Most strains of proteolytic group I
Clostridium botulinum(G1 C. botulinum) and some strains of Clostridium sporogenespossess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bontgene clusters of three major toxin serotypes ( bont/A/B/F) can be found on conjugative plasmids and/or within chromosomal pathogenicity islands. CRISPR-Cas systems enable site-specific targeting of previously encountered mobile genetic elements (MGE) such as plasmids and bacteriophage through the creation of a spacer library complementary to protospacers within the MGEs. To examine whether endogenous CRISPR-Cas systems restrict the transfer of bontgene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinumand C. sporogenesstrains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D casgenes and CRISPR array features were identified in 83% of the strains. Mapping the predicted spacers against the masked strain and RefSeq plasmid dataset identified 56,000 spacer–protospacer matches. While spacers mapped heavily to targets within bont(+) plasmids, no protospacers were identified within the bontgene clusters. These results indicate the toxin is not a direct target of CRISPR-Cas but the plasmids predominantly responsible for its mobilization are. Finally, while the presence of a CRISPR-Cas system did not reliably indicate the presence or absence of a bontgene cluster, comparative genomics across strains indicates they often occupy the same hypervariable loci common to both species, potentially suggesting similar mechanisms are involved in the acquisition and curation of both genomic features.