Methods for the Evasion of Restriction-Modification Systems During Genetic Engineering of Fusobacterium Species

Genetic manipulation of clinical Fusobacterium isolates is severely limited by strain-specific restriction-modification (RM) systems that recognize and degrade foreign DNA. This chapter describes strategies to evade RM defenses and improve transformation efficiencies across diverse Fusobacterium lineages. We employ a sequence-based "RM-silencing" method: in silico removal of nonessential regions and introduction of synonymous or GC-preserving nucleotide substitutions to disrupt RM recognition sites within coding and noncoding regions, respectively. Detailed protocols for high-molecular-weight genomic DNA isolation, computational motif identification, plasmid optimization, anaerobic preparation of electrocompetent cells, and optimized electroporation parameters are provided. Application of these methods has enabled successful delivery of replicative plasmids, linear recombination templates, and transposon cassettes into previously intractable Fusobacterium clinical isolates. Collectively, these techniques constitute a versatile toolkit to overcome RM barriers and advance functional genetic studies in Fusobacterium species.