Protoplast transformation of glutamate-producing bacteria with plasmid DNA

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Abstract
A method for polyethylene glycol(PEG)-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration and PEG-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, C. herculis, Brevibacterium flavum and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (.apprx. 106 transformants/.mu.g of covalently closed circular DNA) but was still 2 orders of magnitude below the frequency of transfection with modified DNA of the bacteriophase .vphi.CG1. The difference was ascribed to the involvement of regeneration in transformation.