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.

FIG. 1.

S. thermophilus CRISPR1 locus overview and newly acquired spacers in various phage-resistant mutants. (A) Repeat/spacer region of strain DGCC7710 and the selected BIM named DGCC7710_φ2972^+S15. Repeats are shown as black diamonds, spacers are numbered in gray boxes, and the leader (L) is shown as a white box. The terminal repeat of CRISPR1 locus is represented with a letter “T” inside the black diamond. (B) The spacer content at the 5′ end of the locus in various phage-resistant mutants is represented. The newly added spacers are indicated in white boxes with a designation containing the prefix S, followed by a number. The 3′ end of CRISPR1 in BIMs is identical to that of the wild-type strain. (C) Repeat/spacer region of S. thermophilus strain SMQ-301 and its derivatives.

FIG. 2.

Nucleotide sequences in wild-type and mutant phages that correspond to the newly acquired spacers by the S. thermophilus strains. The AGAA motif is highlighted in gray. Each mutation is in boldface and underlined. *, Deletion.

FIG. 3.

Schematic representation of the S. thermophilus bacteriophage genomes used in the present study. (A) Distribution of the sequences corresponding to the new spacers in the three phage genomes. The spacers above the genome correspond to the positive strand, while those indicated in lower part correspond to the negative strand. Spacers indicated by an asterisk contain one mismatch with the phage sequence. ORFs connected by a gray box possess more than 70% identity at the amino acid level. (B) Distribution of the AGAAW motif on both strands for the three phages.