Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Riboswitches in eubacteria sense the second messenger c-di-AMP

Nature Chemical Biology volume 9, pages 834–839 (2013)Cite this article

Subjects

Abstract

Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered bacterial second messenger implicated in the control of cell wall metabolism, osmotic stress responses and sporulation. However, the mechanisms by which c-di-AMP triggers these physiological responses have remained largely unknown. Notably, a candidate riboswitch class called ydaO associates with numerous genes involved in these same processes. Although a representative ydaO motif RNA recently was reported to weakly bind ATP, we report that numerous members of this noncoding RNA class selectively respond to c-di-AMP with subnanomolar affinity. Our findings resolve the mystery regarding the primary ligand for this extremely common riboswitch class and expose a major portion of the super-regulon of genes that are controlled by the widespread bacterial second messenger c-di-AMP.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Binding of c-di-AMP by a ydaO motif RNA.
Figure 2: Ligand binding characteristics of WT and variant 165 ydaO RNAs.
Figure 3: Riboswitch regulation of gene expression by c-di-AMP.
Figure 4: The super-regulon for second messenger signaling through c-di-AMP riboswitches.

Similar content being viewed by others

References

  1. Römling, U. Great times for small molecules: c-di-AMP, a second messenger candidate in bacteria and archaea. Sci. Signal. 1, pe39 (2008).

    Article  PubMed  Google Scholar 

  2. Pesavento, C. & Hengge, R. Bacterial nucleotide-based second messengers. Curr. Opin. Microbiol. 12, 170–176 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Sondermann, H., Shikuma, N.J. & Yildiz, F.H. You've come a long way: c-di-GMP signaling. Curr. Opin. Microbiol. 15, 140–146 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Krasteva, P.V., Giglio, K.M. & Sondermann, H. Sensing the messenger: the diverse ways that bacteria signal through c-di-GMP. Protein Sci. 21, 929–948 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sudarsan, N. et al. Riboswitches in eubacteria sense the second messenger c-di-GMP. Science 321, 411–413 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lee, E.R., Baker, J.L., Weinberg, Z., Sudarsan, N. & Breaker, R.R. An allosteric self-splicing ribozyme triggered by a bacterial second messenger. Science 329, 845–848 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Witte, G., Hartung, S., Buttner, K. & Hopfner, K.P. Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol. Cell 30, 167–178 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Oppenheimer-Shaanan, Y., Wexselblatt, E., Katzhendler, J., Yavin, E. & Ben-Yehuda, S. c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis. EMBO Rep. 12, 594–601 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Corrigan, R.M., Abbott, J.C., Burhenne, H., Kaever, V. & Gründling, A. c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLoS Pathog. 7, e1002217 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Smith, W.M. et al. Heat resistance and salt hypersensitivity in Lactococcus lactis due to spontaneous mutation of limg_1816 (gdpP) induced by high temperature growth. Appl. Environ. Microbiol. 78, 7753–7759 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Corrigan, R.M. et al. Systematic identification of conserved bacteria c-di-AMP receptor proteins. Proc. Natl. Acad. Sci. USA 110, 9084–9089 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Baker, J.L. et al. Widespread genetic switches and toxicity resistance proteins for fluoride. Science 335, 233–235 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Barrick, J.E. et al. New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc. Natl. Acad. Sci. USA 101, 6421–6426 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mandal, M. et al. A glycine-dependent riboswitch that uses cooperative binding to control gene expression. Science 306, 275–279 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Winkler, W.C., Nahvi, A., Roth, A., Collins, J.A. & Breaker, R.R. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Roth, A. et al. A riboswitch selective for the queuosine precursor preQ1 contains an unusually small aptamer domain. Nat. Struct. Mol. Biol. 14, 308–317 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Dann, C.E. III et al. Structure and mechanism of a metal-sensing regulatory RNA. Cell 130, 878–892 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Marchais, A., Duperrier, S., Durand, S., Gautheret, D. & Stragier, P. CsfG, a sporulation-specific, small non-coding RNA highly conserved in endospore formers. RNA Biol. 8, 358–364 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Block, K.F., Hammond, M.C. & Breaker, R.R. Evidence for widespread gene control function by the ydaO riboswitch candidate. J. Bacteriol. 192, 3983–3989 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Breaker, R.R. Riboswitches and the RNA world. Cold Spring Harb. Perspect. Biol. 4, a003566 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Meyer, M.M. et al. Challenges of ligand identification for riboswitch candidates. RNA Biol. 8, 5–10 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Breaker, R.R. Prospects for riboswitch discovery and analysis. Mol. Cell 43, 867–879 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Watson, P.Y. & Fedor, M.J. The ydaO motif is an ATP-sensing riboswitch in Bacillus subtilis. Nat. Chem. Biol. 8, 963–965 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Soukup, G.A. & Breaker, R.R. Relationship between internucleotide linkage geometry and the stability of RNA. RNA 5, 1308–1325 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Regulski, E.E. & Breaker, R.R. In-line probing analysis of riboswitches. Methods Mol. Biol. 419, 53–67 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Smith, K.D. et al. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat. Struct. Mol. Biol. 16, 1218–1223 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Shanahan, C.A., Gaffney, B.L., Jones, R.A. & Strobel, S.A. Identification of c-di-GMP derivatives resistance to an EAL domain phosphodiesterase. Biochemistry 52, 365–377 (2012).

    Article  CAS  Google Scholar 

  28. McDaniel, B.A., Grundy, F.J., Artsimovitch, I. & Henkin, T.M. Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA. Proc. Natl. Acad. Sci. USA 100, 3083–3088 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Epshtein, V., Mironov, A.S. & Nudler, E. The riboswitch-mediated control of sulfur metabolism in bacteria. Proc. Natl. Acad. Sci. USA 100, 5052–5056 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sudarsan, N., Wickiser, J.K., Nakamura, S., Ebert, M.S. & Breaker, R.R. An mRNA structure in bacteria that controls gene expression by binding lysine. Genes Dev. 17, 2688–2697 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mehne, F.M. et al. Cyclic di-AMP homeostasis in Bacillus subtilis; both lack and high level accumulation of the nucleotide are detrimental for cell growth. J. Biol. Chem. 288, 2004–2017 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Moriyama, R. et al. Expression of a germination-specific amidase, SleB, of Bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores. J. Bacteriol. 181, 2373–2378 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Gilmore, M.E., Bandyopadhyay, D., Dean, A.M., Linnstaedt, S.D. & Popham, D.L. Production of muramic δ-lactam in Bacillus subtilis spore peptidoglycan. J. Bacteriol. 186, 80–89 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kana, B.D. et al. The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Mol. Microbiol. 67, 672–684 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Kana, B.D. & Mizrahi, V. Resuscitation-promoting factors as lytic enzymes for bacterial growth and signaling. FEMS Immunol. Med. Microbiol. 58, 39–50 (2010).

    Article  CAS  PubMed  Google Scholar 

  36. Klein, D.J., Edwards, T.E. & Ferré-D'Amaré, A.R. Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase. Nat. Struct. Mol. Biol. 16, 343–344 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kang, M., Peterson, R. & Feigon, J. Structural insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA. Mol. Cell 33, 784–790 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Ren, A., Rajashankar, K.R. & Patel, D.J. Fluoride ion encapsulation by Mg2+ ions and phosphates in a fluoride riboswitch. Nature 486, 85–89 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wickiser, J.K., Winkler, W.C., Breaker, R.R. & Crothers, D.M. The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol. Cell 18, 49–60 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Wickiser, J.K., Cheah, M.T., Breaker, R.R. & Crothers, D.M. The kinetics of ligand binding by an adenosine-sensing riboswitch. Biochemistry 44, 13404–13414 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Gilbert, S.D., Stoddard, C.D., Wise, S.J. & Batey, R.T. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. J. Mol. Biol. 359, 754–768 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Lemay, J.-F. et al. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms. PLoS Genet. 7, e1001278 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Haller, A., Soulière, M.F. & Micura, R. The dynamic nature of RNA as key to understanding riboswitch mechanisms. Acc. Chem. Res. 44, 1339–1348 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Frieda, K.L. & Block, S.M. Direct observation of cotranscriptional folding in an adenine riboswitch. Science 338, 397–400 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Edwards, A.L., Reyes, F.E., Héroux, A. & Batey, R.T. Structural basis for recognition of S-adenylhomocysteine by riboswitches. RNA 16, 2144–2155 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wang, J.X., Lee, E.R., Morales, D.R., Lim, J. & Breaker, R.R. Riboswitches that sense S-adenylhomocysteine and activate genes involved in coenzyme recycling. Mol. Cell 29, 691–702 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Woodward, J.J., Iavarone, A.T. & Portnoy, D.A. c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science 328, 1703–1705 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Markley, J.L. et al. New bioinformatics resources for metabolomics. Pac. Symp. Biocomput. 2007, 157–168 (2007).

    Google Scholar 

  49. Kanehisa, M., Goto, S., Sato, Y., Furumichi, M. & Tanabe, M. KEGG for integration and interpretation of large-scale molecular datasets. Nucleic Acids Res. 40, D109–D114 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. Kanehisa, M. & Goto, S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).

    Article  CAS  PubMed  Google Scholar 

  51. Wach, A. PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12, 259–265 (1996).

    Article  CAS  PubMed  Google Scholar 

  52. Vidal-Aroca, F. et al. One-step high-throughput assay for quantitative detection of β-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cells. Biotechniques 40, 433–434 (2006).

    Article  CAS  PubMed  Google Scholar 

  53. Barrick, J.E. & Breaker, R.R. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Nawrocki, E.P., Kolbe, D.L. & Eddy, S.R. Infernal 1.0: inference of RNA alignments. Bioinformatics 25, 1335–1337 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hofacker, I.L. Curr. Prot. Bioinformatics RNA secondary structure analysis using the Vienna RNA package. 26, 12.2 (2009).

    Google Scholar 

  56. Pruitt, K.D., Tatusova, T. & Maglott, D.R. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts, and proteins. Nucleic Acids Res. 35, D61–D65 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Gevers, D. et al. The human microbiome project: a community resource for the healthy human microbiome. PLoS Biol. 10, e1001377 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Markowitz, V.M. et al. IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36, D534–D538 (2008).

    Article  CAS  PubMed  Google Scholar 

  59. Meyer, F. et al. The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9, 386–393 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Sun, S. et al. Community cyberinfrastructure for advanced microbial ecology research and analysis: the CAMERA resource. Nucleic Acids Res. 39, D546–D551 (2011).

    Article  CAS  PubMed  Google Scholar 

  61. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J., Ostell, J. & Wheeler, D.L. Genbank. Nucleic Acids Res. 36, D25–D30 (2008).

    Article  CAS  PubMed  Google Scholar 

  62. Roth, A. et al. A novel class of self-cleaving ribozymes is present in many species of bacteria and eukarya. Nat. Chem. Biol. (in the press).

  63. Gerstein, M., Sonnhammer, E.L.L. & Chothia, C. Volume changes on protein evolution. J. Mol. Biol. 236, 1067–1078 (1994).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Roth and other members of the Breaker laboratory for helpful discussions. We also thank N. Carriero and R. Bjornson for assisting our use of the Yale Life Sciences High Performance Computing Center (NIH grant RR19895-02). K.F. was supported by a Japan Society for the Promotion of Science fellowship for research abroad. This project was supported by US National Institutes of Health grants (GM022778 and DE022340) to R.R.B. Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.

Author information

Author notes

  1. Kazuhiro Furukawa & Joy X Wang

    Present address: Present addresses: Faculty of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan (K.F.); Center for Clinical and Translational Metagenomics, Brigham and Women's Hospital, Boston, Massachusetts, USA (J.X.W.).,

Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    James W Nelson

  2. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

    Narasimhan Sudarsan, Zasha Weinberg & Ronald R Breaker

  3. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Narasimhan Sudarsan, Kazuhiro Furukawa, Zasha Weinberg, Joy X Wang & Ronald R Breaker

  4. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Ronald R Breaker

Authors
  1. James W Nelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Narasimhan Sudarsan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kazuhiro Furukawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zasha Weinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joy X Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ronald R Breaker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.X.W. demonstrated the existence of the ligand in B. subtilis extract and K.F. purified the compound. J.W.N. performed MS, in-line probing and reporter assays. N.S. constructed c-di-AMP reporters and genetic knockouts and conducted the transcription termination assays. Z.W. performed bioinformatics. J.W.N., N.S. and R.R.B. designed experiments and interpreted the data. J.W.N., Z.W. and R.R.B. wrote the paper.

Corresponding author

Correspondence to Ronald R Breaker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–17 and Supplementary Tables 1 and 2. (PDF 2522 kb)

Supplementary Data Set

Locations and gene associations of all known c-di-AMP riboswitches in our database of genomic sequence information (specifically RefSeq and various environmental databases). (PDF 967 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nelson, J., Sudarsan, N., Furukawa, K. et al. Riboswitches in eubacteria sense the second messenger c-di-AMP. Nat Chem Biol 9, 834–839 (2013). https://doi.org/10.1038/nchembio.1363

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.1363

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology