Extended Data Fig. 1: Retron phenotypes are suppressed by inactivating the accessory retron gene, which encodes a cytoplasmic toxin RcaT.

a, ΔrrtT and ΔxseA STm are cold-sensitive. 1536 colony-arrays of the STm gene-deletion library¹⁵ were pinned onto LB plates, and strains were grown at room temperature. Colony sizes⁴⁴ were used to calculate a fitness S-score for each strain⁶⁶. S-scores were calculated from n = 8 biological. Dashed vertical line denotes the mean S-score of all strains (n = 3781); negative or positive S-scores indicate sensitive or resistant mutants. b, Perturbing msDNA biogenesis leads to cold-sensitivity. STm wild-type and retron-deletion strains were serially diluted and spotted on LB plates as in Fig. 1b. Plates were incubated at indicated temperatures (n = 4 biological). c, Retron mutants grow less in anaerobic conditions. Growth curves of STm wild-type and retron-deletion strains were obtained by measuring OD578 in microtiter plates, under anaerobic conditions at 37 °C; n = 11 biological, symbols denote the mean and error bars the standard deviation (not shown if smaller than symbols). d, Retron mutants are not affected in aerobic conditions. Experiment and plotting as in panel c, but strains were grown aerobically (n = 11 biological). e, RNAse H and Exo VII are involved in msDNA biosynthesis. msDNA was extracted from STm wild-type and retron-deletion strains carrying plasmid p-retron-ΔrcaT. Extracted msDNA was electrophoresed in TBE-Polyacrylamide gels (n = 3 biological). f, Deleting rcaT reverts the cold-sensitivity of retron mutants. STm strains were grown and spotted as in Fig. 1d (n = 2 biological). g, Deleting rcaT reverts the anaerobic sensitivity of retron mutants. Growth curves of STm strains were obtained and plotted as in panel c (n = 11 biological). h, RcaT is a soluble protein. Untagged and rcaT-3xFlag STm WT were grown in LB at 37 °C, lysed, and samples were separated into soluble and membrane fractions through ultra-centrifugation steps. Different fractions were analysed by SDS-PAGE and immunoblotting. LpoA and RecA were used as controls for the membrane and soluble fraction, respectively (n = 2 biological). i–j, Suppressors grow like wild type in cold temperatures. Suppressors isolated from cold-sensitive STm mutants (ΔrrtT, ΔxseA, Δmsrmsd; panel i) or the catalytic rcaT-E107Q mutants (panel j) were grown, serially diluted, and spotted on LB plates as in Fig. 1b. Identified suppressor mutations are indicated (n = 2 biological). k–l, Loss-of-function point mutations do not affect RcaT levels. RcaT protein levels were quantified by mass spectrometry in mutant strains (indicated by dashed lines in panels i–j). Y-axis is the ratio of RcaT protein levels (log2 fold-change) in retron deletion strains compared to WT (panel k) or the same ratio in RcaT point mutants compared to the background strain, in which suppressor was isolated in (panel l). The grey dotted line represents no change to RcaT protein levels. Black lines denote the mean (n = 2–4 biological).