| Clostridium botulinum, an important pathogen of humans and animals, produces botulinum neurotoxin (BoNT), the most poisonous toxin known. Historically, the genes encoding BoNTs of serotype A, B, E and F have been believed to be located on the chromosome. I have recently discovered that the BoNT/A3, BoNT/A4 and BoNT/bvB genes are located on large megaplasmids (∼280 kb). The finding of both BoNT/A4 and BoNT/bvB genes being harbored on the same plasmid in the dual neurotoxin producing C. botulinum strain 657Ba led to the hypothesis that similar plasmids may be present in other bivalent C. botulinum strains. Large virulence plasmids were detected in C. botulinum subtype Bf strains that carried both the BoNT/B and BoNT/F genes. These plasmids shared a high degree of sequence homology to the plasmids found in other proteolytic strains of C. botulinum. In contrast, in C. botulinum strain Af 84, both bont/A and bont/F were located on the chromosome, despite the presence of a large (∼240 kb) plasmid in this strain. The presence of different serotype neurotoxin genes residing on highly homologous plasmids suggested that intra-species transfer of these plasmids may occur. Bacterial mating experiments revealed that plasmids found in proteolytic and nonproteolytic C. botulinum strains were conjugatively transferred to other proteolytic C. botulinum strains. Genes homologous to plasmid transfer genes such as TraD, TraG and TraK as well as genes encoding putative type II and type IV secretion system proteins are described in the genome annotations of plasmids found in proteolytic C. botulinum strains CDC-A3 and 657Ba, respectively. Interestingly, the plasmid pCLL of the nonproteolytic C. botulinum strain Eklund 17B was successfully transferred to proteolytic C. botulinum strains. This was a highly significant finding and can be considered an inter-species transfer of virulence plasmids, since nonproteolytic strains are considered a different species. Analysis of the nucleotide sequences of pCLL revealed regions homologous to conjugative virulence plasmids in C. perfringens. The potential transfer of C. botulinum virulence plasmid to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids. |
