ArticleMethods in molecular biology (Clifton, N.J.)2026
Enhanced Transformation Efficiency in Fusobacterium nucleatum by Bypassing Restriction-Modification Systems: A Methylation-Guided Protocol.
Article in Methods in molecular biology (Clifton, N.J.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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