At least 40 toxin subtypes of botulinum neurotoxins (BoNTs), a heterogenous group of bacterial proteins, are produced by seven different clostridial species. A key factor that drives the diversity of neurotoxigenic clostridia is the association of bont gene clusters with various genomic locations including plasmids, phages and the chromosome. Analysis of Clostridium sporogenes BoNT/B1 strain CDC 1632, C. argentinense BoNT/G strain CDC 2741, and Clostridium parabotulinum BoNT/B1 strain DFPST0006 genomes revealed bont gene clusters within plasmid-like sequences within the chromosome or nested in large contigs, with no evidence of extrachromosomal elements. A nucleotide sequence (255,474 bp) identified in CDC 1632 shared 99.5% identity (88% coverage) with bont/B1-containing plasmid pNPD7 of C. sporogenes CDC 67071; CDC 2741 contig AYSO01000020 (1.1 MB) contained a ~140 kb region which shared 99.99% identity (100% coverage) with plasmid pRSJ17_1 of C. argentinense BoNT/G strain 89G; and DFPST0006 contig JACBDK0100002 (573 kb) contained a region that shared 100% identity (99%) coverage with the bont/B1-containing plasmid pCLD of C. parabotulinum Okra. This is the first report of full-length plasmid DNA-carrying complete neurotoxin gene clusters integrated in three distinct neurotoxigenic species: C. parabotulinum, C. sporogenes and C. argentinense.

Botulinum neurotoxins (BoNT) are the most potent toxins in the world. They are produced by a few dozens of strains within several clostridial species. The toxin that they produce can cause botulism, a flaccid paralysis in humans and other animals. With seven established serologically different types and over 40 subtypes, BoNTs are among the most diverse known toxins. The toxin, its structure, its function and its physiological effects on the neural cell and animal hosts along with its diversity have been the subjects of numerous studies. However, many gaps remain in our knowledge about the BoNT toxin and the species that produce them. One of these gaps involves the distribution and extent of variability along the full length of the gene and the protein as well as its domains and subdomains. In this study, we performed an extensive analysis of all of the available 143 unique BoNT-encoding genes and their products, and we investigated their diversity and evolution. Our results indicate that while the nucleotide variability is almost uniformly distributed along the entire length of the gene, the amino acid variability is not. We found that most of the differences were concentrated along the protein’s light chain (LC) domain and especially, the C-terminus of the receptor-binding domain (H_CC). These two regions of the protein are thus identified as the main source of the toxin type differentiation, and consequently, this toxin’s versatility to bind different receptors and their isoforms and act upon different substrates, thus infecting different hosts.