Genetic Systems for Fusobacterium nucleatum

描述：革兰氏阴性菌核梭杆菌(FN)是一种异质性厌氧细菌，在牙菌斑生物膜和感染(包括牙周炎)中具有重要的社会影响。基因组DNA序列可用于三株FN，代表具有不同表型的主要亚种。我们实验室首次报道了FN的转化。最近的进展包括用第二代穿梭质粒可靠地转化了两个FN菌株，并证明了两个FN染色体基因的定点突变。微生物特性研究的最大希望在于在具有已定义基因组的宿主菌株中进行分子操作。我们建议进一步开发FN的遗传转移系统，重点放在具有可用基因组序列的菌株上。在具体目标1中，我们将测试DNA限制阻止或限制FN中有效转化和突变的假设，使用选定的FN限制性内切酶的插入失活，或克隆用于DNA预甲基化的E.COIL中的FN甲基转移酶作为克服限制障碍的策略。在特定的目标2中，我们将检验这样的假设，即天然融合细菌质粒pFN3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。将对pFN3复制子进行表征，并与前面描述的FN复制子进行比较，测试其宿主范围。最后，序列分析表明，天然的融合细菌质粒可以通过接合来动员。在特定的目标3中，我们将检验接合基因转移可以用于FN的假设。这在绕过宿主限制障碍方面可能特别有好处；因为通常对限制性内切酶具有抵抗力的单链DNA被转移到受体菌株。这些研究将能够对一种多样化和重要的本土病原体的代表性菌株进行分子分析，从而为未来的生态学和发病机制研究提供关键的基础。

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.