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

ABSTRACT: 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 transformation 1 ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize these structures in live cells. Here, using the model naturally transformable species Vibrio cholerae and a pilus-labelling 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 double-stranded 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 to DNA via their tip and mediate DNA internalization through retraction during this conserved mechanism of horizontal gene transfer.

SUBMITTER: Ellison CK

PROVIDER: S-EPMC6582970 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Ellison Courtney K CK Dalia Triana N TN Vidal Ceballos Alfredo A Wang Joseph Che-Yen JC Biais Nicolas N Brun Yves V YV Dalia Ankur B AB

Nature microbiology 20180611 7

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 transformation <sup>1</sup> ; however, their mechanism of action has remained unclear owing to an absence of methods to visualize t ...[more]

PMID: 29891864

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Project description:Vibrio cholerae is naturally competent when grown on chitin. It is known that expression of the major regulator of competence, TfoX, is controlled by chitin; however, the molecular mechanisms underlying this requirement for chitin have remained unclear. In the present study, we identify and characterize a membrane-bound transcriptional regulator that positively regulates the small RNA (sRNA) TfoR, which posttranscriptionally enhances tfoX translation. We show that this regulation of the tfoR promoter is direct by performing electrophoretic mobility shift assays and by heterologous expression of this system in Escherichia coli. This transcriptional regulator was recently identified independently and was named "TfoS" (S. Yamamoto et al., Mol. Microbiol., in press, doi:10.1111/mmi.12462). Using a constitutively active form of TfoS, we demonstrate that the activity of this regulator is sufficient to promote competence in V. cholerae in the absence of chitin. Also, TfoS contains a large periplasmic domain, which we hypothesized interacts with chitin to regulate TfoS activity. In the heterologous host E. coli, we demonstrate that chitin oligosaccharides are sufficient to activate TfoS activity at the tfoR promoter. Collectively, these data characterize TfoS as a novel chitin-sensing transcriptional regulator that represents the direct link between chitin and natural competence in V. cholerae. IMPORTANCE Naturally competent bacteria can take up exogenous DNA from the environment and integrate it into their genome by homologous recombination. This ability to take up exogenous DNA is shared by diverse bacterial species and serves as a mechanism to acquire new genes to enhance the fitness of the organism. Several members of the family Vibrionaceae become naturally competent when grown on chitin; however, a molecular understanding of how chitin activates competence is lacking. Here, we identify a novel membrane-bound transcriptional regulator that is required for natural transformation in the human pathogen Vibrio cholerae. We demonstrate that this regulator senses chitin oligosaccharides to activate the competence cascade, thus, uncovering the molecular link between chitin and natural competence in this Vibrio species.

Project description:Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 103 transformants μg-1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 103 transformants μg-1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms.IMPORTANCE Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way that this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here, we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.

Dataset Information

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

Publications

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

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