Sonoporation as a new genetic tool for F. nucleatum

描述(申请人提供：核梭杆菌是一种革兰氏阴性厌氧菌，广泛存在于多种人类感染中。尤其在口腔中含量丰富，在口腔生态学和牙周病的发病机制中发挥着重要作用。它在宫内感染中也非常普遍，与不良妊娠结局有关。由于缺乏有效的遗传工具，对这种有机体的研究一直受到阻碍。最近开发了一种新的遗传工具Sonopation，并用于构建第一个等位基因交换突变体us1(FADA：：ermF-ermAM)，该突变体存在于核盘藻12230中，是一种已知的对遗传操作不敏感的菌株。Sonopation利用超声波瞬间增加细胞膜的通透性。这项技术在将药物和基因输送到哺乳动物和植物细胞方面正变得越来越有用，但直到现在才被应用于细菌。更有趣的是，使用完整的自杀质粒的声波作用产生了双交叉等位基因交换，而不是像电穿孔那样产生单交叉。因此，这项技术有可能发展成为简化突变体构建的强大工具。本探索性研究的重点是利用核盘藻作为模式生物来研究细菌中的声波作用机制。我们推测，声波作用可用于有效地将DNA导入核心菌，并用于一步法双交叉突变体的构建。提出了两个具体目标。在目标1中，将测试不同的声学操作条件，以确定最适合于在核盘藻中进行质粒转化和/或双交叉诱变的条件。在目标2中，我们将研究声波修复介导的DNA进入核盘藻的机制。我们的长期目标是将声学操作发展成为一种强大的微生物基因操作工具

DESCRIPTION (provided by applicant: Fusobacterium nucleatum is a gram-negative anaerobe prevalent in many forms of human infections. It is particularly abundant in the oral cavity, playing an important role in the oral ecology and the pathogenesis of periodontal disease. It is also highly prevalent in intrauterine infections, associated with adverse pregnancy outcomes. Studies of this organism have been hindered by the lack of effective genetic tools. A novel genetic tool, sonoporation, was recently developed and used to construct the first allelic exchange mutant, US1 (fadA::ermF-ermAM), in F. nucleatum 12230, a strain known to be refractory to genetic manipulations. Sonoporation utilizes ultrasound to transiently increase the cell membrane permeability. This technology is becoming increasingly useful for drug and gene delivery into mammalian and plant cells, but had never been applied to bacteria until now. Even more interesting is that sonoporation using intact suicide plasmids produced double-crossover allelic exchanges, rather than single crossovers as would by electroporation. Thus, this technology has the potential to be developed into a powerful tool to streamline mutant construction. The focus of this exploratory study is to use F. nucleatum as a model organism to investigate the mechanism of sonoporation in bacteria. We hypothesize that sonoporation can be used for effective DNA delivery into F. nucleatum and for one-step double-crossover mutant construction. Two specific aims are proposed. In aim 1, different sonoporation conditions will be tested to identify those optimal for plasmid transformation and/or double-crossover mutagenesis in F. nucleatum. In aim 2, the mechanism of sonoporation-mediated DNA delivery into F. nucleatum will be investigated. Our long term goal is to develop sonoporation into a robust tool for genetic manipulation of microorganisms