Extended Data Fig. 6: Retron-Eco9 is similar to Retron-Sen2.

a, Retron-Eco9 has a similar operon structure to Retron-Sen2. Retron-Eco9 contains msr, msd, rcaT and rrtT regions, 85%, 58%, 43%, and 49% identical to the corresponding Retron-Sen2 regions (first two nucleotide, last two protein level); rrt = retron reverse transcriptase. b, msDNA-Eco9 is similar to msDNA-Sen2. Models of msDNAs-Sen2 and -Eco9⁶⁷. c, msDNA-Eco9 biosynthesis requires ExoVII and RNase H. msDNA was extracted from E. coli BW25113 strains (wild-type, ΔxseA, ΔxseB, ΔrnhA) carrying plasmid PBAD-msrmsd-RT-Eco9 and electrophoresed in a TBE-Polyacrylamide gel (n = 2 biological). d, RcaT-Eco9 inhibition requires RNAse H and Exo VII in E. coli. E. coli BW25113 strains (wild-type, ΔxseA, ΔxseB, ΔrnhA) carrying plasmid Ptac-msrmsd-Eco9, along with either PBAD-RT-Eco9 (−), or PBAD-RT-RcaT-Eco9 (+), were grown for 5-6 h at 37 °C in LB with appropriate antibiotics, serially diluted, spotted on antibiotics-LB plates, with/without arabinose, and plates were incubated overnight at 37 °C (n = 2 biological). e, Non-cognate RT-msrmsd pairs produce msDNA. msDNA were extracted from E. coli BW25113 co-expressing the RT of Retrons-Sen2 (Se) or -Eco9 (Ec) (PBAD-RT; Se or Ec – arabinose induction), and Ec/Se msrmsd (Ptac-msrmsd or an empty vector (-)). Extracted msDNA were electrophoresed in a TBE-polyacrylamide gel (n = 2 biological). f, Ec RT cannot act as antitoxin at basal msrmsd (Se) levels, but can when latter is induced (Fig. 2e), in line with lower capacity for Ec RT to reverse transcribe the Se msrmsd (panel e). Experiment conducted as in Fig. 2e (n = 2 biological).