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

Contaminants at Arctic Formerly Used Defense Sites

This study was conducted in order to determine if the source of contaminants at formerly used defense sites (FUDS) in Alaska were deposited as a result of military occupancy or from long-distance transport. This determination largely influences whether remediation will occur, and, if so, to what extent. For this reason, plant samples (rinsed and unrinsed) and sediment cores were collected at military and remote sites on St. Lawrence Island (SLI) and Norton Sound, Alaska during the summers of 2002, 2006, and 2007 and analyzed for persistent organic pollutants. On St. Lawrence Island sediment core samples were collected at the Northeast Cape FUDS, also a traditional fishing/hunting camp, and were sectioned and analyzed for concentrations of Polychlorinated biphenyl (PCB) congeners, Mirex, Dichlorodiphenyldichloroethylene (DDE), Hexachlorobenzene (HCB), Mercury and Cesium-137 (137Cs). Differences in the total concentrations and distributions of PCB congeners, Mirex, DDE and Mercury in sediment cores and in plants collected from the two SLI and three Norton Sound mainland formerly used defense sites indicate the majority of the contaminants found can be temporally related to releases during military occupancy and subsequent redistribution of contaminants. Contaminants in plant samples at the SLI sites are elevated relative to the two remote sites located on St. Lawrence Island and the three mainland Norton Sound FUDS at Elim, Unalakleet, and Wales. The concentrations, lateral and vertical distribution of the total PCBs, and congener-specific differences in sediments and plants readily differentiate locally derived from globally transported contaminants. The relative contaminant concentrations in sediment cores and between rinsed and unrinsed plants collected from the NEC FUDS indicate contaminants were remobilized and redistributed during recent site remediation activities

Retraction of DNA-bound type IV competence pili initiates DNA uptake during natural transformation in Vibrio cholerae

Natural transformation is a broadly conserved mechanism of horizontal gene transfer in bacterial species that can shape evolution and foster the spread of antibiotic resistance determinants, promote antigenic variation, and lead to the acquisition of novel virulence factors. Surface appendages called competence pili promote DNA uptake during the first step of natural transformation1 , however, their mechanism of action has remained unclear due to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus labeling method, we define the mechanism for type IV competence pilus-mediated DNA uptake during natural transformation. First, we show that type IV competence pili bind to extracellular doublestranded DNA via their tip and demonstrate that this binding is critical for DNA uptake. Next, we show that type IV competence pili are dynamic structures and that pilus retraction brings tip-bound DNA to the cell surface. Finally, we show that pilus retraction is spatiotemporally coupled to DNA internalization and that sterically obstructing pilus retraction prevents DNA uptake. Together, these results indicate that type IV competence pili directly bind DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfe