Transformation of Bacillus licheniformis protoplasts by plasmid DNA

Summary: A simple and efficient protoplast regeneration and PEG-induced transformation procedure was developed for Bacillus licheniformis. The protoplast stabilizing osmoticum was 0.5 M sucrose in the regenerating media, and the restoration of cell wall was carried out at pH 8.4. The mean frequency of regeneration was 83%. The transformation efficiency was studied with four different strains and five plasmids, and ranged from 3.5×105to 7.2×106transformants per μg plasmid DNA depending on the plasmid, and on the donor and recipient strains used. Endonuclease digested and ligated plasmid DNA could also be used to transform the protoplasts, but at a lower frequency.