Isolation and Characterization of Forespores from Bacillus megaterium

Abstract

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6hr (t₆) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t₆, t₁₀, t₁₂, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t₆ and t₈ were extremely heat labile (D₈₀ of 9.5 and 21.5min, respectively) relative to mature spores (D₈₀ of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO₃, D-glucose, and L-leucine. Forespores at t₆ were more sensitive to KNO₃-induced germination than those at t₁₀, t₁₂, and mature spores when measured by reduction in the optical density of cell suspension.