Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens.
A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA. Transformation of vegetative cells of C. perfringens strain 13 with the 7.9-kb Escherichia coli-C. perfringens shuttle plasmid pHR 106 required pretreatment with lysostaphin (2 to 20 micrograms/ml) for 1 h at 37 degrees C. Cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases. The transformation frequency increased with the DNA concentration, to a saturating level at 5 to 10 micrograms DNA/ml. The transformation frequency was proportional to the field strength and time constant of the electroporation pulse; however, the field strength was a far more important parameter. A cell density between 1 x 10(8) and 5 x 10(8) cells/ml proved to be optimal for transformation. The procedure was capable of generating up to 3.0 x 10(5) transformants per micrograms DNA. The potential value of the method for the cloning of C. perfringens genes was demonstrated by the cloning of the clostridial tetracycline-resistance determinant, tetP, from the E. coli recombinant plasmid pJIR71, into C. perfringens strain 13.
Scott
P T
PT
Department of Microbiology, Monash University, Victoria, Australia.
Rood
J I
JI
D001431:Bacteriological Techniques; D003016:Clostridium perfringens / Q000187:drug effects / Q000235:genetics*; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D008248:Lysostaphin / Q000494:pharmacology*; D013268:Stimulation, Chemical; D014169:Transformation, Bacterial / Q000187:drug effects*
D008248:Lysostaphin