The crude extract was obtained as described previously (Patil et al., 2011 (link)). Briefly, 2 × 500 mL of culture were incubated for 24 or 48 h at 30 and 37°C (only for WT), shaking at 180 rpm. Cells were harvested by centrifugation at 3900 rpm for 60 min. Pellets were washed four times with 40 mL acidified ethanol (1% v/v HCl 37% in absolute ethanol). The supernatants were dried under low pressure with a rotatory evaporator (Heidolph Laborota 4000) below 40°C.
Prodigiosin hydrochloride (HPLC purity ≥90%, CAS N°: 56144-17-3; Sigma-Aldrich) was dissolved to 0.2 mg/mL in methanol and used without further purification. HPLC samples were prepared from extracts diluted to 10 mg/mL in methanol and 0.22 μm-filtered. For each sample, 10 μL was injected in a Varian 920-LC system equipped with a UV-VIS detector and a photodiode array detector (C18 Hypersil Gold column, Thermo Fisher Scientific, 3 μm, 2.1 × 150 mm, flow rate 0.7 mL/min). All samples were analyzed using a linear gradient of H2O/CH3CN/formic Acid (98:2:0.1 to 2:98:0.1) and detection was performed at 208, 254, 280, and 532 nm.
Prodigiosin hydrochloride (HPLC purity ≥90%, CAS N°: 56144-17-3; Sigma-Aldrich) was dissolved to 0.2 mg/mL in methanol and used without further purification. HPLC samples were prepared from extracts diluted to 10 mg/mL in methanol and 0.22 μm-filtered. For each sample, 10 μL was injected in a Varian 920-LC system equipped with a UV-VIS detector and a photodiode array detector (C18 Hypersil Gold column, Thermo Fisher Scientific, 3 μm, 2.1 × 150 mm, flow rate 0.7 mL/min). All samples were analyzed using a linear gradient of H2O/CH3CN/formic Acid (98:2:0.1 to 2:98:0.1) and detection was performed at 208, 254, 280, and 532 nm.