Endogenous CRISPR-Cas Systems in Group I Clostridium botulinum and Clostridium sporogenes Do Not Directly Target the Botulinum Neurotoxin Gene Cluster

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.

Genomic Diversity, Competition, and Toxin Production by Group I and II Clostridium botulinum Strains Used in Food Challenge Studies

“…Whole genome sequencing of an increasing number of C. botulinum strains is changing the landscape of the C. botulinum and BoNT research field. The sequences for over 1000 strains of C. botulinum are currently available, and bioinformatics analyses are revealing previously uncovered insights into aspects of strain relatedness, lateral gene transfer, immune defenses, evolution, and physiology of the strains [ 13 , 14 , 39 , 51 , 59 , 61 , 65 ]. These data are of particular interest for food safety studies, which for decades have relied on assays utilizing select strains to represent the entire species.…”

“…The resulting core genome analysis was used to construct SNP (single nucleotide polymorphism) phylogenies from non-duplicated core genes present in all species present in each species group (GI/sporo: 726, GII: 1778). The GI/sporo strains include 241 Group I C. botulinum and C. sporogenes that were identified via an MLST scheme described previously [ 50 , 51 ], the 7 Group I strains sequenced as part of this project, and strains NCIB 4301 (GCA_011017885.1), 53B (GCA_011014035.1), and 69A (GCA_000439655.1) which were included due to prior use in food challenge studies [ 30 , 32 ]. The only previously available assembly of strain 69A, GCA_000439655.1, was included in the core genome SNP (cgSNP) analysis due to study relevance despite possessing an elevated pseudogene count.…”

Identification of divergent botulinum neurotoxin homologs in Paeniclostridium ghonii

“…BoNTs are produced not only by four groups of C. botulinum (Group I strains are human-associated and Group III are animal-associated), but also by strains of C. noyvi, C. baratii, C. sporogenes , and C. butyricum . BoNTs are encoded chromosomally, on plasmids, or on phages depending on the subtype and strain [6]. Related non-toxigenic strains are also common [7].…”

Specific Isolation of Clostridium botulinum Group I Cells by Phage Lysin Cell Wall Binding Domain with the Aid of S-Layer Disruption

“…C. botulinum Group I possesses two distinct clades, one consisting mainly of toxinogenic C. botulinum Group I strains and the other consisting mainly of nontoxinogenic C. sporogenes strains. Both clades also harbor sporadic nontoxinogenic and toxinogenic strains, respectively [ 3 , 4 ]. To address the two clades together, we hereafter refer to them as C. botulinum Group I sensu lato .…”

“…The rigid host specificity of CBO1751 led us to hypothesize that its CBD, when isolated from the lytic domain, could be used as a biological probe for the specific isolation of viable cells of C. botulinum Group I. A recent report shows a number of CBO1751 homologs (annotated as N-acetylmuramoyl-L-alanine amidase) in both C. botulinum Group I and C. sporogenes to be targets of endogenous CRISPR systems [ 4 ], which could indicate that CBO1751 and its homologs constitute promising candidates for specifically targeting C. botulinum Group I and C. sporogenes .…”