Transformation of Vibrio cholerae by plasmid DNA

Abstract

The lack of an efficient transformation system in Vibrio cholerae was a handicap in the genetic manipulation of this important human pathogen. Since V. cholerae cells secrete DNases, this may interfere with the uptake of DNA. The present report describes the approaches taken for transforming V. cholerae cells with plasmid DNA, by overcoming this DNase barrier. The partial success of transforming DNase-negative mutants confirmed the role of DNase in the nontransformability of the wild-type cells. Successful transformation was carried out following removal of DNases from the periplasmic space. This was achieved by treating the cells with Mg²⁺ and Ca²⁺ ions to allow the DNase to be released, and then holding them under conditions where the remaining DNase activity was minimized before adding DNA to the competent cells. Transformation efficiencies of the order of 10^-5 per recipient cell were observed.