Summary
Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistaiice plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 × 1013 colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 μg of pNG2 dna was mixed with 400-μl amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 μg of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of ≈ 4 × 104 transformants/μg of pNG2 dna. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 dna was varied, the average total number of transformants increased through a concentration of 30 μg/ml, but the efficiency of transformation was highest at the lowest dna concentration. Transformants contained unmodified pNG2.