Abstract

Classified as the genospecies Clostridium novyi sensu lato and distributed into four lineages (I–IV), Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum are clostridial pathogens that cause animal diseases. Clostridium novyi sensu lato contains a large mobilome consisting of plasmids and circular bacteriophages. Here, we explored clustered regularly interspaced short palindromic repeats (CRISPR) arrays and their associated proteins (Cas) to shed light on the link between evolution of CRISPR-Cas systems and the plasmid and phage composition in a study of 58 Clostridium novyi sensu lato genomes. In 55 of these genomes, types I-B (complete or partial), I-D, II-C, III-B, III-D, or V-U CRISPR-Cas systems were detected in chromosomes as well as in mobile genetic elements (MGEs). Type I-B predominated (67.2%) and was the only CRISPR type detected in the Ia, III, and IV genomic lineages. Putative type V-U CRISPR Cas14a genes were detected in two different cases: next to partial type-IB CRISPR loci on the phage encoding the botulinum neurotoxin (BoNT) in lineage Ia and in 12 lineage II genomes, as part of a putative integrative element related to a phage-inducible chromosomal island (PICI). In the putative PICI, Cas14a was associated with CRISPR arrays and restriction modification (RM) systems as part of an accessory locus. This is the first time a PICI containing such locus has been detected in C. botulinum. Mobilome composition and dynamics were also investigated based on the contents of the CRISPR arrays and the study of spacers. A large proportion of identified protospacers (20.2%) originated from Clostridium novyi sensu lato (p1_Cst, p4_BKT015925, p6_Cst, CWou-2020a, p1_BKT015925, and p2_BKT015925), confirming active exchanges within this genospecies and the key importance of specific MGEs in Clostridium novyi sensu lato.

Highlights

  • Clostridium novyi sensu lato is a genospecies, containing the closely related species Clostridium botulinum, Clostridium novyi, and Clostridium haemolyticum (Skarin et al, 2011; Skarin and Segerman, 2014)

  • Six different clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) system types belonging to class 1 and 2 CRISPR loci were identified in 55 out of the 58 Clostridium novyi sensu lato strains included in our study, located either on the chromosome or on mobile genetic elements (MGEs) (BoNT phages, plasmids p2BKT015925, pCLG2/pCN2; Table 1; Figure 1)

  • In silico-identified Protospacer-adjacent motif (PAM) sequences associated with type I-B, I-D, and II-C CRISPRs matched with canonical PAMs (Kieper et al, 2018; Figure 1)

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Summary

Introduction

Clostridium novyi sensu lato is a genospecies, containing the closely related species Clostridium botulinum (group III), Clostridium novyi, and Clostridium haemolyticum (Skarin et al, 2011; Skarin and Segerman, 2014) This proposed genospecies (Skarin et al, 2011) is based on genomic comparisons: their chromosome is highly conserved and the differences between these three species can be attributed to the content of their mobile genetic elements (MGEs; Skarin and Segerman, 2011). The beta toxin produced by C. haemolyticum is responsible for bacillary hemoglobinuria affecting ruminants (Skarin and Segerman, 2014) These virulence genes are all carried on MGEs (plasmids and phages) as part of their mobilome. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), together making up the CRISPR-Cas system, which records MGEs past encounters, could help to shed light on a putative larger mobilome within Clostridium novyi sensu lato

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