HicA Toxin-Based Counterselection Marker for Allelic Exchange Mutations in Fusobacterium nucleatum

The study of fusobacterial virulence factors has dramatically benefited from the creation of various genetic tools for DNA manipulation, including based counterselection for in-frame deletion mutagenesis in Fusobacterium nucleatum, which was recently developed. However, this method requires a host lacking the gene, which is an inherent limitation. To circumvent this limitation, we explored the possibility of using the gene that encodes a toxin consisting of a HicAB toxin-antitoxin module in Fusobacterium periodonticum as a new counterselective marker. Interestingly, the full-length gene is not toxic in F. nucleatum, but a truncated gene version lacking the first six amino acids is functional as a toxin. The toxin expression is driven by an promoter and is controlled at its translational level by using a theophylline-responsive riboswitch unit. As a proof of concept, we created markerless in-frame deletions in the fusobacterial adhesin gene within the F. nucleatum operon and the gene that encodes the tryptophanase for indole production. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of colonies grown on theophylline-added plates and resulted in in-frame deletions in 50% of the screened isolates. This -based counterselection system provides a robust and reliable counterselection in wild-type background F. nucleatum and should also be adapted for use in other bacteria. Fusobacterium nucleatum is an indole-producing human oral anaerobe associated with periodontal diseases, preterm birth, and several cancers. Little is known about the mechanisms of fusobacterial pathogenesis and associated factors, mainly due to the lack of robust genetic tools for this organism. Here, we showed that a mutated gene from Fusobacterium periodonticum expresses an active toxin and was used as a counterselection marker. This -based in-frame deletion system efficiently creates in-frame deletion mutations in the wild-type background of F. nucleatum. This is the first report to use the gene as a counterselection marker in a bacterial genetic study.