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.

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.

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3′ UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.