Analysis of CRISPR systems of types II-A, I-E and I-C in strains of Lacticaseibacillus

Abstract

A total of 49 strains of Lacticaseibacillus were screened for the presence of CRISPR-Cas systems. Type II-A CRISPR-Cas loci were sequenced for 8 Lacticaseibacillus paracasei and 3 Lacticaseibacillus rhamnosus strains. They contained 21 to 49 spacers, a few of them with similarity to plasmids. Cas9 identity was well correlated with spacer content of the strains. Only strain L. paracasei 85 contained a type I-E CRISPR locus, uncommon in this species, with highly conserved Cas proteins and 79 spacers not previously reported, a high percentage of them (23%) matching L. paracasei phage genomes. PAM sequences were 5′-NGAA-3´ (type II-A) and 5′-aAA-3´ (type I-E). Protospacer selection in type I-E was highly biased towards genes involved in lysis and morphogenesis. Our results improve the knowledge about distribution and diversity of CRISPR-Cas systems in Lacticaseibacillus strains of industrial interest, not profoundly studied despite the economic impact of phage infections.

Introduction

Phage infections represent the main microbiological problem in industrial dairy fermentations driven by lactic acid bacteria (LAB). Consequences range from a slight delay to a complete arrest of the acidification process, which is clearly evidenced by pH monitoring (Pujato, Quiberoni, & Mercanti, 2019). However, in the case of adjunct cultures, phage infections might not be easily detected (Mercanti, Carminati, Reinheimer, & Quiberoni, 2011; Mercanti et al., 2016). Lacticaseibacillus (recently reclassified from the former genus Lactobacillus; Zheng et al., 2020) is present in the gastrointestinal tract of animals and fermented products, for example in ripened cheeses where they contribute to flavour development. In addition, numerous strains possess well-established probiotic features and are added to dairy products as adjunct cultures. As any other LAB of dairy interest, Lacticaseibacillus are prone to phage infection during industrial fermentations (Capra, Binetti, Mercanti, Quiberoni, & Reinheimer, 2009; Mercanti et al., 2016).

Bacteria can fight off phages through diverse, well-known mechanisms, such as inhibition of adsorption, restriction/modification (R/M), abortive infection (Abi) and CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and associated genes) (Doron et al., 2018; Horvath et al., 2009; Labrie, Samson, & Moineau, 2010), as well as by other anti-phage mechanisms discovered later: Argonaute (Swarts et al., 2014), BREX (bacteriophage exclusion) (Goldfarb et al., 2015) and DISARM (Defence Island System Associated with Restriction–Modification) (Ofir et al., 2018). More recently, several new mechanisms have been detected by metagenomic analysis (Doron et al., 2018), though they have not been fully elucidated yet.

Of all these mechanisms, CRISPR-Cas systems have been particularly well studied during the last decade; they are widespread in prokaryotes, present in almost half of bacterial genomes currently available in public databases (Crawley, Henriksen, Stout, Brandt, & Barrangou, 2018; Makarova et al., 2020). CRISPR-Cas are modular systems constituted by a CRISPR locus, which is an array of a variable number of conserved, short DNA sequences (spacers), interspaced with similarly short but highly conserved sequences (repeats), and cas (CRISPR-associated) genes adjacent to this array (Hille & Charpentier, 2016). All CRISPR-Cas systems act as prokaryotic immune systems with three stages, eventually degrading foreign DNA that enters the cell. During the first stage (adaptation), short fragments of foreign DNA (called protospacers) are incorporated as new spacers in the CRISPR array, and then transcribed and processed into individual small CRISPR RNAs (crRNAs) in the second stage (expression). In the last stage (interference), invading DNA molecules complementary to crRNAs are targeted and destroyed by specific nucleases (Makarova et al., 2020). The correct completion of each stage relies on the activity of proteins encoded by cas genes. CRISPR-Cas systems are highly diverse regarding cas genes content and locus architecture, DNA sequences of repeats and representative cas genes, and frequently shuffle adaptation and interference modules. Based on these features, CRISPR-Cas systems have been classified into Class I (comprising types I, III and IV) and Class II (types II, V and VI), each type with numerous subtypes based in minor differences.

Concerning CRISPR in LAB, Streptococcus thermophilus has been the best studied species, due to its importance for the manufacture of cheese, yoghurt and fermented milks worldwide, and because most strains possess at least one active type II-A system (Achigar, Magadán, Tremblay, Pianzzola, & Moineau, 2017; Hynes et al., 2017; Mills et al., 2010). Comparatively, Lacticaseibacillus have received less attention, though significant advances have been made over the last few years (Crawley et al., 2018; Yang et al., 2020). The analysis of available genomes of Lacticaseibacillus showed that type II is the most common in this genus, followed by type I, while type III was not found (Crawley et al., 2018). Although CRISPR-Cas systems became well-known after Cas9 was used for the first time as a tool for genome edition, the study of their original function in nature as a defence against mobile genetic elements (MGE) has receive less attention. This is especially true for Lacticaseibacillus, despite the wide distribution of CRISPR-Cas systems among their genomes.

In this study, 49 strains of Lacticaseibacillus casei, Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus were investigated regarding the presence and distribution of types II-A, I-E and I–C CRISPR-Cas systems. The content and array of spacers, as well as the distribution of Cas proteins were also analysed.