Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts.

Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.