Abstract
Most strains of proteolytic group I Clostridium botulinum (G1 C. botulinum) and some strains of Clostridium sporogenes possess genes encoding botulinum neurotoxin (BoNT), a potent neuroparalytic agent. Within G1 C. botulinum, conserved bont gene 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 bont gene clusters across strains we conducted a bioinformatic analysis profiling endogenous CRISPR-Cas systems from 241 G1 C. botulinum and C. sporogenes strains. Approximately 6,200 CRISPR spacers were identified across the strains and Type I-B, III-A/B/D cas genes 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 bont gene 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 bont gene 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.
Highlights
Botulinum neurotoxins are potent proteinaceous toxins that are horizontally distributed throughout multiple species of Clostridium, a genus of anaerobic, Gram-positive bacteria (Collins and East, 1998)
A single strain, C. sporogenes CDC 1632, the most extreme outlier within the phylogeny, possessed a chromosomally integrated bont/B1 gene cluster (Figure 2A). These data indicate that bont gene clusters are broadly present throughout G1 C. botulinum and frequently chromosomally integrated, while bont gene clusters in C. sporogenes are limited to bont/B and usually plasmid-borne
To investigate whether predicted CRISPR-Cas systems in G1 C. botulinum and C. sporogenes could potentially modulate the range of bont gene transfer through immunity by either directly targeting the bont gene cluster or associated mobile genetic element (MGE), we investigated the CRISPR array encoded spacers and identified their predicted cognate targets
Summary
Botulinum neurotoxins are potent proteinaceous toxins that are horizontally distributed throughout multiple species of Clostridium, a genus of anaerobic, Gram-positive bacteria (Collins and East, 1998). A recent study has demonstrated that ISs potentially play a major role in transferring virulence associated genes from conjugative plasmids to the chromosome (Che et al, 2021) Both intact and degraded ISs are known to occur within the vicinity of and, in some cases, flanking bont gene clusters (Smith et al, 2007, 2015; Hill et al, 2009; Dover et al, 2014). CRISPR arrays represent a library of past encounters with horizontally mobile entities including phage, plasmids, and other MGEs. Previous studies have identified type I and type III CRISPR-Cas systems in a selection of G1 C. botulinum strains, and none have examined their presence in C. sporogenes (Hatoum-Aslan and Marraffini, 2014; Carter et al, 2016; Woudstra et al, 2016; Negahdaripour et al, 2017). C. sporogenes strains indicate that the two species possess and utilize the same types of CRISPR-Cas systems, which do not directly target the bont gene clusters but target bont(+) conjugative plasmids
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