The Regulatory Network of Natural Competence and Transformation of Vibrio cholerae

The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed “natural competence for genetic transformation.” Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae.

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

Regulatory elements involved in the expression of competence genes in naturally transformable Vibrio cholerae

Melanie Blokesch, Mirella Lo Scrudato

BackgroundThe human pathogen Vibrio cholerae normally enters the developmental program of natural competence for transformation after colonizing chitinous surfaces. Natural competence is regulated by at least three pathways in this organism: chitin sensing/degradation, quorum sensing and carbon catabolite repression (CCR). The cyclic adenosine monophosphate (cAMP) receptor protein CRP, which is the global regulator of CCR, binds to regulatory DNA elements called CRP sites when in complex with cAMP. Previous studies in Haemophilus influenzae suggested that the CRP protein binds competence-specific CRP-S sites under competence-inducing conditions, most likely in concert with the master regulator of transformation Sxy/TfoX.ResultsIn this study, we investigated the regulation of the competence genes qstR and comEA as an example of the complex process that controls competence gene activation in V. cholerae. We identified previously unrecognized putative CRP-S sites upstream of both genes. Deletion of these motifs significantly impaired natural transformability. Moreover, site-directed mutagenesis of these sites resulted in altered gene expression. This altered gene expression also correlated directly with protein levels, bacterial capacity for DNA uptake, and natural transformability.ConclusionsBased on the data provided in this study we suggest that the identified sites are important for the expression of the competence genes qstR and comEA and therefore for natural transformability of V. cholerae even though the motifs might not reflect bona fide CRP-S sites.

BioMed Central2014

A transcriptional regulator linking quorum sensing and chitin induction to render Vibrio cholerae naturally transformable

Melanie Blokesch, Mirella Lo Scrudato

The human pathogen Vibrio cholerae is an aquatic bacterium associated with zooplankton and their chitinous exoskeletons. On chitinous surfaces, V. cholerae initiates a developmental programme, known as natural competence, to mediate transformation, which is a mode of horizontal gene transfer. Competence facilitates the uptake of free DNA and recombination into the bacterial genome. Recent studies have indicated that chitin surfaces are required, but not sufficient to induce competence. Two additional regulatory pathways, i.e. catabolite repression and quorum sensing (QS), are components of the regulatory network that controls natural competence in V. cholerae. In this study, we investigated the link between chitin induction and QS. We show that the major regulators of these two pathways, TfoX and HapR, are both involved in the activation of a gene encoding a transcriptional regulator of the LuxR-type family, which we named QS and TfoX-dependent regulator (QstR). We demonstrate that HapR binds the promoter of qstR in a site-specific manner, indicating a role for HapR as an activator of qstR. In addition, epistasis experiments indicate that QstR compensates for the absence of HapR. We also provide evidence that QstR is required for the proper expression of a small but essential subset of competence genes and propose a new regulatory model in which QstR links chitin-induced TfoX activity with QS.

Oxford University Press2013

Mechanistic aspects of DNA uptake in naturally competent Vibrio cholerae

Patrick Martin Seitz

EPFL2014