Protoplast Production and Regeneration in Propionibacterium

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Abstract
A procedure for the production and regeneration of protoplasts of Propionibacterium freudenreichii is described. A cell population that consisted of >99.9% protoplasts was produced by incubation of midexponential cells with lysozyme in a buffer that contained .5 M sucrose, 100 mM Tris-HCl, and 10 mM MgCl2. Regeneration occurred in sodium lactate agar supplemented with .5 M sucrose and 2.5% gelatin. The protoplast suspension was spread onto the surface of regeneration agar and overlaid with soft regeneration agar. Regeneration of the protoplasts took 21 to 25 d. The use of a soft agar overlay was required for efficient regeneration, as was the presence of sucrose and gelatin in the regeneration medium. Regeneration frequencies were generally in the range of 10 to 30%, although frequencies as high as 85% were obtained.