Cultivation and protoplast preparation of E. faecalis

E. faecalis NBRC 100480 was cultivated and protoplasts were prepared as previously described [23 (link)]. The protoplasts were centrifuged at 7000 rpm for 5 min and resuspended in DMB (5 g/L peptone, 1 g/L yeast extract, 0.1 g/L ferric citrate, 19.45 g/L NaCl, 5.9 g/L MgCl₂, 3.24 g/L MgSO₄, 1.8 g/L CaCl₂, 0.55 g/L KCl, 0.16 g/L NaHCO₃, 0.08 g/L KBr, 34 mg/L SrCl₂, 22 mg/L H₃BO₃, 8 mg/L Na₂HPO₄, 4 mg/L Na₂SiO₃, 2.4 mg/L NaF, and 1.6 mg/L NH₄NO₃ [BD, Franklin Lakes, NJ]) containing 300 µg/mL penicillin G. The resulting suspension (5 µL) was diluted with 1 mL of DMB containing 300 µg/mL penicillin G (Wako, Osaka) and incubated at 24 °C.