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The human pathogen and aquatic bacterium  Vibrio cholerae  belongs to the group of naturally competent bacteria. This developmental program allows the bacterium to take up free DNA from its surrounding followed by a homologous recombination event, which allows integration of the transforming DNA into the chromosome. Taking advantage of this phenomenon we genetically engineered  V. cholerae  using natural transformation and FLP recombination. More precisely, we adapted the T7 RNA polymerase/promoter system in this organism allowing expression of genes in a T7 RNA polymerase-dependent manner. We naturally transformed  V. cholerae  by adding a T7-specific promoter sequence upstream the toxin-coregulated pilus ( tcp ) gene cluster. In a  V. cholerae  strain, which concomitantly produced the T7 RNA polymerase, this genetic manipulation resulted in the overexpression of downstream genes. The phenotypes of the strain were also in line with the successful production of TCP pili. This provides a proof-of-principle that the T7 RNA polymerase/promoter system is functional in  V. cholerae  and that genetic engineering of this organism by natural transformation is a straightforward and efficient approach.

plos one

Overexpression of the tcp Gene Cluster Using the T7 RNA Polymerase/Promoter System and Natural Transformation-Mediated Genetic Engineering of Vibrio cholerae