Development of a Conditional Plasmid for Gene Deletion in Non-Model Fusobacterium nucleatum strains

Fusobacterium nucleatum is an opportunistic pathogen with four subspecies: nucleatum (FNN), vincentii (FNV), polymorphum (FNP), and animalis (FNA), each with distinct disease potentials. Research on fusobacterial pathogenesis has mainly focused on the model strain ATCC 23726 from FNN. However, this narrow focus may overlook significant behaviors of other FNN strains and those from other subspecies, given the genetic and phenotypic diversity within F. nucleatum. While ATCC 23726 is highly transformable, most other Fusobacterium strains exhibit low transformation efficiency, complicating traditional gene deletion methods that rely on non-replicating plasmids. To address this, we developed a conditional plasmid system in which the RepA protein, essential for replication of a pCWU6-based shuttle plasmid, is controlled by an inducible system combining an fdx promoter with a theophylline-responsive riboswitch. This system allows plasmid replication in host cells upon induction and plasmid loss when the inducer is removed, forcing chromosomal integration via homologous recombination in the presence of the antibiotic thiamphenicol. We validated this approach by targeting the galK gene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related species Fusobacterium periodonticum. Incorporating a sacB counterselection marker in this conditional plasmid enabled the deletion of the radD gene in non-model strains. Interestingly, while radD deletion in 23726, 10953, and 21_1A abolished coaggregation with Actinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrant F. nucleatum strains, enhancing our understanding of this pathogen. IMPORTANCEFusobacterium nucleatum is implicated in various human diseases, including periodontal disease, preterm birth, and colorectal cancer, often linked to specific strains and reflecting the species genetic and phenotypic diversity. Despite this diversity, most genetic research has centered on the model strain ATCC 23726, potentially missing key aspects of other strains pathogenic potential. This study addresses a critical gap by developing a novel conditional plasmid system that enables gene deletion in genetically recalcitrant strains of F. nucleatum. We successfully deleted genes in the clinical strain CTI-2, the FNA strain 21_1A, and F. periodonticum for the first time. Our findings, particularly the varying behavior of the radD gene production in coaggregation across strains, underscore the complexity of F. nucleatum and the need for broader genetic studies. This work advances our understanding of F. nucleatum virulence at the strain level and provides a valuable tool for future bacterial genetics research.