Extended Data Fig. 2: Titration of toxin and antitoxin expression levels, and sensitive identification of toxin substitutions which do not disrupt toxicity.

a, Cartoon illustration of the expression system. IPTG induces antitoxin, arabinose induces toxin. b, Growth rate of cells harbouring wild-type toxin ParE3 without antitoxin at different arabinose induction levels in arabinose-titratable E. coli strain BW27783. c, d, Growth rate of cells harbouring wild-type toxin–antitoxin ParE3/ParD3 under different antitoxin induction levels modulated with IPTG and 0.00012% arabinose induction (c) or 0.0008% arabinose induction (d). e, Distribution of ∆growth rates(T*-T) for all toxin single substitutions under different arabinose inducer concentrations, with positive ∆growth rate(T*-T) values indicating loss of toxin function. The set of ‘most toxic’ toxin substitutions (n = 310) is coloured in light blue, the set of ‘toxic’ substitutions (n = 781) is coloured in green (see Methods). Other classes of substitutions are indicated. The dynamic range (difference between 0 and the truncated toxin mutants) shrinks, as expected, for lower expression levels that do not fully inhibit growth with the wild-type toxin, and a higher fraction of mutants show loss of toxicity (higher ∆growth rates) under lower expression conditions. The toxin substitution A28Q is highlighted (dark blue) as an example that shows no growth rate difference relative to wild-type toxin at high expression conditions, but is not as toxic as wild-type toxin at lower expression conditions. f, Schematic illustrating loss of toxicity detection using growth rate measurements in different expression regimes. g, Mean ∆growth rates(T*-T) of residue positions mapped onto the ParE3 toxin structure. Values shown for 0.00012% [arabinose] inducer. h, The mean ∆growth rates(T*-T) of a residue are correlated with the relative solvent accessibility of the residue (Pearson r = −0.66). Values shown for 0.00012% [arabinose] inducer. i,j, Distribution of ∆growth rate(T*-T) for all toxin substitutions (black) or top 10 coevolving residue substitutions (purple) in the toxin in absence of antitoxin (g) or presence of antitoxin (h). Values shown for 0.00012% [arabinose] inducer, and antitoxin is induced with 10 µM IPTG. k, The ∆growth rate(T*-T) values of each substitution at any position along the toxin ParE3. Green highlights the top 10 covarying positions between toxin and antitoxin in natural homologues. Values shown for 0.00012% [arabinose] inducer.