Bacterial strains, media, and growth conditions
Acidithiobacillus ferrooxidans ATCC 23270 was obtained from ATCC. The GFP expressing AF-GFP strain was derived from the type strain as described previously (Kernan et al., 2016). A. ferrooxidanscultures were grown at 30 °C under shaking at 200 rpm in 100 mL of filter sterilized AFM1 medium [0.8 g/L (NH4)2SO4, 2.0 g/L MgSO4·7H2O, 0.1 g/L K2HPO4, 5 mL/L trace mineral solution (MD-TMS) and 20.0 g/L (72 mM) FeSO4·7H2O] adjusted to pH 1.8 using concentrated sulfuric acid. For variants of AFM1 medium containing different concentrations of ferrous iron, the amount of FeSO4·7H2O was changed appropriately. For the medium variant containing no iron, ferrous sulfate was completely omitted, and MD-TMS solution prepared without FeSO4·7H2O was used. For the medium variant containing ferric iron instead of ferrous iron, 72 mM ferric iron was added using ferric sulfate hydrate. The different sulfur preparations were added at the noted concentrations to each culture before incubation using aseptic techniques. Both strains were maintained throughout the experiments by weekly sub-culture into AFM1 medium without any addition of sulfur. Cells were harvested by centrifugation and washed with AFM1 basal salts (AFM1 medium without ferrous sulfate).