Strain and Cultivation for DNA Extraction

Haloarcula hispanica JCM8911 was obtained from the Japan Collection of Microorganisms (JCM, Tsukuba, Ibaraki, Japan). The strain was grown in Medium 307, which contained 2 g casamino acid (BD, Franklin Lakes, NJ, USA), 2 g Bacto yeast extract (BD), 1 g sodium glutamate, 3 g trisodium citrate, 10 g MgSO₄⋅7H₂O, 1 g CaCl₂⋅2H₂O, 1 g KCl, 200 g NaCl, 0.36 mg FeCl₂⋅4H₂O, and 0.36 mg MnCl₂⋅4H₂O per liter of distilled water. After the pH of the medium was adjusted to 7.0, the medium was sterilized by filtration with a polyethersulfone membrane filter (pore size, 0.22 μm; Thermo Fisher Scientific, Waltham, MA, USA) and autoclaving at 121°C for 20 min. Exactly 20 ml of the medium was injected into autoclaved 60-ml screw-cap test tubes and inoculated with cells of H. hispanica in exponential growth phase in pre-culture, and the cultures were incubated in the darkness with shaking at 180 rpm at 37°C. The cultures were centrifuged at 6230 × g for 3 min. The pelleted cells were stored at -25°C until DNA extraction.