Proteins in the culture supernatant of Staphylococcus aureus PS 47 were subjected to Sephadex chromatography. In the early stage of the cultivation, staphylokinase appeared to have a molecular weight of 15,000, and in the later stage it appeared to have a molecular weight of 320,000. The staphylokinase having a lower molecular weight (type A) converted into one having a higher value (type B) during the course of cultivation. It was demonstrated that conversion of type A into type B took place in vitro (monitored by gel filtration after the two types of staphylokinases were mixed), and it was observed that type B reverted to type A when type B was treated with KCl or detergent. Type B seems to be a complex form of type A and some high-molecular-weight substance.

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Protoplasts of Staphylococcus aureus 209P and Cowan I were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature.Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCI) formed typical L-form colonies. Horse serum (0.25%) and M g2 + (lOa msr) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan I, M g2 + was not required.The active factor(s) in horse serum was heat-resistant and protein in nature.L-Phase variant induction in Staphylococcus aureus has been reported by many workers since the initial success of Diens and Sharp (2). The essential and common procedure for inducing the variants in various bacterial species is removal of the cell wall by a digesting enzyme (9) or inhibition of cell wall synthesis by penicillins (2) and the addition of an osmotic stabilizer and 5-10% serum to the culture medium. The generally used stabilizer is sucrose or sodium chloride. These are necessary to protect the fragile cells from cell bursting due to increased osmotic pressure. The addition of serum is also indispensable, but its function in the growth of L-phase variants still remains obscure.The induction of protoplast fusion by polyethylene glycol, which was first demonstrated in plant protoplasts by Kao and Michayluk (5), was extended to various microbial protoplasts (3, 7) including Staphylococcus aureus (4, 6). The cell fusion method will provide an excellent opportunity for studying genetic recombination and chromosomal mapping in Staphylococcus aureus, concerning which there has been little work done. Before investigation of protoplast fusing, however, it is necessary to determine the conditions that will allow highly efficient induction of protoplasts and L-phase variants.In this communication, a preliminary experiment on cell conversion to L-phase variants with high efficiency in Staphylococcus aureus is described. Some characteristics of the serum factor are also mentioned.

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