Cleavage and isolation of the outer sheath ofTreponema phagedenisby treatment withN,N′-dicyclohexylcarbodiimide

Abstract

Treatment of the cells of Treponema phagedenis biotype Reiter with 50 μM N,N′-dicyclohexylcarbodiimide (DCCD) in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M NaCl partially cleaved the outer sheath and exposed the protoplasmic cylinder and periplasmic flagella. The wall-membrane complex appeared to be more resistant to DCCD than the outer sheath. The outer sheath, which had been partially cleaved and released from the cells into the isosmotic medium by treatment with DCCD, was isolated and purified by differential centrifugation followed by centrifugation on a linear sucrose density gradient. The purified outer sheath preparation, which consisted of vesicles with a triple-layered membranous structure, was composed of protein (40.2%), phospholipid (39.7%) and carbohydrate (11.4%). The purified outer sheath vesicles were seen to have a polygonal array upon electron microscopic observation of negatively stained preparations.