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Most Group I  Clostridium botulinum  strains harbor botulinum neurotoxin ( bont ) genes on their chromosome, while some carry these genes (including  bont/a ,  bont/b , and  bont/f ) on large plasmids. Prior work in our laboratory demonstrated that Group I BoNT plasmids were mobilized to  C. botulinum  recipient strains containing the Tn 916  transposon. Here, we show that Tn 916  is nonessential for plasmid transfer. Relying on an auxotrophic donor phenotype and a plasmid-borne selectable marker, we observed the transfer of pCLJ, a 270 kb plasmid harboring two  bont  genes, from its host strain to various clostridia. Transfer frequency was greatest to other Group I  C. botulinum  strains, but the plasmid was also transferred into traditionally nontoxigenic species, namely  C. sporogenes  and  C. butyricum . Expression and toxicity of BoNT/A4 was confirmed in transconjugants by immunoblot and mouse bioassay. These data indicate that conjugation within the genus  Clostridium  can occur across physiological Groups of  C. botulinum , supporting horizontal gene transfer via  bont -bearing plasmids. The transfer of plasmids possessing  bont  genes to resistant  Clostridium  spp. such as  C. sporogenes  could impact biological safety for animals and humans. These plasmids may play an environmental role in initiating death in vertebrates, leading to decomposition and nutrient recycling of animal biomass.

Botulinum neurotoxin–encoding plasmids can be conjugatively transferred to diverse clostridial strains