Identification and diversity analysis of CRISPR-Cas systems in the pathogenic strains of Clostridium botulinum to create eco-friendly phage preparations

The article presents a bioinformatic study of the diversity of CRISPR-Cas systems in the genomes of Clostridium botulinum and the phages they detect, with the aim of their targeted screening. The subject matter of the study was 49 complete chromosomal sequences of bacteria obtained from the GenBank database. Cas genes were identified employing the MacSyFinder tool with the use of HMM profiles from the PFAM and TIGRFAM databases. The identification and analysis of CRISPR cassettes were performed using three independent programs: CRISPRFinder, PILER-CR, and CRISPR Recognition Tool, which ensured high accuracy in determining the cassette structure. Protospacers were identified using the CRISPRTarget tool and the BLASTn algorithm against RefSeq-Viral viral databases. The study involved comparing spacer sequences and phage genomes in order to identify complementary sites. A phage immunity analysis revealed a predominance of Cellulophaga phages (19%), which can be attributed to the environmental characteristics of Clostridium botulinum, as well as a significant proportion of Aeromonas and Bacillus phages (12.5%). Another group of phages (predominantly intestinal) included Enterococcus, Escherichia, and Lactococcus species (6–10%). Also, the protospacers of rare phages (3% each) were found: Acidianus filamentous, Prochlorococcus, Pseudoalteromonas, Stenotrophomonas, and Synechococcus. The obtained results indicate complex CRISPR-Cas systems in Clostridium botulinum, evolving under the impact of different ecological niches.

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