Sonoporation as a new genetic tool for F. nucleatum

描述（由申请人提供）：具核梭杆菌是一种革兰氏阴性厌氧菌，在多种人类感染形式中普遍存在。 它在口腔中含量特别丰富，在口腔生态和牙周病的发病机制中起着重要作用。 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，对这种生物的研究受到阻碍。 声孔效应是一种新的遗传工具，最近被开发并用于构建F. nucleatum 12230，一种已知对遗传操作难治的菌株。 声穿孔利用超声瞬时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中变得越来越有用，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穿孔产生双交换等位基因交换，而不是电穿孔产生的单交换。 因此，该技术有可能发展成为一个强大的工具，以简化突变体的建设。 本探索性研究的重点是利用F. nucleatum作为模式生物，以研究细菌中的声穿孔机制。 我们假设声致穿孔可以用于有效地将DNA递送到F。nucleatum和用于一步双交换突变体构建。 提出了两个具体目标。 在目标1中，将测试不同的声致孔条件以鉴定在F中质粒转化和/或双交换诱变的最佳条件。核质 目的二是探讨声穿孔介导DNA导入F.将研究核质。 我们的长期目标是将声致孔技术发展成为微生物遗传操作的有力工具

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