Application of protoplast fusion technique to the genetic studies of antibiotic producing micromonospora spp.

Abstract

Despite the importance of $\mbox{\underline{Micromonospora}}$ for its production of invaluable antibiotics, very little is known about its genetics. Due to their non-conidia forming properties, mycelial fragment and protoplast were used for the studies. By the assessment of the mutation of $\mbox{\underline{M.}}$ $\mbox{\underline{rosaria}}$ to streptomycin-resistancy, the best conditions and effective procedures for the mutagenesis by a chemical mutagen, MNNG, have been determined. Mutation was efficiently induced when mycelial fragments were treated with MNNG at the concentration of 0.3 to 0.5 mg/ml in the reaction buffer of pH 7.0. Optimal treatment time was 30-40 min. Ampicillin treatment was very effective for concentrating the portion of auxotrophs. By the determined procedure, more than 30 mutants have been isolated, characterized, and used for this study. When protoplasts were treated with MNNG, as much as 4\% of the resultant colonies were auxotrophic mutants. Conditions for efficient formation and regeneration of $\mbox{\underline{M.}}$ $\mbox{\underline{rosaria}}$ protoplasts were investigated. The state of inoculum, culture stage and growth in a medium containing partially growth-inhibiting concentration of glycine have significant effects on protoplasting. It seems that growth stage as well as the growth rate are most critical factors for the efficient protoplasting. A high frequency of regeneration (up to 30\%) was accomplished with a hypertonic regeneration agar medium. A slight difference in the optimal culture age for the formation and regeneration of protoplasts was found. Using these optimal conditions, protoplast fusion was carried out. When single auxotrophs were used in protoplast fusion by PEG 1,000, the recombinant frequency varied from 1.3 to 5.9\%. Using two double auxotrophs, MR 28 and MR 217 whose genotypes are $\mbox{\underline{ros}}^-$ $\mbox{\underline{his}}$ $\mbox{\underline{trp}}$ and $\mbox{\underline{ros}}^+$ $\mbox{\underlin...