Extended Data Fig. 7: TAC low-IPTG, reproducibility, hits heatmap and validation.

a-b, TAC results from low-IPTG induction. Experiments as described in Fig. 3b, c, but with low-IPTG plasmid-library induction for MOB (panel a; p1)²⁶ and TransBac (panel b; p2)²⁷. Prophage gene fold enrichment (FE) and p values (one-tailed Fisher’s exact test) are shown on top. c–d, TAC reproducibility in control and experiment plates. Unprocessed colony opacity values of strains from control (ctrl) or experiment (exp) plates, derived from replicate plates 1 and 2, were plotted against each other. The overexpression library and IPTG concentration contained in each plate-pair are denoted above the plots. Results shown for MOB (panel c; p1)²⁶ and TransBac (panel d; p2)²⁷. Replication correlation (R²) is shown for each reproducibility plot. Green-colored dots correspond to the hits shown in panels a-b and in Fig. 3b, c. e, Non-parametric comparison of identified triggers across overexpression libraries. Trigger-genes identified from the MOB²⁶ (p1) and the TransBac²⁷ (p2) overexpression libraries were rank-ordered in percentiles based on their mean z-score difference value, as calculated from TAC screens conducted in low, or high-IPTG induction. Not available (NA) denotes genes for which measurements were flagged as problematic (see Methods) in the respective library/IPTG-concentration. Absent denotes genes which are missing from one library. Prophage and phage-related trigger-genes are in bold and underlined, respectively. f, Plasmids (p1/p2-trigger gene in green) were conjugated into E. coli BW25113 carrying p-empty, p-retron, p-retron-ΔrcaT, or p-rcaT. 384-colony-arrays of the transconjugants were pinned on LB plates containing appropriate antibiotics, arabinose, and IPTG concentrations (IPTG concentrations with strongest effect shown here). The y-axis represents the triggering-degree of each plasmid, measured as the colony opacity ratio of strains (p-retron-ΔrcaT + p1/p2-trigger) divided by the (p-retron + p1/p2-trigger). p1-control values were derived by measuring the same colony opacity ratio, for p1-blocker genes (n = 72 – 36 biological X 2 technical replicates), as a negative control. Ratios were calculated from n = 10 – 5 biological X 2 technical replicates for p1-trigger genes (except for p1-yfbO; n = 8 – 4 biological X 2 technical replicates), and from n = 24 – 12 biological X 2 technical replicates for p2-trigger genes. Representative colonies of strains carrying p1/p2-trigger plasmids are shown below the graphs. Horizontal lines denote the average fitness ratio, and error bars denote the standard deviation. The p1-control fitness data are shown in grey and p1-dam was plotted separately, to avoid compressing the scores of the rest of the trigger-genes. g, Plasmids (p1/p2-trigger gene in green, and p1-empty/p2-empty) from conjugation donor strains were conjugated with E. coli BW25113 carrying plasmids p-retron-ΔrcaT and p-retron. Transconjugants were grown for 5 h in LB with appropriate antibiotics, serially diluted, spotted on LB plates containing antibiotics, arabinose, and IPTG (no IPTG, low, or high), and incubated at 37 °C. Triggers specifically inhibit growth by activating RcaT, and triggering depends on induction levels.