Melanie Blokesch

Chitin Induces Natural Competence in Vibrio cholerae

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.

Maturation of Hydrogenases

Enzymes possessing the capacity to oxidize molecular hydrogen have developed convergently three class of enzymes leading to: [FeFe]-, [NiFe]-, and [FeS]-cluster-free hydrogenases. They differ in the composition and the structure of the active site metal centre and the sequence of the constituent structural polypeptides but they show one unifying feature, namely the existence of CN and/or CO ligands at the active site Fe. Recent developments in the analysis of the maturation of [FeFe]- and [NiFe]- hydrogenases have revealed a remarkably complex pattern of mostly novel biochemical reactions. Maturation of [FeFe]-hydrogenases requires a minimum of three auxiliary proteins, two of which belong to the class of Radical-SAM enzymes and other to the family of GTPases. They are sufficient to generate active enzyme when their genes are co-expressed with the structural genes in a heterologous host, otherwise deficient in [FeFe]-hydrogenase expression. Maturation of the large subunit of [NiFe]-hydrogenases depends on the activity of at least seven core proteins that catalyse the synthesis of the CN ligand, have a function in the coordination of the active site iron, the insertion of nickel and the proteolytic maturation of the large subunit. Whereas this core maturation machinery is sufficient to generate active hydrogenase in the cytoplasm, like that of hydrogenase 3 from Escherichia coli, additional proteins are involved in the export of the ready-assembled heterodimeric enzyme to the periplasm via the twin-arginine translocation system in the case of membrane-bound hydrogenases. A series of other gene products with intriguing putative functions indicate that the minimal pathway established for E. coli [NiFe]-hydrogenase maturation may possess even higher complexity in other organisms.

The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer

Killing, sex, and gene swaps in bacteria The bacterial type VI secretion system (T6SS) is used by bacteria to inject toxins into neighboring cells to eliminate competition. This molecular machine is thus considered to be a mechanism by which bacteria can exert social control in complex microbial communities. Borgeaud et al. have discovered that in Vibrio cholerae, T6SS genes are co-regulated with genes involved in DNA uptake. Hence, T6SS-dependent killing of other bacteria is directed at neighboring cells, which release their DNA to be taken up by the killer, which can then integrate valuable genes and rapidly evolve, leading to antibiotic resistance or virulence. Science, this issue p. 63 A water-borne pathogen seeks out and captures its neighbors’ genes. Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae.

Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria

Bacterial genomics is flourishing, as whole-genome sequencing has become affordable, readily available and rapid. As a result, it has become clear how frequently horizontal gene transfer (HGT) occurs in bacteria. The potential implications are highly significant because HGT contributes to several processes, including the spread of antibiotic-resistance cassettes, the distribution of toxin-encoding phages and the transfer of pathogenicity islands. Three modes of HGT are recognized in bacteria: conjugation, transduction and natural transformation. In contrast to the first two mechanisms, natural competence for transformation does not rely on mobile genetic elements but is driven solely by a developmental programme in the acceptor bacterium. Once the bacterium becomes competent, it is able to take up DNA from the environment and to incorporate the newly acquired DNA into its own chromosome. The initiation and duration of competence differ significantly among bacteria. In this review, we outline the latest data on representative naturally transformable Gram-negative bacteria and how their competence windows differ. We also summarize how environmental cues contribute to the initiation of competence in a subset of naturally transformable Gram-negative bacteria and how the complexity of the niche might dictate the fine-tuning of the competence window.

Reassessment of the 2010–2011 Haiti cholera outbreak and rainfall-driven multiseason projections

Mathematical models can provide key insights into the course of an ongoing epidemic, potentially aiding real-time emergency management in allocating health care resources and by anticipating the impact of alternative interventions. We study the ex post reliability of predictions of the 2010–2011 Haiti cholera outbreak from four independent modeling studies that appeared almost simultaneously during the unfolding epidemic. We consider the impact of different approaches to the modeling of spatial spread of Vibrio cholerae and mechanisms of cholera transmission, accounting for the dynamics of susceptible and infected individuals within different local human communities. To explain resurgences of the epidemic, we go on to include waning immunity and a mechanism explicitly accounting for rainfall as a driver of enhanced disease transmission. The formal comparative analysis is carried out via the Akaike information criterion (AIC) to measure the added information provided by each process modeled, discounting for the added parameters. A generalized model for Haitian epidemic cholera and the related uncertainty is thus proposed and applied to the year-long dataset of reported cases now available. The model allows us to draw predictions on longer-term epidemic cholera in Haiti from multiseason Monte Carlo runs, carried out up to January 2014 by using suitable rainfall fields forecasts. Lessons learned and open issues are discussed and placed in perspective. We conclude that, despite differences in methods that can be tested through model-guided field validation, mathematical modeling of large-scale outbreaks emerges as an essential component of future cholera epidemic control.

Network of Hydrogenase Maturation in Escherichia coli: Role of Accessory Proteins HypA and HybF

We have studied the roles of the auxiliary protein HypA and of its homolog HybF in hydrogenase maturation. A mutation in hypA leads to the nearly complete blockade of maturation solely of hydrogenase 3 whereas a lesion in hybF drastically but not totally reduces maturation and activity of isoenzymes 1 and 2. The residual level of matured enzymes in the hybF mutant was shown to be due to the function of HypA; HybF, conversely, was responsible for a minimal residual activity of hydrogenase 3 in the mutant hypA strain. Accordingly, a hypA DeltahybF double mutant was completely blocked in the maturation process. However, the inclusion of high nickel concentrations in the medium could restore limited activity of all three hydrogenases. The results of this study and of previous work (M. Blokesch, A. Magalon, and A. Böck, J. Bacteriol. 189:2817-2822, 2001) show that the maturation of the three functional hydrogenases from Escherichia coli is intimately connected via the activity of proteins HypA and HypC and of their homologs HybF and HybG, respectively. The results also support the suggestion of Olson et al. (J. W. Olson, N. S. Mehta, and R. J. Maier, Mol. Microbiol. 39:176-182, 2001) that HypA cooperates with HypB in the insertion of nickel into the precursor of the large hydrogenase subunit. Whereas HypA is predominantly involved in the maturation of hydrogenase 3, HybF takes over its function in the maturation of isoenzymes 1 and 2.

Maturation of [NiFe]-hydrogenases in Escherichia coli: The HypC Cycle

Carbamoyl phosphate (CP) has been implicated as an educt for the synthesis of the CO and CN ligands of the metal centre of [NiFe]-hydrogenases in Escherichia coli, since CP synthetase mutants (carAB) are unable to generate active hydrogenases due to a block in enzyme maturation. Citrulline, when added to the growth medium in high concentrations, compensated for the phenotype of the mutants. It is now shown that overexpression of the argI gene lowered the effective concentration of citrulline, thus proving that the amino acid serves as a source for CP. The DeltaCarAB mutant accumulated a complex consisting of the hydrogenase maturation proteins HypC and HypD. This complex was resolved upon citrulline addition and followed-up by the appearance of a complex between HypC and the precursor of the large subunit of hydrogenase 3, preHycE. In the absence of the hycE gene, the HypC-HypD complex did not disappear upon addition of citrulline but developed into a form migrating slower in a non-denaturing polyacrylamide gel, providing strong evidence for the notion that the HypC-HypD complex is the intermediate in hydrogenase maturation where CP or its products are added to the iron atom of the metal centre. This step precedes nickel insertion, since extracts of carAB cells that had been cultivated in the absence of citrulline are unable to process preHycE after the addition of nickel. Complex formation between HypC and HypD, and between HypC and preHycE display dependence on identical primary structure elements of HypC. On the basis of the results, a cycle of HypC activity is proposed whose function is to transfer the iron atom that has been liganded at the HypC-HypD complex to the precursor of the large hydrogenase subunit.

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.

Prediction of the spatial evolution and effects of control measures for the unfolding Haiti cholera outbreak

Here we propose spatially explicit predictions of the residual progression of the current Haiti cholera outbreak accounting for the dynamics of susceptible and infected individuals within different local human communities, and for the redistribution among them of Vibrio cholerae, the causative agent of the disease. Spreading mechanisms include the diffusion of pathogens in the aquatic environment and their dissemination due to the movement of human carriers. The model reproduces the spatiotemporal features of the outbreak to date, thus suggesting the robustness of predicted future developments of the epidemic. We estimate that, under unchanged conditions, the number of new cases in the whole country should start to decrease in January. During this month the epidemic should mainly involve the Ouest department (Port-au-Prince) while fading out in northern regions. Our spatially explicit model allows also the analysis of the effectiveness of alternative intervention strategies. To that end our results show that mass vaccinations would have a negligible impact at this stage of the epidemic. We also show that targeted sanitation strategies, providing clean drinking water supply and/or staging educational campaigns aimed at reducing exposure, may weaken the strength of the residual evolution of the infection. Citation: Bertuzzo, E., L. Mari, L. Righetto, M. Gatto, R. Casagrandi, M. Blokesch, I. RodriguezIturbe, and A. Rinaldo (2011), Prediction of the spatial evolution and effects of control measures for the unfolding Haiti cholera outbreak, Geophys. Res. Lett., 38, L06403, doi: 10.1029/2011GL046823.

DNA-uptake machinery of naturally competent Vibrio cholerae

Significance Transformation allows naturally competent bacteria to take up DNA from the environment and integrate the DNA into the chromosome by recombination. In Gram-negative bacteria, the DNA-uptake machinery shuttles the incoming DNA across the outer membrane, the periplasmic space, and the inner membrane. This study investigates the DNA-uptake complex of the human pathogen Vibrio cholerae, using a cellular biology-based approach. We visualized different components of this multicomponent complex, including a type IV pilus appendage, determined their (co)localization within the bacterial cell, and conducted an analysis of competence-gene mutants. We conclude that the uptake of DNA occurs via (at least) a two-step process. Natural competence for transformation is a mode of horizontal gene transfer that is commonly used by bacteria to take up DNA from their environment. As part of this developmental program, so-called competence genes, which encode the components of a DNA-uptake machinery, are expressed. Several models have been proposed for the DNA-uptake complexes of competent bacteria, and most include a type IV (pseudo)pilus as a core component. However, cell-biology–based approaches to visualizing competence proteins have so far been restricted to Gram-positive bacteria. Here, we report the visualization of a competence-induced pilus in the Gram-negative bacterium Vibrio cholerae. We show that piliated cells mostly contain a single pilus that is not biased toward a polar localization and that this pilus colocalizes with the outer membrane secretin PilQ. PilQ, on the other hand, forms several foci around the cell and occasionally colocalizes with the dynamic cytoplasmic-traffic ATPase PilB, which is required for pilus extension. We also determined the minimum competence regulon of V. cholerae, which includes at least 19 genes. Bacteria with mutations in those genes were characterized with respect to the presence of surface-exposed pili, DNA uptake, and natural transformability. Based on these phenotypes, we propose that DNA uptake in naturally competent V. cholerae cells occurs in at least two steps: a pilus-dependent translocation of the incoming DNA across the outer membrane and a pilus-independent shuttling of the DNA through the periplasm and into the cytoplasm.