A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Laurenceau, Raphaël; Péhau‐Arnaudet, Gérard; Baconnais, Sonia; Gault, Joseph; Malosse, Christian; Dujeancourt, Annick; Campo, Nathalie; Chamot‐Rooke, Julia; Cam, Eric Le; Claverys, Jean‐Pierre; Fronzes, Rémi

doi:10.1371/journal.ppat.1003473

Cited by 148 publications

(224 citation statements)

References 44 publications

(61 reference statements)

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“…4). The model combines data from this work with those of studies of other transformable bacteria and their Tfp and presents evidence for several important mechanisms that have not been previously addressed experimentally (6)(7)(8)(25)(26)(27)(28)(29)(30)(31). In the center of the model is a Tfp appendage with PilA as the major pilin subunit (as demonstrated in Fig.…”

Section: Discussionmentioning

confidence: 75%

DNA-uptake machinery of naturally competent Vibrio cholerae

Seitz

Blokesch

2013

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

157

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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Section: Discussionmentioning

confidence: 75%

DNA-uptake machinery of naturally competent Vibrio cholerae

Seitz

Blokesch

2013

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

157

247

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“…To improve the reliability of the complementation experiments, we used two independently derived clones for each target strain. We initially complemented TH880 with the plasmid containing comGC (pTH5053) because it carried a single nucleotide insertion in comGC, a late com gene encoding the major subunit of the transformation pilus (59). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

et al. 2016

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h Natural genetic transformation of Streptococcus pneumoniae, an important human pathogen, mediates horizontal gene transfer for the development of drug resistance, modulation of carriage and virulence traits, and evasion of host immunity. Transformation frequency differs greatly among pneumococcal clinical isolates, but the molecular basis and biological importance of this interstrain variability remain unclear. In this study, we characterized the transformation frequency and other associated phenotypes of 208 S. pneumoniae clinical isolates representing at least 30 serotypes. While the vast majority of these isolates (94.7%) were transformable, the transformation frequency differed by up to 5 orders of magnitude between the least and most transformable isolates. The strain-to-strain differences in transformation frequency were observed among many isolates producing the same capsule types, indicating no general association between transformation frequency and serotype. However, a statistically significant association was observed between the levels of transformation and colonization fitness/virulence in the hypertransformable isolates. Although nontransformable mutants of all the selected hypertransformable isolates were significantly attenuated in colonization fitness and virulence in mouse infection models, such mutants of the strains with relatively low transformability had no or marginal fitness phenotypes under the same experimental settings. This finding strongly suggests that the pneumococci with high transformation capability are "addicted" to a "hypertransformable" state for optimal fitness in the human host. This work has thus provided an intriguing hint for further investigation into how the competence system impacts the fitness, virulence, and other transformation-associated traits of this important human pathogen. Streptococcus pneumoniae (the pneumococcus) is a commensal in the upper airway of humans and an opportunistic pathogen for numerous infections, including pneumonia, otitis media, and meningitis (1). Genetic plasticity of S. pneumoniae mediated by horizontal gene transfer is a primary driving force behind the success of this pathogen in its adaptation to the increasingly hostile environment in humans, the only known natural host (2). S. pneumoniae is capable of horizontal gene transfer via transduction, transformation, conjugation, and specialized transduction (or pseudotransduction) (3). Natural genetic transformation, referred to as natural transformation herein, alters the bacterial genome via uptake of foreign DNA and subsequent integration of new sequences into recipient genomes by homologous recombination (4). Although natural transformation appears to be the sole mechanism of transferring chromosomal DNA in S. pneumoniae, conjugation mobilizes the transfer of integrative and conjugative elements (ICEs) between pneumococcal cells or between S. pneumoniae and other Gram-positive bacteria (3).Natural transformation has been attributed to the acquisition of exogenous genes for immune ev...

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“…First, the T4P "fishes" for DNA at the extracellular side and by retraction through depolymerization, it takes the DNA along into the periplasm (5,6,43). Extended competence-associated T4P polymers have been found in Streptococcus pneumoniae and Vibrio cholerae but not in B. subtilis (27,44,45). A second scenario assumes the formation of an alternative DNAuptake complex by T4P proteins.…”

Section: Discussionmentioning

confidence: 99%

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

Hepp

Maier

2016

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

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Horizontal gene transfer can speed up adaptive evolution and support chromosomal DNA repair. A particularly widespread mechanism of gene transfer is transformation. The initial step to transformation, namely the uptake of DNA from the environment, is supported by the type IV pilus system in most species. However, the molecular mechanism of DNA uptake remains elusive. Here, we used singlemolecule techniques for characterizing the force-dependent velocity of DNA uptake by Neisseria gonorrhoeae. We found that the DNA uptake velocity depends on the concentration of the periplasmic DNA-binding protein ComE, indicating that ComE is directly involved in the uptake process. The velocity-force relation of DNA uptake is in very good agreement with a translocation ratchet model where binding of chaperones in the periplasm biases DNA diffusion through a membrane pore in the direction of uptake. The model yields a speed of DNA uptake of 900 bp·s −1 and a reversal force of 17 pN. Moreover, by comparing the velocityforce relation of DNA uptake and type IV pilus retraction, we can exclude pilus retraction as a mechanism for DNA uptake. In conclusion, our data strongly support the model of a translocation ratchet with ComE acting as a ratcheting chaperone.

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A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Cited by 148 publications

References 44 publications

DNA-uptake machinery of naturally competent Vibrio cholerae

DNA-uptake machinery of naturally competent Vibrio cholerae

Addiction of Hypertransformable Pneumococcal Isolates to Natural Transformation for In Vivo Fitness and Virulence

Kinetics of DNA uptake during transformation provide evidence for a translocation ratchet mechanism

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