Formation and Reversion of Streptomycete Protoplasts: Cultural Condition and Morphological Study

Abstract

SUMMARY: Cultural conditions for forming and stabilizing protoplasts of Streptomyces griseus and S. venezuelae were studied by reference to the number of protoplasts formed, leakage from protoplasts and reversion rate. Effective formation and stabilization of the protoplasts was accomplished by using a hypertonic medium containing 10 mm-MgCl2 and 25 mm-CaCl2. Electron microscopy showed that the protoplasts of S. griseus, when prepared in the above medium, formed vesicles on the cytoplasmic membrane or out of the protoplasts, but when prepared in a medium with 3 mm-MgCl2 and 3 mm-CaCl2 they provided few such vesicles. The reversion of protoplasts to the normal filamentous state was examined by the growth on various synthetic agar media. A high reversion rate was obtained by incubating the protoplasts on a hypertonic agar medium containing 20 or 50 mm-MgCl2, 50 or 20 mm-CaCl2, 0·44 or 0·22 mm-phosphate and 0·01 % Casamino acids. Combinations of appropriate concentrations of MgCl2 and CaCl2 were significantly effective in the reversion as well as in the stability of protoplasts. The concentration of phosphate should be adjusted to 0·44 or 0·22 mm. Casamino acids enhanced both growth and reversion rates. The reversion of protoplasts of Streptomyces griseus was followed by using phase-contrast microscopy. The protoplasts incubated on medium R1 enlarged to about 40 µm and then generated filamentous hyphae from the periphery.