Sonoporation as a new genetic tool for F. nucleatum

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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