A Conditional Plasmid System for Markerless Gene Deletion in Genetically Recalcitrant Fusobacterium nucleatum subsp....

Fusobacterium nucleatum subsp. animalis (FNA) plays a prominent role in oral microbial ecology and is increasingly implicated in systemic diseases such as colorectal cancer. However, its genetic intractability has significantly hindered functional studies to understand its pathogenic mechanisms. To address this challenge, we developed a conditional plasmid system that enables efficient, markerless gene deletion in FNA strains. This system features inducible control of plasmid replication via a theophylline-responsive riboswitch regulating repA, and counterselection via the MazF toxin under the control of an anhydrotetracycline-inducible promoter. In this chapter, we provide a detailed, step-by-step protocol for implementing this system using FNA strain 7_1 as a model. We illustrate the procedure by deleting the luxS gene, which encodes S-ribosylhomocysteine lyase-an enzyme involved in AI-2 quorum sensing and biofilm regulation. Our protocol provides a powerful and adaptable tool for advancing genetic studies in this genetically recalcitrant subspecies.