CRISPR immunity drives rapid phage genome evolution in Streptococcus thermophilus

Abstract

Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems.

Importance: Phages remain an enigmatic part of the biosphere. As predators, they challenge the survival of host bacteria and archaea and set off an "arms race" involving host immunization countered by phage mutation. The CRISPR-Cas system is adaptive: by capturing fragments of a phage genome upon exposure, the host is positioned to counteract future infections. To investigate this process, we initiated massive deep-sequencing experiments with a host and infective phage and tracked the coevolution of both populations over hundreds of days. In the present study, we found that CRISPR immunity drives the accumulation of phage genome rearrangements (which enable longer phage survival) and escape mutations, establishing CRISPR as one of the fundamental drivers of phage evolution.

FIG 1

Host-phage dynamics and CRISPR spacer acquisition. (A) Fluctuations in the numbers of host bacteria (in CFU per milliliter) and phage (in PFU per milliliter) in the serial transfer experiment, over time (days), for four experimental series with different MOIs (host:phage ratios of 1:2 [MOI-2A and MOI-2B], 1:10 [MOI-10], and a no-phage control [MOI-0]). Samples were transferred daily as a 1% (vol/vol) inoculum. Time points for which samples were subjected to deep-sequencing analyses are indicated by green dots. (B) Number of unique CRISPR spacer types (defined by sequence) in the CRISPR1 and CRISPR3 systems, oth type IIA systems, and CRISPR4 locus, classified as a type IE system.

FIG 2

Phage genome sampling and mutations in response to CRISPR immunization. (A) The frequency of CRISPR spacer incorporation events from the phage 2972 genome, across the three experimental series, measured by the sequencing of novel spacers acquired in the host active CRISPR loci. Note that the x axis is the same for panels A and B. (B) Mutations observed in the surviving phage population at the last time point, sampled in the three experimental series. (C) Examples of mutations in the surviving phage population that enable the phage to evade CRISPR immunization. SNPs are localized in the proto-spacer (underlined sequences) and the PAM (boxed sequences). Perfect PAMs are represented in boxes with solid lines. An imperfect hypothetical PAM is shown in a box with dashed lines. Examples of fixed SNPs circumventing targeting by CRISPR1 (orange) or CRISPR3 (blue) are shown. Proto-spacers that extend off the ends of the figure were truncated. (D) Screen capture of Strainer program (31) of assembled scaffolds (white) mapped to the reference 2972 phage genome. The region displayed corresponds to the same showed in panel C with the same color code. Colored bases indicate differences in the nucleotide sequence from that of 2972 (SNPs).

FIG 3

Phage genome rearrangements to escape CRISPR targeting. Blocks of hypervariability in the phage genome reflect recombination events that have eliminated wild-type sequences heavily targeted by active CRISPR1 and CRISPR3 loci in the host. (Top) Proto-spacers targeted by CRISPR loci in the 23.0- to 23.6-kb region of the reference phage 2972 genome. (Bottom) Proto-spacers targeted by CRISPR loci in the 23.0- to 23.6-kb region of the phage 2766 genome. PAMs and proto-spacers are colored according to loci (brown square and blue line for CRISPR1, and black square and red line for CRISPR3). The regions in blue correspond to the wild-type 2972 phage (top), and the regions in yellow correspond to the variant phage 2766 (bottom), from which blocks were acquired through recombination events. Sequence that is identical in both genotypes is shown in green. (Left) Experimental series from which the chimeric phages were isolated. (Right) Time points (days) at which the observed chimera were sequenced and assembled. Blocks of conserved sequences are shown in similar colors.

FIG 4

Phage genome re-arrangements to escape CRISPR targeting and recombination events between phages. (A) The surviving phage genome at transfer 232 in the MOI-2B series. The red line reflects coverage consensual with the WT 2972 phage, whereas the blue line reflects coverage consensual with the 2766 phage, from which sequences were acquired through recombination events to generate the chimeric phage (green). (B) Assembly of the surviving phage genome at transfer 195 in the MOI-10 series. Color codes are as used in panel (A). Baseline coverage reflects boundaries of recombination events (i.e. 28k-33k in the lower panel). (C) Screen capture of Strainer program 29 of sequencing reads (in white) mapped to the reference phage genome. Arrows mark sequences that are hybrid between the reference genome and the mutated phage genomes. These patterns suggest that recombination events introduced polymorphic blocks. Colored tick marks indicate differences in nucleotide sequence from the reference. Blue, red, purple, and green ticks indicate substitutions for the bases A, C, T and G respectively. Ticks of half height indicate extra bases in a read sequence, and missing bases are colored black.

FIG 5

Phylogenetic tree of the phage genotypes. Whole-genome phylogenetic tree (using Phyml over multiple Muscle Alignment, with phage 2972 as the reference) showing the relatedness of the various dominant chimeric phages across the experimental series over time, and their similarity to the WT 2972 and 2766 phage (both in red). In orange, MOI-2A series; Blue: MOI-2B series; Purple: MOI-10 series. The last digit for each sample indicates the time point from which dominant phage genomes were assembled (in case there were 2 dominant phage in a time point, they will be shown as A or B). Noteworthy, all genomes from the first time point in each series cluster with the WT 2972 phage, whereas the phage reconstructed from the last time point in each series (MOI-2A_28; MOI-2B_232; MOI-10_195) are less phylogenetically related.

FIG 6

Host-phage population dynamics in re-inoculation experiments. Fluctuation in the number of host (colony forming units per ml) and phage (plaque forming units per ml) in the serial transfer experiment, over time (days). The “asterisked” series represent re-inoculation of the samples at defined time points, after 3 and 21 transfers in the MOI-2A series (3* and 21*, respectively –top panel); after 210 and 216 transfers in the MOI-2B series (210* and 216*, respectively –centered panel); and after 172 and 180 transfers in the MOI-10 series (172* and 180*, respectively –bottom panel). Data shows that a tipping point has been reached for phage loss over time at the latest re-inoculated time point in each series (shown in red in each panel).