ELECTROTRANSFORMATION OF STREPTOCOCCUS-AGALACTINE WITH PLASMID DNA

A protocol for efficient electrotransformation of Streptococcus agalac tiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Es cherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying th e ermB gene for resistance to erythromycin was used as donor DNA. Froz en 'electrocompetent' cells were prepared by repeated washes in 10% gl ycerol. A 50-mu l aliquot containing about 5 X 10(9) colony forming un its of bacteria was subjected to the electric pulse. Optimal condition s for electrotransformation were determined using different media, har vesting cells at different points of the growth curve, and using diffe rent field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied bet ween 0.01 and 3 mu g. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu mu g(-1). The trans formation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococ cal DNA in S. agalactiae.