Electrotransformation of Bordetella

Abstract

Whooping cough (pertussis) is a disease of the upper respiratory tract caused by a localized infection by B. pertussis. The absence of a technique for high-frequency transformation has limited the genetic manipulation of this organism. Weiss and Falkow (1982) described that B. pertussis cells cannot be transformed by classical methods such as calcium chloride treatment but that a cold shock or freezing of the cells results in transformation by plasmids of the P and W incompatibility groups. The transformation frequency obtained was less than 10³/µg of DNA however and a restriction system in B. pertussis prevents the introduction of plasmids containing the Hind III recognition sequence. A modified form of this technique has been used to transform B. bronchiseptica at a frequency of 10⁴/µg of DNA (Lax, 1987). Exposure of living cells to brief pulses of electric current results in a reversible permeabilisation of the biomembranes and has been termed electroporation. This transient membrane permeability then allows DNA uptake by the cells. We describe here the applicability of electroporation to the transformation of Bordetella pertussis and B. parapertussis. Electrotransformation routinely yields > 10⁶ transformants per µg of plasmid DNA.