Fig. 2. Addition of iron to growth media increases the yield of pulcherrimin, but not the activity of its biosynthetic genes.

a Comparison of B. subtilis wild-type cell suspensions grown overnight under shaking in regular LBGM broth and in LBGM supplemented with 0.05 or 0.2 mM FeCl₃. As the concentration of FeCl₃ increases, so does the amount of reddish pigment. b Pellets obtained after harvesting cell suspensions of the wild type (left) and the pulcherrimin mutant (right) grown overnight in LBGM broth without or with the addition of 0.2 mM FeCl₃. Pellets contained a mix of cells and pulcherrimin, which co-precipitate after centrifugation. c Results of indirect pulcherrimin quantification from cell pellets obtained after centrifugation of wild-type overnight cell suspensions from a. There is a significant increase in pulcherrimin production in samples supplemented with FeCl₃ to a final concentration of 0.05 mM compared with the ones without FeCl₃ supplementation (p = 0.0151, *); significantly higher pulcherrimin production was also observed when comparing samples supplemented with 0.2 mM FeCl₃ to the ones without FeCl₃ supplementation (p = 0.0002, ***). Averages are representative of three biological replicates. Error bars represent standard deviation. d, e Results of β-galactosidase assays for the pulcherrimin reporter P_yvmC-lacZ under shaking (24 h, d) and pellicle (48 h, e) conditions in LBGM broth without or with FeCl₃ supplementation at 0.05 or 0.1 mM. Averages are representative of three biological replicates. Error bars represent standard deviation. ns: statistically not significant. M.U. Miller units.