Figure 4. PhageMaP identifies anti-phage defense counters and triggers in Bas63 phage.

(A) Plot showing all identified uncorrected significant hits (Welch’s t-test, p<0.05) and significant hits after multiple testing correction with the Benjamini-Hochberg method (q<0.1, denoted by an asterisk in each tile) with a score that is at least a difference of 0.5 from the control. The color of the tiles represents the difference in Z-normalized fitness scores between with defense and the control. Black boxes indicate targets deem to be potential counters to a defense system and subjected for secondary validation. Red boxes indicate targets deem to be potential triggers for a defense system and subjected for secondary validation. Asterisks at the end of gene names indicate genes which were manually annotated after BLASTp search. (B) Efficiency of plating (EOP) measurements for Bas63 knockouts with the Sir2-HerA host. Average titers on the control strain were first determined (3 technical replicates). Each point on the plot represents a technical replicate for EOP using those calculated averages as the denominator for each titer measurement on the defense host. Bars represent the mean of three replicates, and error bars denote the standard deviation. Hypothesis testing was performed pairwise with each knockout and WT. P-values (Welch’s t-test) and means are annotated above the bars. Complementation host contained the defense system and a plasmid expressing deleted gene. (C) Mechanisms of defense and counter-defense for Sir2-HerA. (D) EOPs for Ec86 retron calculated the same way as (B). (E) Proposed mechanisms of defense and counter-defense for Ec86 retron. (F) EOPs for Ec67 retron calculated the same way as (B). (G) Proposed mechanisms of defense and counter-defense for Ec67 retron. (H) EOPs for qatABCD calculated the same way as (B). (I) Proposed mechanisms of defense and counter-defense for qatABCD. (J) EOPs for ppl calculated the same way as (B). Since plaques were too small to count, an ‘S’ was annotated in lieu of a plaquing efficiency. The plotted efficiency is the highest Bas63Δ130 dilution for which zones of clearing were observable and clear on a spot plate. (K) Proposed mechanisms of defense and counter-defense for ppl. (L) Growth curves for expression of g40 in cells containing no exogenous defense or the hhe system. 1 x 10⁷ cells were either repressed with 0.2% glucose (uninduced) or induced with 0.1% L-arabinose and grown for ~20hrs. Central line represents the average of 3 replicates and the shaded flanking regions represent the standard deviation. (M) Growth curves for expression of g123 in cells containing no exogenous defense or the Ec67 retron system. Experiment was performed and plot was constructed the same way as (H).