Isolation of Spheroplast Membranes and Stability of Spheroplasts of Bacillus stearothermophilus

Abstract

Spheroplasts were prepared by lysozyme digestion of the cell wall and ruptured by suspension in 0.15 m NaCl, followed by centrifugation at 30,900 × g for 35 min, and by a final suspension in 0.05 m NaCl for 12 to 16 hr at 5 C. The membrane ghosts were washed four times in tris(hydroxylmethyl)aminomethane (Tris) magnesium buffer and once in distilled water. The intact membranes resembled empty sacs with narrow slits in which the cytoplasm was extruded. A 92% recovery of cell membrane was obtained with all membrane preparations. The spheroplasts do not require a stabilizing medium to keep them from rupturing, and they are stable for 2 to 3 hr when exposed to a temperature of 65 C. The membrane content of the cell increases with age of culture (mid-log, 16.5%; late-log, 17.0%; and stationary, 17.6%) and temperature of growth (55 C, 16.5%; and 65 C, 17.8%), and it is unaffected by composition of the growth medium. The ratio of the protein to lipid content of the membrane increases with the complexity of the medium, age of culture (mid-log, 3.65; late-log, 3.91; and stationary, 4.15), and temperature of growth (55 C, 3.65; and 65 C, 5.22). The ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) content of the membranes was 9.0 to 13.7% and 0.3 to 0.8%, respectively. Reducing sugar (determined as glucose) amounts to 0.9 to 1.0% of the membrane weight and did not significantly vary for the different membrane preparations. Medium composition, age of culture, and temperature of growth have no significant effect on the amount of each amino acid in the membrane. Aspartic acid, glutamic acid, alanine, leucine, and lysine are present in the greatest amount and represent 12.9 to 14.1%, 10.4 to 11.3%, 9.6 to 10.3%, 7.7 to 8.8%, and 7.6 to 8.5% of the membrane peptide, respectively. Prior to the rupture of the spheroplasts, 25.0, 15.7, and 50.0% of the protein, RNA, and DNA, respectively, is lost. In potassium phosphate-magnesium buffer without sucrose, 90% of the protein and RNA and 95% of the DNA is lost from the spheroplasts. In the presence of sucrose, the leakage of RNA and DNA is similar to that observed for spheroplasts suspended in Tris magnesium buffer; however, the leakage of protein is 2.4 times greater.