Abstract
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 Tn916 transposon. Here, we show that Tn916 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.
Highlights
Clostridium is a diverse genus whose members are gram-positive, spore-forming, anaerobic bacteria
Prior experiments demonstrated the transfer of pCLJ to C. botulinum strains containing Tn916, a conjugative transposon that confers tetracycline resistance[32]
After multiple attempts to chemically mutagenize C. botulinum strain 657Ba (Table 1) with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a single lysine auxotroph was isolated from approximately 200 screened colonies
Summary
Clostridium is a diverse genus whose members are gram-positive, spore-forming, anaerobic bacteria. Multi-domain homologues of BoNTs were newly identified in a separate class of Firmicutes, raising questions about the origin and distribution of the neurotoxin[7] Expression of one such homolog, BoNT/Wo, reveals that the novel toxin possesses a metalloprotease domain similar to BoNT/B but cleaves VAMP at a unique site[8]. These discoveries, in addition to the well-documented prophage-borne BoNT/C and /D in Group III C. botulinum[25] and plasmid-borne BoNT/G in Group IV26, strongly imply a role for horizontal gene transfer in shaping the modern species. Movement of BoNT-encoding plasmids into nontoxigenic clostridial strains, species with more resistant phenotypes, would pose real threats to health and safety, especially given the wide prevalence of clostridia in the environment and in the mammalian gastrointestinal tract
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