Fig. 6: Relative fitness costs of evolved clones containing fusA and sbmA mutations.

Each evolved clone tagged with mars was competed with wildtype tagged with GFP at a 1/1 ratio in the absence (a) and presence (b) of 5x AMK MIC. In order to match the evolution protocol, in (a) wt/mutants LB overnight cultures were mixed, diluted to OD 0.02 and grown in LB for 24 h before flow cytometer analysis. In b wt/mutants LB overnight cultures were mixed, diluted to OD 2, treated for 24 h by 5x AMK MIC, washed, diluted 1/100 and regrow in LB for 24 h before flow cytometer analysis. The fitness index of the comparison of wildtype tagged with mars to wildtype tagged with GFP is taken as 1 (dotted line). Detailed information on the different analysed clones can be found in supplementary Data 4. It should be noted that clones 853, 863 and 891 differ, respectively, from the ancestral strain by a single G604V, G676C and P610L fusA mutation, and clones 236 and 501 differ, respectively, from the ancestral strain by a single Q192L and (17/1221 nt) (T)6 → 7 sbmA mutation, and are otherwise isogenic. The data corresponded to n = 3 biological replicates for each clone and are represented as the mean ± SD. Statistics correspond to unpaired two-tailed t-test with Welch’s correction comparing each condition to wildtype/wildtype fitness. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001.