Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated genes are linked to a mechanism of acquired resistance against bacteriophages. Bacteria can integrate short stretches of phage-derived sequences (spacers) within CRISPR loci to become phage resistant. In this study, we further characterized the efficiency of CRISPR1 as a phage resistance mechanism in Streptococcus thermophilus. First, we show that CRISPR1 is distinct from previously known phage defense systems and is effective against the two main groups of S. thermophilus phages. Analyses of 30 bacteriophage-insensitive mutants of S. thermophilus indicate that the addition of one new spacer in CRISPR1 is the most frequent outcome of a phage challenge and that the iterative addition of spacers increases the overall phage resistance of the host. The added new spacers have a size of between 29 to 31 nucleotides, with 30 being by far the most frequent. Comparative analysis of 39 newly acquired spacers with the complete genomic sequences of the wild-type phages 2972, 858, and DT1 demonstrated that the newly added spacer must be identical to a region (named proto-spacer) in the phage genome to confer a phage resistance phenotype. Moreover, we found a CRISPR1-specific sequence (NNAGAAW) located downstream of the proto-spacer region that is important for the phage resistance phenotype. Finally, we show through the analyses of 20 mutant phages that virulent phages are rapidly evolving through single nucleotide mutations as well as deletions, in response to CRISPR1.

CRISPR — a widespread system that provides acquired resistance against phages in bacteria and archaea

“…Further sequencing of the phage genomes revealed that the phage had mutated so that their sequence was no longer identical to the spacers 21 . Resistant phage having sequences identical to spacers were also isolated, but the AGAA downstream recognition motif was mutated in their genome, further strengthening the hypothesis that this motif is important for CRISPR function 49 . The selective pressure imposed by CRISPR on the phage therefore leads to rapid changes in their genome, and provides a glimpse into how CRISPR might be involved in driving the extremely high evolutionary rates observed in phage.…”

“…Two recent studies have reported on a short motif present in phage genomes 1-2 nucleotides downstream to spacer-matching sequences [49][50] . This motif was hypothesized to be important for recognition, or cleavage, of phage sequences by the CRISPR system.…”

“…Bolotin et al also reported on a negative correlation between the sensitivity of bacteria to phage infection and the number of CRISPR spacers in their genome 20 . Recently, Barrangou and colleagues have confirmed this hypothesis experimentally by showing that new spacers acquired following phage challenge confer resistance against the phage 21,[49][50] . Their discovery is discussed in more detail below.…”

CRISPR–Cas encoding of a digital movie into the genomes of a population of living bacteria

“…In the E. coli Type I-E CRISPR-Cas system, DNA from invading viruses is inserted into a genomic CRISPR array in 33-base units termed spacers 11 . The sequences from which spacers are derived are termed protospacers 12 . Here, we began with an image (Extended Data Fig.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…Additionally, the specificity of the resistance was demonstrated to be directly related to the sequence of the spacers, which are derived from DNA strands of phage open reading frames [7]. This was further supported by Deveau and colleagues, through studies of phage responses in Streptococcus thermophilus which demonstrated that CRISPR confers phage resistance [36].…”