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

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 Q

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

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

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

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

Molecular keys to the Janthinobacterium and Duganella spp. Interaction with the plant pathogen Fusarium graminearum

Janthinobacterium and Duganella are well-known for their antifungal effects. Surprisingly, almost nothing is known on molecular aspects involved in the close bacterium-fungus interaction. To better understand this interaction, we established the genomes of 11 Janthinobacterium and Duganella isolates in combination with phylogenetic and functional analyses of all publicly available genomes. Thereby, we identified a core and pan genome of 1058 and 23,628 genes. All strains encoded secondary metabolite gene clusters and chitinases, both possibly involved in fungal growth suppression. All but one strain carried a single gene cluster involved in the biosynthesis of alpha-hydroxyketone-like autoinducer molecules, designated JAI-1. Genome-wide RNA-seq studies employing the background of two isolates and the corresponding JAI-1 deficient strains identified a set of 45 QS-regulated genes in both isolates. Most regulated genes are characterized by a conserved sequence motif within the promoter region. Among the most strongly regulated genes were secondary metabolite and type VI secretion system gene clusters. Most intriguing, co-incubation studies of J. sp. HH102 or its corresponding JAI-1 synthase deletion mutant with the plant pathogen Fusarium graminearum provided first evidence of a QS-dependent interaction with this pathogen

The emergence of Vibrio pathogens in Europe: ecology, evolution, and pathogenesis

Global change has caused a worldwide increase in reports of Vibrio-associated diseases with ecosystem-wide impacts on humans and marine animals. In Europe, higher prevalence of human infections followed regional climatic trends with outbreaks occurring during episodes of unusually warm weather. Similar patterns were also observed in Vibrio-associated diseases affecting marine organisms such as fish, bivalves and corals. Basic knowledge is still lacking on the ecology and evolutionary biology of these bacteria as well as on their virulence mechanisms. Current limitations in experimental systems to study infection and the lack of diagnostic tools still prevent a better understanding of Vibrio emergence. A major challenge is to foster cooperation between fundamental and applied research in order to investigate the consequences of pathogen emergence in natural Vibrio populations and answer federative questions that meet societal needs. Here we report the proceedings of the first European workshop dedicated to these specific goals of the Vibrio research community by connecting current knowledge to societal issues related to ocean health and food security

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

Glucose- but Not Rice-Based Oral Rehydration Therapy Enhances the Production of Virulence Determinants in the Human Pathogen Vibrio cholerae

Despite major attempts to prevent cholera transmission, millions of people worldwide still must address this devastating disease. Cholera research has so far mainly focused on the causative agent, the bacterium Vibrio cholerae, or on disease treatment, but rarely were results from both fields interconnected. Indeed, the treatment of this severe diarrheal disease is mostly accomplished by oral rehydration therapy (ORT), whereby water and electrolytes are replenished. Commonly distributed oral rehydration salts also contain glucose. Here, we analyzed the effects of glucose and alternative carbon sources on the production of virulence determinants in the causative agent of cholera, the bacterium Vibrio cholerae during in vitro experimentation. We demonstrate that virulence gene expression and the production of cholera toxin are enhanced in the presence of glucose or similarly transported sugars in a ToxR-, TcpP- and ToxT-dependent manner. The virulence genes were significantly less expressed if alternative non-PTS carbon sources, including rice-based starch, were utilized. Notably, even though glucose-based ORT is commonly used, field studies indicated that rice-based ORT performs better. We therefore used a spatially explicit epidemiological model to demonstrate that the better performing rice-based ORT could have a significant impact on epidemic progression based on the recent outbreak of cholera in Haiti. Our results strongly support a change of carbon source for the treatment of cholera, especially in epidemic settings

Correlation among HapR protein levels, <i>hapA</i> gene expression, and the nuclease Dns.

<p>Panel A and C: Proteins of the indicated strains, each containing artificially inducible <i>tfoX</i> on the chromosome, were separated by SDS-PAGE. After blotting, the relative abundance of proteins HapR (panel A) or Dns (panel C) were determined by detection with protein-specific antibodies. For each sample 6 µg (panel A) and 12 µg total protein (panel C), respectively, were applied per lane. Strains were tested under non-competence-inducing and competence-inducing conditions as indicated above each image. Panel B: HapR-dependent expression of <i>hapA</i> promoter-driven gene expression was quantified as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002778#pgen-1002778-g003" target="_blank">Figure 3</a> for <i>comEA</i>/<i>pilA</i>. The growth conditions were as described for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002778#pgen-1002778-g004" target="_blank">Figure 4</a>. Averages of three independent experiments are indicated. Error bars indicate standard deviations. Statistically significant differences were calculated using the Student's <i>t</i> test. * <i>P</i><0.05, ** <i>P</i><0.01, <i>n.s.</i> = not significant.</p