Figure 1.
Impact of acr Genes on Phage Population Dynamics and Evolution of CRISPR Resistance during Infection of the Initially Sensitive WT Host Population
(A and B) Phage (solid lines) and bacterial (dashed lines) populations dynamics upon individual infections of the CRISPR-KO (A) or the WT host by Acr-negative or Acr-positive phages with different Acr (B).
(C) Frequency of spacer acquisition in CRISPR arrays 1 and 2 at 3 dpi in the evolved WT PA14 populations, as determined by deep sequencing.
(D) Frequencies of reads containing 1, 2, 3, 4, or 5 additional spacers.
(E) Protospacer distributions. Newly acquired spacers were extracted from read sequences and corresponding protospacers were mapped back to phage genomes, on positive and negative strands. Observed distributions are consistent with primed spacer acquisition.
(F) Relative fitness of WT PA14 and CRISPR-KO strains at 3 dpi in the presence or absence of indicated phages at an initial MOI of 0.01. One-sample t tests indicate significant differences from 1 for “Acr(−)” (p = 0.0004 and t5 = 8.3) and no significant differences for F1 (p = 0.42 and t5 = 0.87), F4 (p = 0.52 and t5 = 0.52), and No phage control (p = 0.61 and t5 = 0.54).
(G) Distribution of phage-resistance mechanisms that evolved at 3 dpi (based on analysis of 24 clones per replicate). CRISPR-Cas, detection of additional spacers assessed by PCR; Sm, surface mutant; Und., undetermined. These are clones for which the results were inconclusive.
In all panels, data shown are the mean of 6 biological replicates per treatment. Shaded areas and error bars represent 95% confidence intervals (CIs). F1, DMS3vir-acrIF1; F4, DMS3vir-acrIF4; and Acr(−), DMS3vir.