Function of plasmids in the production of aureothricin. I. Elimination of plasmids and alteration of phenotypes caused by protoplast regeneration in Streptomyces kasugaensis.

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Abstract
The spontaneous mutant 18a derived from S. kasugaensis MB273 exhibited pleiotropic effects such as loss of aerial mycelium formation, aureothricin (AT) production and citrullin biosynthesis, and changes in plasmids; the mutant required cystine for production of aureothricin. An improved method of protoplast regeneration was applied to S. kasugaensis MB 273-18a and a regeneration efficiency of 90% or more was obtained. Of the colonies regenerated from the 18a protoplasts, 60-90% exhibited reversion of the pleiotropic mutation in 18a. Of 13 regenerated strains which showed these drastic phenotypic variations, it was found that their plasmid types varied. These types could be divided into 2 groups; the RI type (5 strains) which contained a large amount of pSK2, a small amount of pSK3 and no pSK1, and the RII type (8 strains) in which no closed-circular DNA was detected. Plasmid curing in RII type strains and also the variation of plasmid copy in the RI type strains occurred as the result of protplast regeneration. The structural genes for biosynthesis af AT probably exist on chromosome. Regeneration of 18a protoplasts causes the reversion of pleiotropic mutation with high frequency. A working hypothesis was proposed to explain these complex phenomena.

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