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

Nature Biotechnology volume 30, pages 1232–1239 (2012)Cite this article

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A Corrigendum to this article was published on 10 June 2013

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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.

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Figure 1: Extensive kinetic variation detected in C227-11.
Figure 2: Identification and annotation of the MTases targeting the different sequence motifs in the C227-11 genome.
Figure 3: Φ104 encodes a functional restriction-modification system.
Figure 4: The RM system associated with M.

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Change history

  • 03 May 2013

    In the version of this article initially published, on p. 1235, line 32, the wrong MTases were given for the motif GATC. Instead of “…GATC (for M.EcoGI and M.EcoGII)…,” it should have read, “…GATC (for M.EcoGV and M.EcoGVII)….” The error has been corrected for the PDF and HTML versions of this article.

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Acknowledgements

This study was supported in part by a US National Science Foundation grant IIS0916439 (G.F. and V.K.) and NIH R37 AI-42347 and HHMI (M.K.W.).

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Author notes

  1. Gang Fang and Diana Munera: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Gang Fang & Vipin Kumar

  2. Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Diana Munera, Anjali Mandlik, Michael C Chao, Brigid M Davis & Matthew K Waldor

  3. Howard Hughes Medical Institute, Boston, Massachusetts, USA

    Diana Munera, Anjali Mandlik, Michael C Chao, Brigid M Davis & Matthew K Waldor

  4. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA

    David I Friedman

  5. Pacific Biosciences, Menlo Park, California, USA

    Onureena Banerjee, Tyson A Clark, Khai Luong, Jonas Korlach & Steve W Turner

  6. Department of Statistics, Stanford University, Stanford, California, USA

    Zhixing Feng

  7. Tsinghua National Laboratory for Information Science and Technology, Tsinghua University, Beijing, China

    Zhixing Feng

  8. Department of Automation, Tsinghua University, Beijing, China

    Zhixing Feng

  9. Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, New York, USA

    Bojan Losic, Milind C Mahajan, Omar J Jabado, Gintaras Deikus, Alona Keren-Paz, Andrew Chess & Eric E Schadt

  10. New England Biolabs, Inc., Ipswich, Massachusetts, USA

    Iain A Murray & Richard J Roberts

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Contributions

G.F., D.M., M.K.W. and E.E.S. designed the experiments; G.F., D.M., D.I.F., A.M., M.C.C., O.B., Z.F., I.A.M., A.K.-P., A.C., R.J.R., J.K., S.W.T., V.K., M.K.W. and E.E.S. designed the methods; D.M., D.I.F., A.M., M.C.C., M.C.M., O.J.J., G.D., T.A.C., K.L., I.A.M., A.K.-P. and A.C. carried out all sample-preparation experiments, all sequencing runs and all validation experiments; G.F., D.M., D.I.F., A.M., M.C.C., O.B., Z.F., B.L., I.A.M., B.M.D., A.K.-P., A.C., R.J.R., V.K., M.K.W. and E.E.S. jointly analyzed the data sets; and G.F., D.M., D.I.F., A.M., M.C.C., M.C.M., O.J.J., T.A.C., B.M.D., A.K.-P., A.C., R.J.R., J.K., M.K.W. and E.E.S. wrote the manuscript.

Corresponding authors

Correspondence to Matthew K Waldor or Eric E Schadt.

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Competing interests

O.B., T.A.C., K.L., J.K., S.W.T. and E.E.S. are employees or consultants for and own stock in Pacific Biosciences.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–2 and Supplementary Figures 1–15 (PDF 1923 kb)

Supplementary Table 3

Genes annotated in the C227-11 genome (PDF 836 kb)

Supplementary Table 4

C227-11 versus C227-11-delRM gene expression signature (PDF 552 kb)

Supplementary Table 5

Pathway enrichments for signature genes for different comparisons between strains that contain the RM.EcoGIII system and strains that do not contain this system (PDF 198 kb)

Supplementary Table 6

C227-11 versus 55989 gene expression signature (PDF 637 kb)

Supplementary Table 7

K12/K37 versus k12+Stx104 phage gene expression signature (PDF 507 kb)

Supplementary Table 8

Genes from Supplementary Table 4 ordered in the way in which they occur in the heatmap in Figure 4b of the main text (PDF 94 kb)

Supplementary Table 9

Synthetic oligonucleotides used for PCR amplification and subcloning of E.coli KV methyltransferase genes (PDF 44 kb)

Supplementary Tables 3, 4, 6, 7 and 8 (XLSX 6398 kb)

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Fang, G., Munera, D., Friedman, D. et al. Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing. Nat Biotechnol 30, 1232–1239 (2012). https://doi.org/10.1038/nbt.2432

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