Characterization and Exploitation of CRISPR Loci in Bifidobacterium longum.

Claudio Hidalgo-Cantabrana,Rodolphe Barrangou,Alexandra B Crawley,Borja Sanchez

doi:10.3389/fmicb.2017.01851

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.

Highlights

Bifidobacteria are one of the first commensal microorganisms that colonize the human gut, making them the dominant intestinal bacteria in infants and one of the main inhabitants in healthy adults (Arboleya et al, 2016)
Type I systems are present in the subspecies B. longum subsp. longum, B. longum subsp. infantis, and B. longum subsp. suis, the Type II system was only detected in B. longum subsp. longum and only represented by subtype II-C
The presence of subtype II-C in B. longum was previously described for the strain DJO10A (Horvath et al, 2009) it was not found in a large data set with other species of bifidobacteria (Briner et al, 2015), mainly due to the use of a unique strain as a representative of each species

Summary

Introduction

Bifidobacteria are one of the first commensal microorganisms that colonize the human gut, making them the dominant intestinal bacteria in infants and one of the main inhabitants in healthy adults (Arboleya et al, 2016). Bifidobacterium longum is the species most prevalence in healthy adults and widely commercialized in probiotic products. Despite new regulations for health claims of probiotics, many products still misidentify the taxonomic classification of their strains based on 16S sequencing or are manufactured with low amounts of the stated microorganisms (Lewis et al, 2016; Morovic et al, 2016). In this regard, new methodologies should be applied for correct taxonomy together with internal quality control. The use of high-throughput sequencing has been suggested as a reliable methodology for correct identification (Morovic et al, 2016) as well as the use of glycolysis genes for correct taxonomy (Brandt and Barrangou, 2016)

Methods

Results

Conclusion