Isolation, characterization, and in vitro assembly of the tetragonally arrayed layer of Bacillus sphaericus

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Abstract
The tetragonally arranged cell wall layer (T-layer) of B. sphaericus NTCC 9602 was isolated and characterized. Parallel studies were made on a spontaneous variant of the wild-type strain which had a T-layer subunit of altered MW. A purification method for the T-layers was devised which involved separation of the cell walls from the cytoplasmic contents urea dissociation of the T-layer from the cell walls, removal of soluble contaminants by differential centrifugation, and finally selective adsorption of uncleaved subunits to sacculi. The purified subunits retained the capacity to form an assembly in vitro with the same lattice parameters as that observed on whole cells or cell walls and could readsorb to the cell walls from which they were extracted. Both the wild-type and the variant subunits behaved as single, homogeneous polypeptide chains. Carbohydrate assay and isoelectric point determinations revealed that both subunit types were acidic glycoproteins. Values obtained for the buoyant density, isoelectric point and extinction coefficient differed minimally; major differences were observed in the MW and the characteristic width of cylinders formed by in vitro-assembled T-layer of the wild-type and variant. Assembled T-layer was subject to alkaline or acid dissociation and in acid titration dissociated at its isoelectric point.