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

Abstract

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 m~-MgCl, and 25 m~-CaCl,. Electron microscopy showed that the ])rotoplasts of 5'. 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 m~-MgCl, and 3 mM-CaCI, 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-MgCl,, 50 or 20 m~-CaCl,, 0.44 or 0-22 mM-phosphate and 0.01 % Casamino acids. Combinations of appropriate concentrations of MgCl, and CaC1, 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 R I enlarged to about 40 ,um and then generated filamentous hyphae from the periphery.

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp

“…R2YE soft-agar overlays contained R2YE medium plus 0.6% Bacto-Agar. Protoplast buffer (P buffer), a hypertonic medium used for protoplast preparation and transformation, was described previously (33). E. coli strains were grown at 37°C on Luria-Bertani (LB) medium (32) or LB medium supplemented with 1.5% BactoAgar for growth on solid medium.…”

Studies of Protoplast Fusion in Streptomyces chrysomallus

“…After cultivation for 2 d in medium S , with or without glycine, cells were collected from 3 4 flasks and washed twice with 0.3 M-Sucrose and then suspended in medium P (Okanishi et al, 1974) to give 50 mg mycelium ml-l in the presence of 2.5 mg lysozyme ml-l. After incubation (3 h at 30 "C) with occasional careful shaking, mycelia were disintegrated. When grown in the presence of 0.5 or 1 % (w/v) glycine, various strains of S. chrysomallus were converted into protoplasts much faster than when grown in the absence of glycine (1 h).…”

“…Changes in the composition of R2 affecting optimal cell regeneration from protoplasts and optimal recombination frequencies are described in Results and Discussion. Liquid growth medium was medium S (Okanishi et al, 1974).…”

Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

“…Modified medium R2 was as described by Okanishi, Suzuki & Umezawa (1974) except that 1.8 g L-asparagine was substituted for proline as the nitrogen source. Medium P was as described by Okanishi et al (1974).…”

“…Protoplasts of S. fradiae and S. griseofuscus were formed by a modification of the glycine-lysozyme-lytic enzyme method (Okanishi, Suzuki & Umezawa, 1974). Regeneration of cells from protoplasts was monitored throughout the growth cycle and was most efficient when cells of either S. fradiae or S. griseofuscus were taken from the transition phase between the exponential and stationary growth phases.…”