Extended Data Fig. 2: Retron-Sen2 functions as a toxin-antitoxin system in E. coli and the antitoxin does not shut down RcaT expression.

a, RcaT can be inhibited by msrmsd-rrtT in trans. E. coli carrying binary combinations of plasmids p-rcaT, p-retron-ΔrcaT, and empty vectors, were grown in LB with appropriate antibiotics, serially diluted and spotted as in Fig. 1f (n = 3 biological). b, RcaT inhibition requires both msrmsd and rrtT in E. coli. E. coli with plasmids carrying retron-components were grown and spotted as in Fig. 1f. Only the intact retron could restore growth. RcaT expression is sufficient to inhibit growth (n = 3 biological). c, RcaT inhibition requires RNAse H and Exo VII in E. coli. E. coli strains (WT, ΔxseA, ΔxseB, ΔrnhA) carrying plasmid p-retron-ΔrcaT or p-retron were grown, serially diluted and spotted as in panel b (n = 2 biological). d, RcaT levels are only slightly affected by antitoxin deletions. rcaT-3xFlag STm strains (WT and retron-deletions) and the STm untagged strain (native) were either grown in LB at 37 °C, or shifted to 20 °C for 5 h. Protein samples from strains were analysed by SDS-PAGE and immunoblotting. LpoA levels (α-LpoA antibody) were used as loading control. Bars and error bars indicate mean and standard deviation, respectively (n = 5 biological). e, Quantification of RcaT-3xFlag signal from 37 °C immunoblots in panel d using ImageJ (pixel-density). Bars and error bars depict the mean and standard deviation, respectively (n = 5 biological).