Fig. S3.

Growth of C. glutamicum WT (black squares) and the indicated C. glutamicum mutant strains in media containing different iron concentrations. Cells were cultivated in CGXII minimal medium with 4% (wt/vol) glucose containing 36 µM FeSO₄ (A, B, D, and E) or 1 µM FeSO₄ (C, F, G, and H). In the experiment in H, addition of 10 µM FeSO₄ to a culture of the C. glutamicum Δpup mutant (blue upright triangles) 6 h after inoculation was able to abolish the growth defect. Similarly, induction of pup expression in strain C. glutamicum Δpup pVWEx1-pup (dark blue tilted triangle) by IPTG addition after 6 h improved growth compared with a control culture without IPTG (green diamonds). These results confirm that the growth defect of the Δpup mutant is caused by iron limitation and the absence of pup, respectively. The figure shows mean values of three independent biological replicates. Cultivation was performed in 500-mL shake flasks containing 50 mL medium.