Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum

Abstract

Diverse CRISPR-Cas systems provide adaptive immunity in many bacteria and most archaea, via a DNA-encoded, RNA-mediated, nucleic-acid targeting mechanism. Over time, CRISPR loci expand via iterative uptake of invasive DNA sequences into the CRISPR array during the adaptation process. These genetic vaccination cards thus provide insights into the exposure of strains to phages and plasmids in space and time, revealing the historical predatory exposure of a strain. These genetic loci thus constitute a unique basis for genotyping of strains, with potential of resolution at the strain-level. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in the genomes of various Bifidobacterium longum strains across three sub-species. Specifically, we analyzed the genomic content of 66 genomes belonging to B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis, and identified 25 strains that carry 29 total CRISPR-Cas systems. We identify various Type I and Type II CRISPR-Cas systems that are widespread in this species, notably I-C, I-E, and II-C. Noteworthy, Type I-C systems showed extended CRISPR arrays, with extensive spacer diversity. We show how these hypervariable loci can be used to gain insights into strain origin, evolution and phylogeny, and can provide discriminatory sequences to distinguish even clonal isolates. By investigating CRISPR spacer sequences, we reveal their origin and implicate phages and prophages as drivers of CRISPR immunity expansion in this species, with redundant targeting of select prophages. Analysis of CRISPR spacer origin also revealed novel PAM sequences. Our results suggest that CRISPR-Cas immune systems are instrumental in mounting diversified viral resistance in B. longum, and show that these sequences are useful for typing across three subspecies.

Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

“…In many incomplete sets, we observed the occurrence of transposases, which have been previously observed in CRISPR loci (41,42). The coexistence of several CRISPR-Cas systems in the same genome has been previously described in several gut lactobacilli and bifidobacteria (41,43), as well as in Streptococcus thermophilus starter cultures (44,45). Overall, we determined widespread occurrence of CRISPR-Cas systems in L. crispatus, notably complete type I-E systems ( Fig.…”

Comparative Genomics Analysis of Lactobacillus mucosae from Different Niches

“…Previous studies have identified phylogeny of Cas1 as one of the key factors to subtype classification [68]. Additionally, the tree showed the aggregation of the Cas1 sequences was on the same big branch according to subtype IE, IIA, and IIIA, which was roughly the same as the phylogeny of the DR sequence, confirming the trend of co-evolution of components in the immune system [29]. The most frequent repeats were usually defined as typical repeats.…”

“…The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region and the CRISPR-related protein (Cas) were predicted using the CRISPR CasFinder [28], and neighbor-joining trees based on Cas protein were built [29]. The sequence of conserved direct repeats (DRs) were visualized by WebLogo [30].…”

Genomic insights and probiotic characteristics of Bifidobacterium gallinarum CACC 514 isolated from canine

“…CRISPR-Cas Type II immune systems (II-A, II-C) are fairly rare and occur in only 5% of bacteria, but they occur at a much higher frequency in the Bifidobacterium genus [24]. Characterization of Type II elementsmay provide opportunities to use the molecular genome-editing tool for the development of next-generation probiotic bacteria [25]. The B. gallinarum CACC 514 CRISPR-Cas system may also provide a platform for various potential biotechnological and ecological uses as probiotic bacteria.…”