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Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

ABSTRACT: Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

SUBMITTER: Fang G

PROVIDER: S-EPMC3879109 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Fang Gang G Munera Diana D Friedman David I DI Mandlik Anjali A Chao Michael C MC Chao Michael C MC Banerjee Onureena O Feng Zhixing Z Losic Bojan B Mahajan Milind C MC Jabado Omar J OJ Deikus Gintaras G Clark Tyson A TA Luong Khai K Murray Iain A IA Davis Brigid M BM Keren-Paz Alona A Chess Andrew A Roberts Richard J RJ Korlach Jonas J Turner Steve W SW Kumar Vipin V Waldor Matthew K MK Schadt Eric E EE

Nature biotechnology 20121108 12

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at ea ...[more]

PMID: 23138224

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Project description:BACKGROUND: Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain chi7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. RESULTS: Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain chi7122. CONCLUSION: Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.

Dataset Information

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Publications

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Similar Datasets

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