Application of protoplasts to the studies of micromonospora species

Abstract

The possibility of the mutagenesis using M. rosaria protoplasts were investigated. The optimized condition for mutagenesis and practical mutant selection were described in this paper. The maximum yield of mutants was obtained by treating protoplasts for 15 to 30 min with 50 $\mu$g/ml of NTG in P buffer at pH 7.6. Under that condition, more than 4\% of the survivors are auxotrophs and survival is of the order of 40\%. An intercalating agent, acriflavin, was successfully employed for curing Micromonospora purpurea using mycelial fragments as well as protoplasts. Up to 90\% of colonies were shown to have lost their antibiotic producing ability after treatment with acriflavin at the concentration of 0.5 $\mu$g/ml. A plasmid DNA band was detected by an agrose gel electrophoresis from the parental strain. Some intermediates and intermediate analogs were supplemented to the antibiotic-nonproducing cured strain and the results were analyzed. The responses of each strain were somewhat different, and the results indicated that complex phenomena might be caused by acriflavin treatment, including loss of a plasmid which is involved in gentamicin production. This result is somewhat different from those obtained by testing some Streptomyces spp. producing other aminoglycoside antibiotics, which indicated simple loss of plasmids by curing agent.