Fig. S1. The toxic effect of RelE2Spn on E. coli cells could be alleviated by co-expression of the RelB2Spn antitoxin. E. coli cells were streaked on TY plates containing 0.2% arabinose and supplemented or not, as indicated, with 2 mM IPTG. Cells harboured the following plasmids: pLB404 and pYE304 (streaks 1), pL204 (streaks 2), pY104 (streaks 3), pL204 and pYE304 (streaks 4), and pYE304 (streaks 5). Plates were incubated overnight at 37°C.

Fig. S2. Disassociation of the pneumococcal toxin and its antitoxin counterpart could be visualized as a decrease in the BRET signal. E. coli TOP10 cells (auxotroph for leucine) were cotransformed with plasmids pB404 (pneumococcal antitoxin fused to the Rluc) and pYE304 (pneumococcal toxin fused to the EYFP). Cells were grown in LB (rich medium) in the presence of Km and Ap. Cells were diluted (1:500) in the same medium and grown until they reached an OD600 of 0.3. Then, cells were centrifuged and washed twice either with media LB or M9 (minimal medium, lacking amino acids or glucose), and resuspended in the corresponding medium at the same optical density in the presence of arabinose 0.2% and IPTG 2 mM. Samples were withdrawn at the indicated times and BRET assays performed for each medium in the same conditions as described in Methods. The results of these experiments are shown in the figure below and are expressed as the % of the BRET ratio of the cells growing in M9+arabinose+IPTG in comparison to the same cells growing in LB+arabinose+IPTG (100%) at every assayed time. It has to be pointed out that the BRET ratio of cells in LB barely changed in the time course of the assay. Assays were performed in triplicates.

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