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ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells.

ABSTRACT: The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.

SUBMITTER: Seitz P

PROVIDER: S-EPMC3879209 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells.

Seitz Patrick P Pezeshgi Modarres Hassan H Borgeaud Sandrine S Bulushev Roman D RD Steinbock Lorenz J LJ Radenovic Aleksandra A Dal Peraro Matteo M Blokesch Melanie M

PLoS genetics 20140102 1

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Usin ...[more]

PMID: 24391524

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Project description:The physiological state of natural competence for transformation allows certain bacteria to take up free DNA from the environment and to recombine such newly acquired DNA into their chromosomes. However, even though conserved components that are required to undergo natural transformation have been identified in several naturally competent bacteria, our knowledge of the underlying mechanisms of the DNA uptake process remains very limited. To better understand these mechanisms, we investigated the competence-mediated DNA transport in the naturally transformable pathogen Vibrio cholerae. Previously, we used a cell biology-based approach to experimentally address an existing hypothesis, which suggested the competence protein ComEA plays a role in the DNA uptake process across the outer membrane of Gram-negative bacteria. Here, we extended this knowledge by investigating the dynamics of DNA translocation across both membranes. More precisely, we indirectly visualized the transfer of the external DNA from outside the cell into the periplasm followed by the shuttling of the DNA into the cytoplasm. Based on these data, we conclude that for V. cholerae, the DNA translocation across the outer and inner membranes is spatially but not temporally coupled.As a mode of horizontal gene transfer, natural competence for transformation has contributed substantially to the plasticity of genomes and to bacterial evolution. Natural competence is often a tightly regulated process and is induced by diverse environmental cues. This is in contrast to the mechanistic aspects of the DNA translocation event, which are most likely conserved among naturally transformable bacteria. However, the DNA uptake process is still not well understood. We therefore investigated how external DNA reaches the cytosol of the naturally transformable bacterium V. cholerae. More specifically, we provide evidence that the DNA translocation across the membranes is spatially but not temporally coupled. We hypothesize that this model also applies to other competent Gram-negative bacteria and that our study contributes to the general understanding of this important biological process.

Project description:In Vibrio cholerae, high intracellular cyclic di-GMP (c-di-GMP) concentration are associated with a biofilm lifestyle, while low intracellular c-di-GMP concentrations are associated with a motile lifestyle. c-di-GMP also regulates other behaviors, such as acetoin production and type II secretion; however, the extent of phenotypes regulated by c-di-GMP is not fully understood. We recently determined that the sequence upstream of the DNA repair gene encoding 3-methyladenine glycosylase (tag) was positively induced by c-di-GMP, suggesting that this signaling system might impact DNA repair pathways. We identified a DNA region upstream of tag that is required for transcriptional induction by c-di-GMP. We further showed that c-di-GMP induction of tag expression was dependent on the c-di-GMP-dependent biofilm regulators VpsT and VpsR. In vitro binding assays and heterologous host expression studies show that VpsT acts directly at the tag promoter in response to c-di-GMP to induce tag expression. Last, we determined that strains with high c-di-GMP concentrations are more tolerant of the DNA-damaging agent methyl methanesulfonate. Our results indicate that the regulatory network of c-di-GMP in V. cholerae extends beyond biofilm formation and motility to regulate DNA repair through the VpsR/VpsT c-di-GMP-dependent cascade.IMPORTANCEVibrio cholerae is a prominent human pathogen that is currently causing a pandemic outbreak in Haiti, Yemen, and Ethiopia. The second messenger molecule cyclic di-GMP (c-di-GMP) mediates the transitions in V. cholerae between a sessile biofilm-forming state and a motile lifestyle, both of which are important during V. cholerae environmental persistence and human infections. Here, we report that in V. cholerae c-di-GMP also controls DNA repair. We elucidate the regulatory pathway by which c-di-GMP increases DNA repair, allowing this bacterium to tolerate high concentrations of mutagens at high intracellular levels of c-di-GMP. Our work suggests that DNA repair and biofilm formation may be linked in V. cholerae.

Dataset Information

ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells.

Publications

ComEA is essential for the transfer of external DNA into the periplasm in naturally transformable Vibrio cholerae cells.

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