Autolytic Mechanism for Spheroplast Formation in Bacillus cereus and Escherichia coli

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Abstract
Spheroplasts of B. cereus strain-T and E. coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr. at 30 and 37 C, respectively. Upon incubation in 0.05 M tris(hydroxymethyl)amino-methane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 M sodium acetate buffer osmotically stabilized with 1.6 M sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined; B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromato-graphic analysis of cell-wall hydrolysates from B. cereus and E. coli indicated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopimelic acid, glutamic acid and muramic acid.