A New Method for Gene Deletion to Investigate Cell Wall Biogenesis in Fusobacterium nucleatum

Controlled septal peptidoglycan hydrolysis is vital for bacterial cell division, preserving cellular integrity and facilitating proper daughter cell separation. In Escherichia coli, the FtsEX ABC system governs cell wall hydrolase activation by regulating the EnvC activator. However, the processes underlying cell division in the Gram-negative oral bacterium Fusobacterium nucleatum are poorly understood, mainly due to its well-known genetic intractability. Herein, we provide a step-by-step procedure for a new gene deletion method in F. nucleatum, focusing on the ftsX gene as a target. This novel approach exploits the HicAB toxin-antitoxin system, using HicA as a counter-selective marker to enable efficient and precise gene deletion. By implementing this technique, we successfully demonstrated its applicability in F. nucleatum, providing new insights into this important microorganism's cell division process. Furthermore, this advanced gene deletion technique offers a valuable resource for future investigation into the functional characterization of genes involved in cell wall biogenesis in F. nucleatum and other genetically intractable microorganisms.