Directed polar secretion of protease from single cells of
            <i>Vibrio cholerae</i>
            via the type II secretion pathway

Scott, Maria E.; Dossani, Zain Y.; Sandkvist, Maria

doi:10.1073/pnas.241411198

Cited by 83 publications

(90 citation statements)

References 48 publications

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“…Previous studies with the Vibrio cholerae PulG homolog EpsG indicated that it clusters at the cell pole, consistent with the facts that type II secretion occurs at the pole in this bacterium and that other secreton components also accumulate there (53). In contrast, GFP-PrePulG and PulG-mRFP were evenly distributed throughout the E. coli inner membrane, consistent with previous data showing similar patterns of GFP-PulM and GFP-PulL distribution (10).…”

Section: Discussionsupporting

confidence: 89%

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

et al. 2007

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The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The ␤-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

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Section: Discussionsupporting

confidence: 89%

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

et al. 2007

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“…Interestingly, a recent study on the type II secretion system of V. cholerae also showed discrete fluorescent foci along the cell periphery for different components of this macromolecular complex (37). This finding is in contrast to the polar localization pattern described earlier (22), which most likely resulted from an overexpression artifact (37). Indeed, Lybarger et al concluded that "chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins" (ref.…”

Section: Discussionmentioning

confidence: 88%

“…we could show that the pilus was able to localize to either of the two poles (e.g., the old or the new pole) (22) (Fig. S3).…”

Section: Construction Of Competence-gene Mutants and Assessment Of Theirmentioning

confidence: 98%

DNA-uptake machinery of naturally competent Vibrio cholerae

Seitz

Blokesch

2013

Proc. Natl. Acad. Sci. U.S.A.

158

247

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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.N atural competence for genetic transformation is one of three modes of horizontal gene transfer (HGT) in prokaryotes and is often tightly regulated (1-3). Large pieces of DNA containing a series of genes can be transferred by natural transformation without the need for direct interaction with other microbes or mobile genetic elements. This process can foster rapid evolution, and HGT is known to be involved in the spread of antibiotic resistance, adaptation to new environmental niches, and the emergence of new pathogens.Many bacterial species are able to enter a state of natural competence, including the human pathogen Vibrio cholerae. In this bacterium, competence is induced upon growth on chitinous surfaces (3, 4), the natural habitat of V. cholerae (5). Although we have gained a reasonably clear understanding of the regulatory network driving competence induction in this organism (for a review, see ref.3), almost nothing is known about its DNAuptake machinery. Indeed, the sophisticated DNA-uptake complexes used by naturally competent bacteria during transformation are still poorly characterized (6), especially in Gram-negative bacteria in which the transforming DNA (tDNA) must cross two membranes and the periplasmic space (including the peptidoglycan layer) to enter the cytoplasm and recombine with the chromosome (the latter step is not required if the tDNA consists of plasmid DNA). Interestingly, the majority of competence-protein localization studies using cellu...

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“…This observation leads to a particularly attractive model for the role of the PilA-containing ChiRP in the V. cholerae chitin utilization program that could explain why type IV pilus assembly complex genes reside in the same regulon with genes that encode chitin catabolic functions. This model incorporates the fact that the eps type II chitinase secretion apparatus and most type IV pilus assembly complexes localize to the cell pole (24). In view of these topographical features, the model predicts that the assembly complex for the ChiRP localizes to the same pole as the the eps type II secretion apparatus.…”

Section: Discussionmentioning

confidence: 99%

The Vibrio cholerae chitin utilization program

Meibom

Nielsen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

492

663

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Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms. Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc) 2-6, GlcNAc, or the glucosamine dimer (GlcN) 2 identified three sets of differentially regulated genes. We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc) 2-6 gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V. cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN) 2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry. Collectively, these results provide a global portrait of a complex, multistage V. cholerae program for the efficient utilization of chitin.T he agent of Asiatic cholera, Vibrio cholerae O1, causes a dehydrating diarrheal illness and sometimes death. However, outside the human host, V. cholerae is a normal member of natural aquatic environments such as lakes, rivers, estuaries, and the ocean, which serve as the principal reservoir for this organism in nature (1). How it survives in habitats of this kind and the mechanisms by which it periodically emerges as a human pathogen are compelling questions in the ecology of infectious diseases (2). Observational studies in Bangladesh epidemiologically link seasonal phytoplankton and zooplankton blooms with cholera outbreaks. Studies of this epidemiological association identified V. cholerae attached to plankton in the environment, an observation that was confirmed by microcosm coinfection experiments. The interaction of V. cholerae with zooplankton is particularly intriguing because copepods, a subclass of crustacean zooplankton, have been incriminated in the transmission of this agent from aquatic reservoirs to susceptible human hosts (3).Chitin, composed of ␤1,4-linked GlcNAc residues, is the most abundant polysaccharide in nature after cellulose. In the aquatic biosphere alone, Ͼ10 11 metric tons of chitin are produced annually. This vast amount of insoluble, carbon-containing material is recycled mainly by chitinolytic bacteria, including members of the family Vibrionaceae. Chitin is found throughout all kingdoms and is the main component of the cell walls of fungi and of the exoskeletons of crustaceans. Copepods are estimated to produce billions of tons of chitin each year (4).Many Vibrio species that live in aquatic environments are capable of using chitin as the sole carbon source. Studies of the nonpathogenic marine organism Vibrio furnissii have shown that chitin utili...

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Directed polar secretion of protease from single cells of Vibrio cholerae via the type II secretion pathway

Cited by 83 publications

References 48 publications

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

DNA-uptake machinery of naturally competent Vibrio cholerae

The Vibrio cholerae chitin utilization program

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