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

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.

DNA-uptake machinery of naturally competent Vibrio cholerae

Melanie Blokesch, Patrick Martin Seitz

Natural competence for transformation is a mode of horizontal gene transfer that is commonly used by bacteria to take up DNA from their environment. As part of this developmental program, so-called competence genes, which encode the components of a DNA-uptake machinery, are expressed. Several models have been proposed for the DNA-uptake complexes of competent bacteria, and most include a type IV (pseudo)pilus as a core component. However, cell-biology-based approaches to visualizing competence proteins have so far been restricted to Gram-positive bacteria. Here, we report the visualization of a competence-induced pilus in the Gram-negative bacterium Vibrio cholerae. We show that piliated cells mostly contain a single pilus that is not biased toward a polar localization and that this pilus colocalizes with the outer membrane secretin PilQ. PilQ, on the other hand, forms several foci around the cell and occasionally colocalizes with the dynamic cytoplasmic-traffic ATPase PilB, which is required for pilus extension. We also determined the minimum competence regulon of V. cholerae, which includes at least 19 genes. Bacteria with mutations in those genes were characterized with respect to the presence of surface-exposed pili, DNA uptake, and natural transformability. Based on these phenotypes, we propose that DNA uptake in naturally competent V. cholerae cells occurs in at least two steps: a pilus-dependent translocation of the incoming DNA across the outer membrane and a pilus-independent shuttling of the DNA through the periplasm and into the cytoplasm.

National Academy of Sciences2013

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

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

Horizontal gene transfer (HGT) has a major impact on bacterial evolution, leading to acquisition or deletion of genes and gene clusters, including those encoding antibiotic resistances and virulence factors. HGT therefore contributes to pathogen emergence, which can have a major impact on human health. As a mode of HGT, natural competence for transformation allows bacteria to directly acquire DNA from the environment and to integrate parts of this genetic material within their genomes. The human pathogen Vibrio cholerae is naturally competent when it grows in association with the chitinous exoskeletons of zooplankton. Under this condition, the competence state is initiated by the regulatory protein TfoX, which co-induces a molecular killing device known as the type VI secretion system (T6SS). The co-regulation of the T6SS and competence results in the lysis of non-immune neighboring bacteria and the subsequent acquisition of their DNA, which fosters HGT. In V. cholerae, the T6SS is also activated by the TfoX homologue protein, named TfoY. In this study, we aimed at studying the regulatory pathways and the co-regulation of interbacterial predation and DNA uptake in order to provide new insights into the environmental lifestyle of V. cholerae and other vibrios. We first investigated the conserved function of TfoX- and TfoY in V. fischeri, V. alginolyticus, and V. parahaemolyticus using diverse methods such as qRT-PCR, interbacterial killing assays, and motility assessments. We demonstrated that TfoX-mediated T6SS and competence induction and TfoY-induced motility are conserved phenotypes in the tested Vibrio species, whereas the TfoY-mediated T6SS regulation varied. These variations might reflect diverse defense mechanisms, as these different species have adapted to cope with their environmental niches. Next, we tested the outcome of the TfoX-mediated co-regulation of the T6SS and competence in V. cholerae. Under conditions mimicking the bacteriumâs natural habitat, we showed for the first time the extent of DNA that was integrated on V. choleraeâs genome after T6SS-mediated interbacterial predation. Indeed, we demonstrated that T6SS-dependent bacterial predation not only increased the transformation rates but also fostered the exchange of multiple and huge fragments of DNA. We also showed the necessity of an exquisite co-regulation between the T6SS, the competence-related DNA uptake machinery, and the competence-repressed nuclease. Finally, we disclosed that prey-released DNA is not a private good of the attacking bacterium but also accessible to other surrounding cells that have likewise entered the competence state. Altogether, our findings shed light on the conservation of the TfoX- and TfoY-driven phenotypes in several Vibrio species. We also contributed to a better understanding of the interplay between neighbor predation and DNA uptake and the consequences that this interplay has on genome plasticity.

EPFL2019

Two nanomachines drive evolution in diverse Vibrio species

Noémie Matthey

EPFL2019