DNA-uptake machinery of naturally competent Vibrio cholerae

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.

DNA Transport across the Outer and Inner Membranes of Naturally Transformable Vibrio cholerae Is Spatially but Not Temporally Coupled

Melanie Blokesch, Patrick Martin Seitz

The physiological state of natural competence for transformation allows certain bacteria to take up free DNA from the environment and to recombine such newly acquired DNA into their chromosomes. However, even though conserved components that are required to undergo natural transformation have been identified in several naturally competent bacteria, our knowledge of the underlying mechanisms of the DNA uptake process remains very limited. To better understand these mechanisms, we investigated the competence-mediated DNA transport in the naturally transformable pathogen Vibrio cholerae. Previously, we used a cell biology-based approach to experimentally address an existing hypothesis, which suggested the competence protein ComEA plays a role in the DNA uptake process across the outer membrane of Gram-negative bacteria. Here, we extended this knowledge by investigating the dynamics of DNA translocation across both membranes. More precisely, we indirectly visualized the transfer of the external DNA from outside the cell into the periplasm followed by the shuttling of the DNA into the cytoplasm. Based on these data, we conclude that for V. cholerae, the DNA translocation across the outer and inner membranes is spatially but not temporally coupled.

American Society for Microbiology2014

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

The physiological state of competence for natural transformation allows bacteria to take up free DNA from the environment and recombine it into their genome. As one of three modes of horizontal gene transfer, natural transformation has hence contributed substantially to the enormous diversity and plasticity of bacterial genomes. With its potential to promote the spread of antibiotic resistances and virulence factors of a variety of pathogens, natural transformation also has an important impact on human health. Even though conserved components required to undergo natural transformation have been identified in many bacterial species, our knowledge on the underlying mechanism of DNA transport remains very limited. To better understand this aspect, we established a first model of DNA uptake in the naturally competent human pathogen Vibrio cholerae. We showed that at least twenty proteins are required for efficient uptake, protection and recombination of transforming DNA in a non-species-specific manner. 14 of these proteins are needed to produce a type IV pilus, which we were able to visualize for the first time. We showed that this competence pilus is not restricted to cell poles, as has been shown for other competent bacterial species (e.g. Bacillus subtilis). Furthermore, we compared the position of this pilus to the localization of other competence proteins and determined their role in the assembly of a surface exposed pilus fiber. While this type IV pilus is important for efficient DNA uptake, we also observed rare transformation events in its absence. In contrast, three pilus-unrelated proteins that are likely involved in the transport process were absolutely required for transformation. Two of them were implicated in DNA transport across the inner membrane. Deletion of the corresponding genes led to a strong accumulation of transforming DNA within the periplasm. However, one of them, ComEA, was essential for DNA uptake across the outer membrane, independently of competence pili. We further characterized the role of this protein by showing that it is localized to the periplasm of competent V. cholerae cells and that it binds to DNA in vivo. Based on our results, we proposed that ComEA directly contributes to DNA transport. Although we showed that DNA uptake across the outer membrane and translocation of DNA into the cytoplasm are functionally uncoupled, our results suggest that the two events coincide spatially. In summary, these findings allow us to present a first mechanistic model of the DNA uptake process in V. cholerae. Many aspects of this model likely apply to other naturally competent bacteria, and therefore represent an important contribution to the general understanding of this basic biological process.

EPFL2014

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

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

EPFL2014