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.

Reprogramming bacteria to seek and destroy an herbicide

A Retraction to this article was published on 14 February 2014

Abstract

A major goal of synthetic biology is to reprogram cells to perform complex tasks. Here we show how a combination of in vitro and in vivo selection rapidly identifies a synthetic riboswitch that activates protein translation in response to the herbicide atrazine. We further demonstrate that this riboswitch can reprogram bacteria to migrate in the presence of atrazine. Finally, we show that incorporating a gene from an atrazine catabolic pathway allows these cells to seek and destroy atrazine.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Chemical structures and synthetic scheme.
Figure 2: Progress of the SELEX experiment.
Figure 3: Motility selection for riboswitches.
Figure 4: In vivo characterization of atrazine-dependent riboswitches.
Figure 5: Characterization of atrazine-dependent riboswitches.
Figure 6: Motility of reprogrammed E. coli cells expressing GFP.

References

  1. Parkinson, J.S., Ames, P. & Studdert, C.A. Collaborative signaling by bacterial chemoreceptors. Curr. Opin. Microbiol. 8, 116–121 (2005).

    Article  CAS  Google Scholar 

  2. Wadhams, G.H. & Armitage, J.P. Making sense of it all: bacterial chemotaxis. Nat. Rev. Mol. Cell Biol. 5, 1024–1037 (2004).

    Article  CAS  Google Scholar 

  3. Adler, J. Chemotaxis in bacteria. Annu. Rev. Biochem. 44, 341–356 (1975).

    Article  CAS  Google Scholar 

  4. Derr, P., Boder, E. & Goulian, M. Changing the specificity of a bacterial chemoreceptor. J. Mol. Biol. 355, 923–932 (2006).

    Article  CAS  Google Scholar 

  5. Matsumura, I. & Ellington, A.D. In vitro evolution of β-glucuronidase into a β-galactosidase proceeds through non-specific intermediates. J. Mol. Biol. 305, 331–339 (2001).

    Article  CAS  Google Scholar 

  6. Aharoni, A. et al. The “evolvability” of promiscuous protein functions. Nat. Genet. 37, 73–76 (2005).

    Article  CAS  Google Scholar 

  7. Goldberg, S.D., Derr, P., DeGrado, W.F. & Goulian, M. Engineered single- and multi-cell chemotaxis pathways in E. coli. Mol. Syst. Biol. 5, 283 (2009).

    Article  Google Scholar 

  8. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  Google Scholar 

  9. Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).

    Article  CAS  Google Scholar 

  10. Werstuck, G. & Green, M.R. Controlling gene expression in living cells through small molecule-RNA interactions. Science 282, 296–298 (1998).

    Article  CAS  Google Scholar 

  11. Desai, S.K. & Gallivan, J.P. Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation. J. Am. Chem. Soc. 126, 13247–13254 (2004).

    Article  CAS  Google Scholar 

  12. Topp, S. & Gallivan, J.P. Guiding bacteria with small molecules and RNA. J. Am. Chem. Soc. 129, 6807–6811 (2007).

    Article  CAS  Google Scholar 

  13. Lynch, S.A., Desai, S.K., Sajja, H.K. & Gallivan, J.P. A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function. Chem. Biol. 14, 173–184 (2007).

    Article  CAS  Google Scholar 

  14. Topp, S. & Gallivan, J.P. Random walks to synthetic riboswitches—a high-throughput selection based on cell motility. ChemBioChem 9, 210–213 (2008).

    Article  CAS  Google Scholar 

  15. Topp, S. & Gallivan, J.P. Riboswitches in unexpected places—a synthetic riboswitch in a protein coding region. RNA 14, 2498–2503 (2008).

    Article  CAS  Google Scholar 

  16. Lynch, S.A. & Gallivan, J.P. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res. 37, 184–192 (2009).

    Article  CAS  Google Scholar 

  17. Muranaka, N., Sharma, V., Nomura, Y. & Yokobayashi, Y. An efficient platform for genetic selection and screening of gene switches in Escherichia coli. Nucleic Acids Res. 37, e39 (2009).

    Article  Google Scholar 

  18. Muranaka, N., Abe, K. & Yokobayashi, Y. Mechanism-guided library design and dual genetic selection of synthetic OFF riboswitches. ChemBioChem 10, 2375–2381 (2009).

    Article  CAS  Google Scholar 

  19. Kotter, P., Weigand, J.E., Meyer, B., Entian, K.D. & Suess, B. A fast and efficient translational control system for conditional expression of yeast genes. Nucleic Acids Res. 37, e120 (2009).

    Article  Google Scholar 

  20. Hunsicker, A. et al. An RNA aptamer that induces transcription. Chem. Biol. 16, 173–180 (2009).

    Article  CAS  Google Scholar 

  21. Hering, O., Brenneis, M., Beer, J., Suess, B. & Soppa, J. A novel mechanism for translation initiation operates in haloarchaea. Mol. Microbiol. 71, 1451–1463 (2009).

    Article  CAS  Google Scholar 

  22. Weigand, J.E. et al. Screening for engineered neomycin riboswitches that control translation initiation. RNA 14, 89–97 (2008).

    Article  CAS  Google Scholar 

  23. Sharma, V., Nomura, Y. & Yokobayashi, Y. Engineering complex riboswitch regulation by dual genetic selection. J. Am. Chem. Soc. 130, 16310–16315 (2008).

    Article  CAS  Google Scholar 

  24. Weigand, J.E. & Suess, B. A designed RNA shuts down transcription. Chem. Biol. 14, 9–11 (2007).

    Article  CAS  Google Scholar 

  25. Nomura, Y. & Yokobayashi, Y. Dual selection of a genetic switch by a single selection marker. Biosystems 90, 115–120 (2007).

    Article  CAS  Google Scholar 

  26. Hanson, S., Bauer, G., Fink, B. & Suess, B. Molecular analysis of a synthetic tetracycline-binding riboswitch. RNA 11, 503–511 (2005).

    Article  CAS  Google Scholar 

  27. Suess, B., Fink, B., Berens, C., Stentz, R. & Hillen, W. A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res. 32, 1610–1614 (2004).

    Article  CAS  Google Scholar 

  28. Buskirk, A.R., Landrigan, A. & Liu, D.R. Engineering a ligand-dependent RNA transcriptional activator. Chem. Biol. 11, 1157–1163 (2004).

    Article  CAS  Google Scholar 

  29. Thompson, K.M., Syrett, H.A., Knudsen, S.M. & Ellington, A.D. Group I aptazymes as genetic regulatory switches. BMC Biotechnol. 2, 21 (2002).

    Article  Google Scholar 

  30. Win, M.N. & Smolke, C.D. A modular and extensible RNA-based gene-regulatory platform for engineering cellular function. Proc. Natl. Acad. Sci. USA 104, 14283–14288 (2007).

    Article  CAS  Google Scholar 

  31. Jenison, R.D., Gill, S.C., Pardi, A. & Polisky, B. High-resolution molecular discrimination by RNA. Science 263, 1425–1429 (1994).

    Article  CAS  Google Scholar 

  32. Berens, C., Thain, A. & Schroeder, R. A tetracycline-binding RNA aptamer. Bioorg. Med. Chem. 9, 2549–2556 (2001).

    Article  CAS  Google Scholar 

  33. Wallis, M.G., von Ahsen, U., Schroeder, R. & Famulok, M. A novel RNA motif for neomycin recognition. Chem. Biol. 2, 543–552 (1995).

    Article  CAS  Google Scholar 

  34. Wang, Y., Killian, J., Hamasaki, K. & Rando, R.R. RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities. Biochemistry 35, 12338–12346 (1996).

    Article  CAS  Google Scholar 

  35. Wang, L. et al. Biodegradation of atrazine in transgenic plants expressing a modified bacterial atrazine chlorohydrolase (atzA) gene. Plant Biotechnol. J. 3, 475–486 (2005).

    Article  CAS  Google Scholar 

  36. de Souza, M.L., Seffernick, J., Martinez, B., Sadowsky, M.J. & Wackett, L.P. The atrazine catabolism genes atzABC are widespread and highly conserved. J. Bacteriol. 180, 1951–1954 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. de Souza, M.L. et al. Molecular basis of a bacterial consortium: interspecies catabolism of atrazine. Appl. Environ. Microbiol. 64, 178–184 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. de Souza, M.L., Sadowsky, M.J. & Wackett, L.P. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J. Bacteriol. 178, 4894–4900 (1996).

    Article  CAS  Google Scholar 

  39. Scott, C. et al. Catalytic improvement and evolution of atrazine chlorohydrolase. Appl. Environ. Microbiol. 75, 2184–2191 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  42. Mandelbaum, R.T., Allan, D.L. & Wackett, L.P. Isolation and characterization of a Pseudomonas sp. that mineralizes the s-triazine herbicide atrazine. Appl. Environ. Microbiol. 61, 1451–1457 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Clay, S.A. & Koskinen, W.C. Adsorption and desorption of atrazine, hydroxyatrazine, and S-glutathione atrazine on 2 soils. Weed Sci. 38, 262–266 (1990).

    CAS  Google Scholar 

  44. Wackett, L.P., Sadowsky, M.J., Martinez, B. & Shapir, N. Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies. Appl. Microbiol. Biotechnol. 58, 39–45 (2002).

    Article  CAS  Google Scholar 

  45. Adler, J. Chemotaxis in bacteria. Science 153, 708–716 (1966).

    Article  CAS  Google Scholar 

  46. Budrene, E.O. & Berg, H.C. Complex patterns formed by motile cells of Escherichia coli. Nature 349, 630–633 (1991).

    Article  CAS  Google Scholar 

  47. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 1–11 (2006).

    Article  Google Scholar 

  48. Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406–3415 (2003).

    Article  CAS  Google Scholar 

  49. Mathews, D.H., Sabina, J., Zuker, M. & Turner, D.H. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 288, 911–940 (1999).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was supported by the US National Institutes of Health (GM074070 to J.P.G.), the Arnold and Mabel Beckman Foundation and the Herman Frasch Foundation of the American Chemical Society. J.P.G. is a Camille Dreyfus Teacher-Scholar and an Alfred P. Sloan Research Fellow. We thank S. Topp for helpful discussions.

Author information

Authors and Affiliations

Authors

Contributions

J.P.G. conceived the research. J.S. performed the molecular and microbiology experiments; S.J.R. carried out the synthetic chemistry. J.S. and J.P.G. analyzed data and wrote the paper.

Corresponding author

Correspondence to Justin P Gallivan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Figures 1–9 (PDF 1753 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sinha, J., Reyes, S. & Gallivan, J. Reprogramming bacteria to seek and destroy an herbicide. Nat Chem Biol 6, 464–470 (2010). https://doi.org/10.1038/nchembio.369

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.369

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

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