Formation of Staphylococcus aureus Protoplasts by Lytic Enzyme

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The formation of Staphylococcus aureus IFO 3060 protoplasts was investigated, using lysostaphin and lytic enzyme#2 (LE#2), and the preparation method of the protoplasts was examined. Optimum pH was 7.5 for the bacteriolytic activity of lysostaphin and 8.5 to 9.5 for that of LE#2 in reaction mixtures of 0.05M Tris-acetate buffer. A complete cell-lysis against this bacterium was caused by incubating at 37℃ with 2.0μg/ml lysostaphin for 15min and with 100μg/ml LE#2 for 20min in reaction mixtures containing cells of OD_<660>=0.5, hypotonic buffer (0.05M Tris-HCl buffer pH 7.5 containing 0.015M MgSO_4 for lysostaphin assay, 0.05M Tris-HCl buffer pH 8.5 for LE#2 assay) and enzyme. As no cell-lysis was observed in a lytic reaction mixture containing 1.2 to 1.4M sucrose, it was confirmed that this condition was isotonic for the osmotically sensitized cells. The protoplasts could be formed and prepared by the reaction for 90min under such conditions as described above, using the mixture of cell suspension (OD_<660>=1.0 in hypotonic buffer) with the same volume of hypertonic buffer consisting of hypotonic buffer and 2.4M sucrose. This experiment revealed the formation of S. aureus protoplasts by lysostaphin or LE#2, and established the preparation method of them.
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