Enhanced Transformation Efficiency in Fusobacterium nucleatum by Bypassing Restriction-Modification Systems: A Methylation-Guided Protocol.

The genetic intractability of Fusobacterium nucleatum has slowed progress in defining mechanisms of fusobacterial pathogenesis. Transformation is inefficient, and broadly applicable genetic tools are still limited. A major obstacle is the presence of endogenous restriction-modification (RM) systems that cleave incoming DNA lacking appropriate methylation. This chapter presents a concise, reproducible protocol to increase transformation efficiency in F. nucleatum by systematically bypassing RM barriers. The workflow combines methylome mapping by single-molecule real-time (SMRT) sequencing, expression of cognate methyltransferases (MTases) in an intermediary Escherichia coli host to pre-methylate shuttle plasmids, and optimized electroporation with careful anaerobic handling and selective recovery. Using F. nucleatum ATCC 25586 as an example, the method yields a marked improvement in transformation efficiency, and the same logic can be adapted to additional F. nucleatum isolates once their methylation context is defined.