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. Author Summary The human pathogen Vibrio cholerae is an aquatic bacterium often encountered in rivers, estuaries, and coastal regions. Within this environmental niche, the bacterium often associates with the chitinous exoskeleton of zooplankton. Upon colonization of these chitinous surfaces, V. cholerae switches on a developmental program known as natural competence for genetic transformation. Natural competence for transformation is a mode of horizontal gene transfer that allows bacteria to acquire new genes derived from free DNA, which is released by other members within the same habitat. The evolutionary consequences could be that the bacterial recipient becomes better adapted to its environmental niche or, in a worst-case scenario, more pathogenic for man. The results of this study show that, under optimal conditions, the majority of cells within a V. cholerae population express competence genes. However, in an aquatic environment, a combination of different ecological factors might lead to heterogeneity in the competence phenotype. Therefore, we investigated the role of extracellular and intracellular signaling molecules with respect to competence induction. This report illustrates that at least three interconnected signaling cascades are required for competence induction, which are based on bacterial metabolism and group behavior.
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