TransFLP - A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes(1-3). Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons(1,2)). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene(4). The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)(5) (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism(6) and other representative of the genus Vibrio such as V. fischeri(7). Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region(8). Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants. This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences(5). Several optimization steps concerning the initial protocol of chitin-induced natural transformation(6) are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes(8), the donation of PCR-derived DNA as transforming material(9), and the addition of FLP-recombination target sites (FRT)(5). FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase(10).

Protocols for Visualizing Horizontal Gene Transfer in Gram-Negative Bacteria Through Natural Competence

Melanie Blokesch

Horizontal gene transfer (HGT) is widespread in bacteria and archaea and plays a significant role in prokaryotic evolution. One mode of HGT is natural competence for genetic transformation, which allows the organism to take up free DNA from its surroundings and incorporate this DNA into its own genome. Natural transformation has been studied in many diverse organisms since its discovery in the 1920s; however, the majority of these studies were primarily based on population-wide methods. Recent advances in biological imaging, including the use of fluorescent proteins, have revolutionized the field of microbiology, and such imaging techniques allow for the study of important phenomena at the single-cell level. Here, we describe the visualization of the DNA uptake process in naturally competent Vibrio cholerae cells. More precisely, the protocol below provides instructions for the genetic engineering of V. cholerae to generate strains carrying translational fusion constructs between important competence/recombination and fluorescent proteins. Moreover, detailed steps for live cell time-lapse microscopy imaging of these strains under competence-inducing conditions are discussed for direct or indirect visualization of the DNA uptake process.

Humana Press2015

Quorum Sensing Contributes to Natural Transformation of Vibrio cholerae in a Species-Specific Manner

Melanie Blokesch, Patrick Martin Seitz, Gaia Francesca Marianne Suckow

Although it is a human pathogen, Vibrio cholerae is a regular member of aquatic habitats, such as coastal regions and estuaries. Within these environments, V. cholerae often takes advantage of the abundance of zooplankton and their chitinous molts as a nutritious surface on which the bacteria can form biofilms. Chitin also induces the developmental program of natural competence for transformation in several species of the genus Vibrio. In this study, we show that V. cholerae does not distinguish between species-specific and non-species-specific DNA at the level of DNA uptake. This is in contrast to what has been shown for other Gram-negative bacteria, such as Neisseria gonorrhoeae and Haemophilus influenzae. However, species specificity with respect to natural transformation still occurs in V. cholerae. This is based on a positive correlation between quorum sensing and natural transformation. Using mutant-strain analysis, cross-feeding experiments, and synthetic cholera autoinducer-1 (CAI-1), we provide strong evidence that the species-specific signaling molecule CAI-1 plays a major role in natural competence for transformation. We suggest that CAI-1 can be considered a competence pheromone.

American Society for Microbiology2011

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

Melanie Blokesch

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.

Public Library of Science2013