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).