Development of a Conditional Plasmid for Gene Deletion in Non-ModelFusobacterium nucleatumstrains

Abstract

ABSTRACTFusobacterium nucleatumis an opportunistic pathogen with four subspecies:nucleatum(FNN),vincentii(FNV),polymorphum(FNP), andanimalis(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 withinF. nucleatum. While ATCC 23726 is highly transformable, most otherFusobacteriumstrains 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 anfdxpromoter 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 thegalKgene, successfully generating mutants in FNN (ATCC 23726, CTI-2), FNP (ATCC 10953), FNA (21_1A), and the closely related speciesFusobacterium periodonticum. Incorporating asacBcounterselection marker in this conditional plasmid enabled the deletion of theradDgene in non-model strains. Interestingly, whileradDdeletion in 23726, 10953, and 21_1A abolished coaggregation withActinomyces oris, the CTI-2 mutant retained this ability, suggesting the involvement of other unknown adhesins. This work significantly advances gene deletion in genetically recalcitrantF. nucleatumstrains, enhancing our understanding of this pathogen.IMPORTANCEFusobacterium nucleatumis 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 ofF. nucleatum. We successfully deleted genes in the clinical strain CTI-2, the FNA strain 21_1A, andF. periodonticumfor the first time. Our findings, particularly the varying behavior of theradDgene production in coaggregation across strains, underscore the complexity ofF. nucleatumand the need for broader genetic studies. This work advances our understanding ofF. nucleatumvirulence at the strain level and provides a valuable tool for future bacterial genetics research.