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Isolation and Characterization of Forespores from Bacillus megaterium
Author(s) -
Watabe Kazuhito,
Koshikawa Tomihiko,
Yamazoe Mari,
Ogawa Setsuko,
Torii Mitsuo
Publication year - 1987
Publication title -
microbiology and immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0385-5600
DOI - 10.1111/j.1348-0421.1987.tb03073.x
Subject(s) - spore , bacillus megaterium , dipicolinic acid , lysozyme , protoplast , differential centrifugation , sonication , biology , chromatography , germination , centrifugation , density gradient , biochemistry , microbiology and biotechnology , botany , chemistry , bacteria , genetics , physics , quantum mechanics
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 6 hr (t 6 ) 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 t 6 , t 10 , t 12 , and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t 6 and t 8 were extremely heat labile (D 80 of 9.5 and 21.5 min, respectively) relative to mature spores (D 80 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 KNO 3 , d‐glucose, and l‐leucine. Forespores at t 6 were more sensitive to KNO 3 ‐induced germination than those at t 10 , t 12 , and mature spores when measured by reduction in the optical density of cell suspension.