Targeted Mutagenesis in Fusobacterium nucleatum

The Gram-negative anaerobe Fusobacterium nucleatum is a prominent species in dental plaque biofilms, is involved in human infectious diseases including periodontitis and abscesses throughout the body, and is implicated in the occurrence of adverse pregnancy outcomes. Genomic DNA sequence is now available for three strains of F. nucleatum, representing three subspecies. Understanding the role of this species in dental plaque ecology and pathogenesis will be facilitated by genetic manipulations, and the ability to insertionally inactivate chromosomal genes is a critical tool for genetic analyses. Objective: This investigation focused on generating insertions in the F. nucleatum chromosome as a method of targeted mutagenesis. Methods: The plasmid pHS19 was used as an integration vector. This plasmid possesses an erythromycin resistance cassette (erm F-ermB) but lacks a fusobacterial replicon. Transformants were isolated and subcultured on selective media. Chromosomal DNA isolated from the transformants was used in PCR-based and Southern analyses to characterize the site of plasmid integration. Results: A putative exoribonuclease R (rnr) gene was selected for targeted mutagenesis based on its homology to a previously insertionally inactivated gene in an enteric bacterial pathogen. A PCR-amplified fragment of rnr was cloned into pHS19 in E. coli and the resulting plasmid was used to transform F. nucleatum. Transformation with the rnr -integration plasmid yielded 14 putative mutants; 11 of which were assessed by Southern blot and PCR analyses. In all 11 mutants the results confirmed insertion of the plasmid into the homologous site in the F. nucleatum chromosome. Conclusions: These findings demonstrate the feasibility of integration mutagenesis in F. nucleatum. To our knowledge, this is the first report of chromosomal mutagenesis in this species. (Supported by NIH/NIDCR PHS Grant No. DE12639)