Serological Studies on the Formation of Protein Parasporal Inclusions in Bacillus Thuringiensis

A strain of Bacillus thuringiensis has been isolated, and methods have been developed for separation of the crystalline, parasporal inclusions in a pure form. Normal sporulation with concomitant crystal formation takes place when cells are incubated under suitable conditions in a nutrient free medium. Serological techniques have been used to study the origin and development of the crystals. Rabbit antisera have been prepared to a vegetative cell extract, suspensions of crystals, and a solution of crystal protein (obtained by alkali treatment of crystals). Tests have been carried out mainly by the Ouchterlony gel plate technique. Crystal protein solutions were found to be more active than suspensions of intact crystals both in reaction with, and in neutralization of, the crystal antibodies. Antisera to the vegetative cell extract gave no reaction with crystal protein. Ultrasonic extracts of cells taken before or during crystal formation gave no reaction with the crystal antibodies. Tests with alkali extracts of disrupted cells showed that the crystal antigen is absent in vegetative cells but arises during sporulation. The appearance of the antigen can be correlated with the formation and growth of the crystals as followed by examination of disrupted cell preparations under the electron microscope. It can be concluded that the crystalline protein inclusions do not arise from precursors in the same antigenic state.