Extended Data Fig. 9: Dam-mediated retron-TA triggering.

a, Bacterial/phage dam homologues trigger Retron-Sen2. E. coli BW25113 carrying combinations of p-retron-ΔrcaT, p-retron, p1-dam^Ec, p1-dam^STm, p1-dam^P1 and p1-empty, were grown for 5 h in LB with appropriate antibiotics, serially diluted, spotted on LB plates with antibiotics, with/without arabinose, and IPTG (low), and incubated at 37 °C (n = 2 biological). b, Phage P1 dam triggers the chromosomal Retron-Eco9 in E. coli NILS-16. E. coli NILS-16 WT and ΔrcaT-Eco9 strains carrying plasmids p2-dam^P1 or p2-empty, were grown for 5 h in LB-hygromycin, serially diluted, spotted on LB-hygromycin plates, with or without IPTG, and incubated at 37 °C (n = 2 biological). c, Phage P1 Dam is expressed at comparable levels to the p2-dam^P1 plasmid. An E. coli BW25113 ΔlacY strain carrying an empty plasmid was infected with phage P1vir (MOI 2) for 0, 5, or 35 min (uninfected, early, and late infection). The same strain, but carrying a single-copy vector expressing Dam^P1 was grown in LB and the plasmid was induced with 60 μM of IPTG. The levels of the Dam^P1 protein were quantified by proteomics (TMT labeling), and Dam^P1 amounts were compared between phage-infection and plasmid-induction (X-axis). Phage infection or Dam^P1 induction does not alter the expression of endogenous Dam (Table S7), which has diverse enough sequence to be distinguished from Dam^P1 (Dam 39% identical for 266/754 residues of Dam^P1) (n = 2 biological). d, The Dam^P1 protein levels produced by P1vir are enough to trigger Retron-Eco9. The same strains as in panel b were grown in LB-hygromycin at 37 °C with IPTG (0 – 1000 μM), their optical density was measured at 8 h post-induction, and the fitness of the WT was compared to the ΔrcaT-Eco9 strain (X-axis). Vertical lines denote the mean fitness ratio, and horizontal lines denote standard deviation (n = 3 biological). e, Dam overexpression does not impact msDNA levels. msDNA was isolated from STm carrying combinations of plasmids p-retron-ΔrcaT, p1-dam, or p1-empty. Plasmids were co-induced with arabinose and IPTG (low). Extracted msDNA was electrophoresed in a TBE-polyacrylamide gel (n = 4 biological). f, RcaT in p-retron^mut is functional. E. coli BW25113 ΔxseA strains carrying combinations of plasmids p-retron^WT, p-retron^mut, p1-dam, and p1-empty, were grown and spotted as described in panel a (n = 3 biological). g, p-retron^mut produces same levels of msDNA as wild-type retron. msDNA was extracted from STm strains carrying p-retron^WT, or p-retron^mut. Extracted msDNA was electrophoresed in a TBE-polyacrylamide gel (n = 2 biological).