Transformation of Burkholderia pseudomallei by electroporation

Conditions were defined for the successful transformation of the human pathogen Burkholderia pseudomallei 4845 by electroporation, using the incQ plasmid pKT230. We have obtained frequencies of up to 8 x 10(3) transformants per microgram plasmid DNA with freshly prepared electrocompetent B. pseudomallei. The method also allows for easy and reproducible production of frozen cell suspensions which can produce efficiencies up to 2.5 x 10(2) transformants per microgram of plasmid DNA. Kanamycin had to be added to the culture growth medium at a minimum concentration of 20 micrograms ml-1 and a maximum concentration of 50 micrograms ml-1 for the bacteria to become electrocompetent. Bacteria grown in medium containing a final concentration of 30 micrograms ml-1 kanamycin produced the highest numbers of transformants, although the transformation frequency at this concentration was not as efficient as that at 50 micrograms ml-1. Electron microscopy indicated that the kanamycin affects the integrity of the bacterial outer membrane, which becomes loose and wrinkled in appearance. The electrocompetence does not result in a permanent morphological change.