Figure 4. ALA potentiates tellurite toxicity.

(a) Strains BW25113 (upper panel) and EM2 (lower panel) were grown in M9 medium supplemented or not with 25 μg ml⁻¹ ALA, challenged or not with tellurite and CFU ml⁻¹ were quantified at the indicated time points. Strains BW25113 and EM2 were exposed to 10 and 400 μg ml⁻¹ tellurite, respectively. Heme and ALA content were determined from cell extracts. Levels of heme and ALA in strains BW25113 and EM2 were compared with those of strain BW25113 grown without ALA (dotted magenta line, from Fig. 1d). Values represent the mean of three biological replicates. Error bars represent s.d. (b) Strains BW25113 and EM2 were grown in M9 medium supplemented with 25 μg ml⁻¹ ALA and challenged with tellurite (25 and 400 μg ml⁻¹ for strains BW25113 and EM2, respectively) and the porphyrin content was extracted and quantified by HPLC at the indicated time points. Units for proto IX and coproporphyrin III are nanomoles normalized by mg of protein. Values represent the mean of three biological replicates. Error bars represent s.d. (c) Representative HPLC chromatograms of samples from each strain quantified in b. The colour of each chromatogram (indicated at the bottom of the figure) represents the time at which porphyrins were extracted regarding to tellurite addition. C=coproporphyrin III; P=protoporphyrin IX. (d) Representative growth curves of strains BW25113, EM2, ΔhemN and ΔhemF in M9 medium supplemented with 25 μg ml⁻¹ ALA in the presence of the indicated tellurite concentrations. Values represent the mean of three biological replicates. S.d. was removed for clarity (growth curves with s.d. are shown in Supplementary Fig. 5). (e) Representative growth curves of strains BW25113 and EM2 in M9 medium with tellurite (0.5 and 50 μg ml⁻¹, respectively) supplemented with the indicated concentrations of ALA. Values represent the mean of three biological replicates. S.d. was removed for clarity. Growth curves with s.d. are shown in Supplementary Fig. 6). TeO₃²⁻: tellurite.