Transformation of Pseudomonas syringae with nonconjugative R plasmids

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Abstract
Transformation of Pseudomonas syringae strains with plasmid DNA occurs at a frequency of 1 × 10−3 to 4 × 10−9 per recipient cell, depending on the strain, plasmid, and conditions for transformation. R plasmids used successfully in transformation were pR0161 (26 × 106 molecular weight) and RSF1010 (5.5 × 106 molecular weight). Transformation involved growing the recipient cells to approximately 8 × 108 colony-forming units per millilitre in 50 mL of a nutrient broth. After washes with a 150 mM CaCl2 – 10% (v/v) glycerol mixture, cells were concentrated 20-fold and resuspended in this solution. The cells then were incubated with purified plasmid DNA for 1 h prior to a heat pulse at 45 °C for 2 min. Transformants were selected by antibiotic resistance and plasmid presence was verified by agarose gel electrophoresis. With plasmid pCG131 (34 × 106 molecular weight; putatively associated with syringomycin production), transformation of syringomycin-negative P. syringae strains that contained no detectable plasmid or were cured of pCG131 was unsuccessful, whether the plasmid was used alone or in combination with either pR0161 or RSF1010.