Isolation and characterization of the plasma membrane and the outer membrane of Deinococcus radiodurans strain Sark

Deinococcus radiodurans strain Sark, although gram-positive, has a complex cell wall profile that includes an outer membrane-like structure. The outer cell envelope layers formed blebs throughout the growth cycle, which were shed as large vesicles (0.5–3.5 μm in diameter) from approximately 5% of the cell population. Instability of the compartmentalized layer immediately beneath the outer membrane was the cause of the vesicle production. This instability was accentuated by treatment with 10% NaCl, which released the outer membrane from all cells without disrupting the peptidoglycan layer, and provided an outer membrane fraction uncontaminated by plasma membrane. Cells so treated formed protoplasts after sequential treatment with 6 M urea, trypsin, and the supernatant from batch cultures of Lysobacter enzymogenes 495. The plasma membrane was isolated from lysed protoplasts. The absence or presence of catalase activity, and differences in lipid composition, were used to differentiate between plasma membrane and outer membrane.