Fragmentation and solubilization of filamentous appendages of Bacillus cereus spores

Abstract

Filamentous appendages were removed from the surfaces of Bacillus cereus spores by treatment with sodium thioglycolate and purified by filtration through a glass microfiber paper and a membrane filter. The basic structure of the isolated appendages resembled that of the native appendages on the spores. The results of electron microscopical analysis, amino acid analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) attested the purity of the preparation. The purified filamentous appendages were fragmented by treatment with 2N sodium hydroxide at 30C but neither with SDS nor mercaptoethanol, and the fragmentation progressed with the prolongation of the treatment period. When observed under an electron microscope, each fragmented appendage appeared to be rod shaped with an apparent axial hole and helical or disk-like arrangement of subunits. In the agar gel immunodiffusion test, a soluble fraction of the appendages obtained by treatment with sodium hydroxide formed only a single precipitation line against the antibody to the intact appendages. The molecular mass of the subunit separated by SDS-PAGE was approximately 10kDa. The 10-kDa protein was distributed over the surface of the intact appendages in immunoelectron microscopy. The antigenicity of the appendage subunits disappeared upon treatment with 2N sodium hydroxide for longer than 24h. Neither intact appendages nor intact spores agglutinated sheep or guinea-pig erythrocytes.