High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.
Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.
Miller
J F
JF
Department of Medical Microbiology, Stanford University School of Medicine, CA 94305.
Dower
W J
WJ
Tompkins
L S
LS
D001431:Bacteriological Techniques; D002168:Campylobacter fetus / Q000235:genetics*; D002463:Cell Membrane Permeability; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D005821:Genetic Techniques; D010957:Plasmids; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004274:DNA, Recombinant