Dataset Information

Characterization of undermethylated sites in Vibrio cholerae.

ABSTRACT: The activities of DNA methyltransferases are important for a variety of cellular functions in bacteria. In this study, we developed a modified high-throughput technique called methyl homopolymer tail mediated sequencing (methyl HTM-seq) to identify the undermethylated sites in the Vibrio cholerae genome for the two DNA methyltransferases, Dam, an adenine methyltransferase, and VchM, a cytosine methyltransferase, during growth in rich medium in vitro. Many of the undermethylated sites occurred in intergenic regions, and for most of these sites, we identified the transcription factors responsible for undermethylation. This confirmed the presence of previously hypothesized DNA-protein interactions for these transcription factors and provided insight into the biological state of these cells during growth in vitro. DNA adenine methylation has previously been shown to mediate heritable epigenetic switches in gene regulation. However, none of the undermethylated Dam sites tested showed evidence of regulation by this mechanism. This study is the first to identify undermethylated adenines and cytosines genomewide in a bacterium using second-generation sequencing technology.

SUBMITTER: Dalia AB

PROVIDER: S-EPMC3650525 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Characterization of undermethylated sites in Vibrio cholerae.

Dalia Ankur B AB Lazinski David W DW Camilli Andrew A

Journal of bacteriology 20130315 10

The activities of DNA methyltransferases are important for a variety of cellular functions in bacteria. In this study, we developed a modified high-throughput technique called methyl homopolymer tail mediated sequencing (methyl HTM-seq) to identify the undermethylated sites in the Vibrio cholerae genome for the two DNA methyltransferases, Dam, an adenine methyltransferase, and VchM, a cytosine methyltransferase, during growth in rich medium in vitro. Many of the undermethylated sites occurred in ...[more]

PMID: 23504020

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Project description:UnlabelledThe ability to form biofilms is critical for environmental survival and transmission of Vibrio cholerae, a facultative human pathogen responsible for the disease cholera. Biofilm formation is controlled by several transcriptional regulators and alternative sigma factors. In this study, we report that the two main positive regulators of biofilm formation, VpsR and VpsT, bind to nonoverlapping target sequences in the regulatory region of vpsL in vitro. VpsR binds to a proximal site (the R1 box) as well as a distal site (the R2 box) with respect to the transcriptional start site identified upstream of vpsL. The VpsT binding site (the T box) is located between the R1 and R2 boxes. While mutations in the T and R boxes resulted in a decrease in vpsL expression, deletion of the T and R2 boxes resulted in an increase in vpsL expression. Analysis of the role of H-NS in vpsL expression revealed that deletion of hns resulted in enhanced vpsL expression. The level of vpsL expression was higher in an hns vpsT double mutant than in the parental strain but lower than that in an hns mutant. In silico analysis of the regulatory regions of the VpsR and VpsT targets resulted in the identification of conserved recognition motifs for VpsR and VpsT and revealed that operons involved in biofilm formation and vpsT are coregulated by VpsR and VpsT. Furthermore, a comparative genomics analysis revealed substantial variability in the promoter region of the vpsT and vpsL genes among extant V. cholerae isolates, suggesting that regulation of biofilm formation is under active selection.ImportanceVibrio cholerae causes cholera and is a natural inhabitant of aquatic environments. One critical factor that is important for environmental survival and transmission of V. cholerae is the microbe's ability to form biofilms, which are surface-associated communities encased in a matrix composed of the exopolysaccharide VPS (Vibrio polysaccharide), proteins, and nucleic acids. Two proteins, VpsR and VpsT, positively regulate VPS production and biofilm formation. We characterized the structural features of the promoter of the vpsL gene, determined the target sequences recognized by VpsT and VpsR, and analyzed their distribution and conservation patterns in multiple V. cholerae isolates. This work fills a fundamental gap in our understanding of the regulatory mechanisms employed by the master regulators VpsR and VpsT in controlling biofilm matrix production.

Project description:The phage shock protein (Psp) system is a stress response pathway that senses and responds to inner membrane damage. The genetic components of the Psp system are present in several clinically relevant Gram-negative bacteria, including Vibrio cholerae However, most of the current knowledge about the Psp response stems from in vitro studies in Escherichia coli and Yersinia enterocolitica In fact, the Psp response in V. cholerae has remained completely uncharacterized. In this study, we demonstrate that V. cholerae does have a functional Psp response system. We found that overexpression of GspD (EpsD), the type II secretion system secretin, induces the Psp response, whereas other V. cholerae secretins do not. In addition, we have identified several environmental conditions that induce this stress response. Our studies on the genetic regulation and induction of the Psp system in V. cholerae suggest that the key regulatory elements are conserved with those of other Gram-negative bacteria. While a psp null strain is fully capable of colonizing the infant mouse intestine, it exhibits a colonization defect in a zebrafish model, indicating that this response may be important for disease transmission in the environment. Overall, these studies provide an initial understanding of a stress response pathway that has not been previously investigated in V. cholerae IMPORTANCE Vibrio cholerae leads a dual life cycle, as it can exist in the aquatic environment and colonize the human small intestine. In both life cycles, V. cholerae encounters a variety of stressful conditions, including fluctuating pH and temperature and exposure to other agents that may negatively affect cell envelope homeostasis. The phage shock protein (Psp) response is required to sense and respond to such insults in other bacteria but has remained unstudied in V. cholerae Interestingly, the Psp system has protein homologs, principally, PspA, in a number of bacterial clades as well as in archaea and plants. Therefore, our findings not only fill a gap in knowledge about an unstudied extracytoplasmic stress response in V. cholerae, but also may have far-reaching implications.

Dataset Information

Characterization of undermethylated sites in Vibrio cholerae.

Publications

Characterization of undermethylated sites in Vibrio cholerae.

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