn focusing on strains with available genomic sequence. In Specific Aim 1 we will test the hypothesis that DNA restriction prevents or limits efficient transformation and mutagenesis in Fn, using insertional inactivation of selected Fn restriction endonucleases, or cloning of Fn methyltransferases in E. coil for use in pre-methylating DNA as a strategy to overcome restriction barriers. In Specific Aim 2 we will test the hypothesis that the novel replicon of a native fusobacterial plasmid pFN3 will provide the basis for an alternate stable vector system with a distinct host range. The pFN3 replicon will be characterized and its host range tested in comparison to previously described Fn replicons. Finally, sequence analyses suggest that native fusobacterial plasmids may be mobilizable by conjugation. In Specific Aim 3 we will test the hypothesis that conjugative gene transfer can be used in Fn. This may be of particular benefit in bypassing host restriction barriers; because single stranded DNA, typically resistant to restriction endonucleases, is transferred to the recipient strain. These studies will enable molecular analyses in representative strains of a diverse and important indigenous pathogen, and will thus provide a critical foundation for future studies of ecology and pathogenesis.