Isolation of Gas Vesicles from Methanosarcina barken

SUMMARY: A gas-vacuolate strain of Methanosarcina barkeri formed protoplasts in substrate-depleted cultures and gas vesicles were isolated from the protoplasts. Vacuolate protoplasts were separated from unvacuolate ones by flotation and the protoplast membrane was removed by Tween 20, liberating the gas vesicles. The gas vesicles were purified by flotation after initial passage through a 0.45 μm filter to remove contaminating material. Gas vesicle membranes were purified by isopycnic gradient centrifugation and were shown by electron microscopy to have a rib spacing of 4.8 nm.