Porphyrin extraction and quantification

Overnight cultures of the indicated strains grown in M9 medium were diluted (1:100) into 2 l of M9 medium supplemented or not with 25 μg ml⁻¹ ALA and grown aerobically at 37 °C to an OD 600 nm of 0.4. At this point, 25 μg ml⁻¹ tellurite was added to the cultures and porphyrins were extracted from 1 l (cells grown in M9 medium) or 400 ml (cells grown in M9 medium with ALA) of the cultures at the indicated time points as described previously⁵⁴.

Porphyrins were analysed and quantified as described by Tatsumi and Wachi⁴⁵ using a 1,200 series high-performance liquid chromatography (HPLC) system (Agilent Technologies) including a degasser (G1322A), a quaternary pump (G1311A), a variable wavelength detector (G1314B) and a manual sampler. The injection volume was 20 μl. The LC separation was performed on an Agilent Zorbax Eclipse XDB-C18 column (4.6 × 150 mm², 5 μm) eluted for 45 min at a flow rate of 1 ml min⁻¹ with a linear gradient from 50% solvent A (10% acetonitrile in 1 M ammonium acetate; pH 5.1), 50% solvent B (methanol–acetic acid 10:1, v v⁻¹) to 100% solvent B, followed by elution with 100% solvent B for 5 min. The porphyrin composition of each sample was analysed following the absorbance at 300 nm. Commercially available standards of coproporphyrin III and proto IX (Frontier Scientific) were used to identify and quantify each compound from the different samples. Values were normalized by mg of protein as determined by the Bradford method⁵⁵.