Cultivation and Genetic Manipulation of Sulfolobus acidocaldarius

The strains used in this study are listed in Table 1. The S. acidocaldarius pyrimidine-auxotrophic and restriction endonuclease SuaI-deficient strain SK-1 (ΔpyrE ΔsuaI) was used as basic host strain [8 (link)]. This strain and its derivatives were cultivated in xyrose and tryptone (XT) medium (pH 3) [9 ] containing 1× basal salts (3 g K₂SO₄, 2 g NaH₂PO₄, 0.3 g MgSO₄·7 H₂O, and 0.1 g CaCl₂·2H₂O), 20 μL of trace mineral solution (1 mg FeCl₃·6H₂O, 0.1 mg CuCl₂·2 H₂O, 0.12 mg CoSO₄·7 H₂O, 0.1 mg MnCl₂·4 H₂O, and 0.1 mg ZnCl₂), 2 g/L xyrose, and 1 g/L tryptone in 1 L Milli-Q H₂O at 75°C with or without shaking (160 rpm). To solidify plates, identical components of 1× basal salts containing 2.9 g MgSO₄·7 H₂O and 0.5 g CaCl₂·2 H₂O were used. For growth of the uracil-auxotrophic strain, 0.02 g/L uracil was added to XT medium (XTU). XTU medium supplemented with 50 μg/mL 5-FOA was used for counterselection with the pop-out recombination method. For cultivation of the argD mutant, 1 mg/mL agmatine (agmatine sulfate [Tokyo Chemical Industry]) was added to the XTU medium. Escherichia coli strain DH5α, used for general manipulation, was routinely cultivated at 37°C in Luria–Bertani medium supplemented with ampicillin (100 μg/mL).