Abstract
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.
Highlights
A plethora of methods exist today to allow genetic engineering of bacteria
The T7 specific promoter sequence was delivered on a plasmid as well as upstream the tcp operon. The latter was accomplished using chitininduced natural transformation followed by FLP recombination
In conclusion we successfully used the T7 RNA polymerase/ promoter system in V. cholerae. To our knowledge this is the first time that this system was utilized in this organism though it was already suggested in earlier studies that ‘‘comparable T7 expression systems [to those in E. coli] can be developed in other types of cell’’ [12]
Summary
A plethora of methods exist today to allow genetic engineering of bacteria. Many of those methods have been used for the last 30 years or even longer and straightforward protocols are available today to master those techniques. One prominent example is the seminal book Molecular Cloning: A Laboratory Manual published initially in 1982 by Tom Maniatis, Edward Fritsch, and Joseph Sambrook [1] Even though many techniques are available and work well, reducing the time needed for cloning is often desired. We developed a fast protocol [2,3,4] to genetically modify our favorite organism, the human pathogen and aquatic bacterium Vibrio cholerae, using chitin-induced natural transformation [5,6]
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