Characterization and distribution of CRISPR–Cas systems in Lactobacillus sakei
Clustered regularly interspaced palindromic repeats (CRISPR)–Cas (CRISPR-associated) structures, known as prokaryotes ‘immune system’, have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR–Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR–Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR–Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.
KeywordsLactobacillus sakei CRISPR–Cas Prophage Starter culture Differentiation Tracking Genotyping
Part of this work was funded via AiF within the program for promotion of Industrial Collective Research (IGF) of the German Ministry of Economic Affairs and Energy (BMWi), and the research association of the German food industry (FEI) in project 18552 N.
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Conflict of interest
The authors declare that they have no conflict of interest.
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