Purification of Siderophore Chryseochelin A

A total of 450 mL bacterial supernatant was prepared under iron limited conditions as described in the previous section. In a first step, the supernatant was frozen by liquid nitrogen and then lyophilized overnight. The dried residue was reconstituted in 80 mL MeOH and the solution was filtered over a filter paper. Afterward, the organic solvent was evaporated under a gentle stream of nitrogen. The dried extract was then dissolved in 5 mL ultrapure H₂O. This solution containing the siderophore chryseochelin A was centrifuged for 10 min (4°C, 14 000 rpm) and transferred into a new Eppendorf tube. The crude siderophore was then purified twice by reversed-phase high pressure liquid chromatography (RP–HPLC) on a preparative Triat C18 column (150 × 30 mm, 5 μm, YMC-Actus) and then on an analytical CORTECS C18 column (150 × 4.6 mm, 2.7 μm, Waters) while monitoring the absorption wavelength at 270 nm. Detailed chromatographic conditions can be found in the supplementary (Supplementary Figs. S1 and S2). The final pure siderophore (3.1 mg), chryseochelin A, was stored as a white powder at −20°C.