Hydroxycobalamin

The strains carrying various expression vectors were grown overnight at 37 °C on LB-agar plates supplemented with 25 μg ml⁻¹ kanamycin and 100 μg ml⁻¹ ampicillin. M9 minimal medium (47.7 mM Na₂HPO₄ × 12H₂O, 17.2 mM KH₂PO₄, 18.7 mM NH₄Cl, 8.6 mM NaCl) was supplemented with 0.4% glycerol, 2 mM MgSO₄, 0.1 mM CaCl₂, 100 μg ml⁻¹l-arginine, 25 μg ml⁻¹ kanamycin and 100 μg ml⁻¹ ampicillin. A single colony was picked and used to inoculate an M9-medium pre-culture supplemented with 50 μg ml⁻¹l-methionine (Sigma-Aldrich). The pre-culture was grown ~24 h at 37 °C, shaking in tubes with gas-permeable lids (Cellstar), and then used to inoculate the assay medium in a 1:500 ratio. The assay medium was supplemented with 0.00001% l-arabinose (Sigma-Aldrich) and either 50 μg ml⁻¹l-methionine, 0.01 nM, 1 nM and 5 nM cyano-cobalamin (Acros Organics), or 0.1 nM hydroxy-cobalamin (Sigma-Aldrich). Overall, 200 μl medium was added per well of a sterile 96 well plate (Cellstar). Plates were sealed with a sterile and gas-permeable foil (BreatheEasy, Diversified Biotech). The cultures were grown for 1000 min (1250 min for Cbi) in a BioTek Power Wave 340 plate reader at 37 °C, shaking. The OD₆₀₀ was measured every 5 min at 600 nm. All experiments were conducted as technical triplicates from biological triplicate. The displayed growth curves are the averages of all nine curves.

B₁₂ was quantified using mass spectrometry in tandem with liquid chromatography as described elsewhere [21 ]. Calibration standards (all from Sigma-Aldrich; Saint Louis, USA) used were adenosylcobalamin, hydroxycobalamin, and methylcobalamin at concentrations 0.3 ng mL⁻¹, 1.0 ng mL⁻¹, 3.0 ng mL⁻¹, 10 ng mL⁻¹, 30 ng mL⁻¹, 100 ng mL⁻¹ and 300 ng mL⁻¹ in dimethyl sulphoxide (DMSO). Cyanocobalamin (internal standard) was used at 2 μg mL⁻¹. Samples or standards were injected at 100 μL with 10 μL internal standard. Details of the LC-MS setup is provided in section 1.3 of S1 File.