B. theta VPI-5482 were grown in brain heart infusion broth supplemented with hemin (5 μg/ml) and menadione (1 μg/ml) in an anaerobic atmosphere of 90% N2, 5% H2, and 5% CO2. The culture was centrifuged at 10,000 rpm at 4°C. The supernatant was spun down twice and then filtered sequentially through filters of 5, 1, 0.6, and 0.2 μm size to eliminate bacteria. The clarified supernatant was then centrifuged in a Ti45 rotor at 40,000 rpm for 1 hour. The OMV pellet was dissolved in 10 ml of 10 mM Hepes (pH 7.4) buffer and spun down in a Ti70 rotor at 50,000 rpm. The pellet was resuspended in 1 ml of 10 mM Hepes (pH 7.4) buffer, and the protein concentration was determined using Bradford protein reagent.

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