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Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR

Nature Biotechnology volume 24, pages 708–712 (2006)Cite this article

A Corrigendum to this article was published on 01 August 2006

Abstract

The transcription factor LuxR activates gene expression in response to binding the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6HSL), an acyl-HSL with a carbonyl substituent at the third carbon of the acyl chain. We previously described a LuxR variant, LuxR-G2E, that activates gene expression by binding a broader range of acyl-HSLs, including straight-chain acyl-HSLs to which LuxR does not respond1. Here, we use a dual positive-negative selection system to identify a variant of LuxR-G2E that retains the response to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation, R67M, reduces LuxR-G2E's response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain. This specificity-enhancing mutation would not have been identified by positive selection alone. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have or lack the desired response to a given set of acyl-HSL signals. LuxR variants with altered signaling specificities might become useful components for constructing artificial cell-cell communication systems that program population level behaviors.

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Figure 1: Schematic of dual selection system for directed evolution of LuxR variants with altered specificity.
Figure 2: Activation of gfpuv transcription with 3OC6HSL, C10HSL, C6HSL, C8HSL, C12HSL and 3OC12HSL by wild-type LuxR (▴), LuxR-G2E (▪) and LuxR-G2E-R67M (⋄).
Figure 3: Gel mobility shift assays of LuxR, LuxR-G2E and LuxR-G2E-R67M in the presence of 3OC6HSL and C10HSL.
Figure 4: Solid-phase assays show minimal crosstalk between LuxR and LuxR-G2E-R67M.

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References

  1. Collins, C.H., Arnold, F.H. & Leadbetter, J.R. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Mol. Microbiol. 55, 712–723 (2005).

    Article  CAS  Google Scholar 

  2. Taga, M.E. & Bassler, B.L. Chemical communication among bacteria. Proc. Natl. Acad. Sci. USA 100 suppl. Suppl. 2, 14549–14554 (2003).

    Article  CAS  Google Scholar 

  3. Whitehead, N.A., Barnard, A.M.L., Slater, H., Simpson, N.J.L. & Salmond, G.P.C. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol. Rev. 25, 365–404 (2001).

    Article  CAS  Google Scholar 

  4. Fuqua, C. & Greenberg, E.P. Listening in on bacteria: Acyl-homoserine lactone signalling. Nat. Rev. Mol. Cell Biol. 3, 685–695 (2002).

    Article  CAS  Google Scholar 

  5. Schaefer, A.L., Val, D.L., Hanzelka, B.L., Cronan, J.E. & Greenberg, E.P. Generation of cell-to-cell signals in quorum sensing: Acyl homoserine lactone synthase activity of a purified Vibrio fischeri LuxI protein. Proc. Natl. Acad. Sci. USA 93, 9505–9509 (1996).

    Article  CAS  Google Scholar 

  6. Schaefer, A.L., Hanzelka, B.L., Eberhard, A. & Greenberg, E.P. Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178, 2897–2901 (1996).

    Article  CAS  Google Scholar 

  7. Schaefer, A.L., Taylor, T.A., Beatty, J.T. & Greenberg, E.P. Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production. J. Bacteriol. 184, 6515–6521 (2002).

    Article  CAS  Google Scholar 

  8. Basu, S., Gerchman, Y., Collins, C.H., Arnold, F.H. & Weiss, R. A synthetic multicellular system for programmed pattern formation. Nature 434, 1130–1134 (2005).

    Article  CAS  Google Scholar 

  9. Basu, S., Mehreja, R., Thiberge, S., Chen, M.T. & Weiss, R. Spatiotemporal control of gene expression with pulse-generating networks. Proc. Natl. Acad. Sci. USA 101, 6355–6360 (2004).

    Article  CAS  Google Scholar 

  10. Kobayashi, H. et al. Programmable cells: interfacing natural and engineered gene networks. Proc. Natl. Acad. Sci. USA 101, 8414–8419 (2004).

    Article  CAS  Google Scholar 

  11. Weiss, R. & Knight, T.F. Engineered communications for microbial robotics. DNA Computing: 6th International Workshop on DNA-Based Computers, Leiden, The Netherlands, June, 13–17, 2000 (Springer, Berlin/Heidelberg, 2001).

    Google Scholar 

  12. You, L., Cox, R.S., Weiss, R. & Arnold, F.H. Programmed population control by cell-cell communication and regulated killing. Nature 428, 868–871 (2004).

    Article  CAS  Google Scholar 

  13. Weiss, R. Challenges and opportunities in programming living cells. The Bridge 32, 39–46 (2004).

    Google Scholar 

  14. Karig, D. & Weiss, R. Signal-amplifying genetic enables in vivo observation circuit of weak promoter activation in the RhI quorum sensing system. Biotechnol. Bioeng. 89, 709–718 (2005).

    Article  CAS  Google Scholar 

  15. Havranek, J.J. & Harbury, P.B. Automated design of specificity in molecular recognition. Nat. Struct. Biol. 10, 45–52 (2003).

    Article  CAS  Google Scholar 

  16. Yokobayashi, Y. & Arnold, F.H. A dual selection module for directed evolution of genetic circuits. Nat. Comput. 4, 245–254 (2005).

    Article  CAS  Google Scholar 

  17. Shadel, G.S., Young, R. & Baldwin, T.O. Use of regulated cell-lysis in a lethal genetic selection in Escherichia coli-identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC-7744. J. Bacteriol. 172, 3980–3987 (1990).

    Article  CAS  Google Scholar 

  18. Gray, K.M., Passador, L., Iglewski, B.H. & Greenberg, E.P. Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa. J. Bacteriol. 176, 3076–3080 (1994).

    Article  CAS  Google Scholar 

  19. Egland, K.A. & Greenberg, E.P. Quorum sensing in Vibrio fischeri: elements of the luxl promoter. Mol. Microbiol. 31, 1197–1204 (1999).

    Article  CAS  Google Scholar 

  20. Urbanowski, M.L., Lostroh, C.P. & Greenberg, E.P. Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. J. Bacteriol. 186, 631–637 (2004).

    Article  CAS  Google Scholar 

  21. Cormack, B.P., Valdivia, R.H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  Google Scholar 

  22. Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. 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 

  25. Gould, S.M. & Tawfik, D.S. Directed evolution of the promiscuous esterase activity of carbonic anhydrase II. Biochemistry 44, 5444–5452 (2005).

    Article  CAS  Google Scholar 

  26. Bloom, J.D. et al. Evolving strategies for enzyme engineering. Curr. Opin. Struct. Biol. 15, 447–452 (2005).

    Article  CAS  Google Scholar 

  27. Parikh, M.R. & Matsumura, I. Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase. J. Mol. Biol. 352, 621–628 (2005).

    Article  CAS  Google Scholar 

  28. Voziyanov, Y., Stewart, A.F. & Jayaram, M. A dual reporter screening system identifies the amino acid at position 82 in Flp site-specific recombinase as a determinant for target specificity. Nucleic Acids Res. 30, 1656–1663 (2002).

    Article  CAS  Google Scholar 

  29. Horton, R.M. et al. Gene-splicing by overlap extension. Methods Enzymol. 217, 270–279 (1993).

    Article  CAS  Google Scholar 

  30. Cirino, P.C., Mayer, K.M. & Umeno, D. in Directed Evolution Library Creation (eds. Arnold, F.H. & Georgiou, G.G.) 3–9, (Humana Press, Totowa, NJ, 2003).

    Book  Google Scholar 

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Acknowledgements

The authors wish to thank C. Parker and J. Barnet for valuable assistance with the gel shift experiments and M. Surette for assistance with solid-phase fluorescence imaging. This research was supported by the US National Science Foundation (no. CCF-0522831) and the US National Institutes of Health (1 R01 GM074712-01A1).

Author information

Authors and Affiliations

  1. Biochemistry & Molecular Biophysics, California Institute of Technology, Pasadena, 91125, California, USA

    Cynthia H Collins & Frances H Arnold

  2. Environmental Science & Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Jared R Leadbetter

  3. Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, 91125, California, USA

    Frances H Arnold

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  2. Jared R Leadbetter
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Contributions

F.H.A. and C.H.C. conceived the project; C.H.C., F.H.A. and J.R.L. designed the experiments; C.H.C. performed the experiments and analyzed data; C.H.C. and F.H.A. wrote the manuscript.

Corresponding author

Correspondence to Frances H Arnold.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Activation of gfpuv transcription with 3OC6HSL and C6HSL by wild-type LuxR and LuxR-R67M. (PDF 51 kb)

Supplementary Fig. 2

Activation of gfpuv transcription with 3OC12HSL and C12HSL by wild-type LasR and LasR-R61M. (PDF 51 kb)

Supplementary Fig. 3

Protein accumulation of LuxR, LuxR-G2E and LuxR-G2E-R67M is dependent upon acyl-HSL binding. (PDF 17 kb)

Supplementary Figure 4

Maps of plasmids used in this study. (PDF 33 kb)

Supplementary Table 1

Nucleotide and amino acid changes in luxR/LuxR and lasR/LasR mutants (PDF 57 kb)

Supplementary Table 2

Synthetic oligonucleotides used in this study (PDF 11 kb)

Supplementary Note

Characterization of the dual-selection system (PDF 75 kb)

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Collins, C., Leadbetter, J. & Arnold, F. Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR. Nat Biotechnol 24, 708–712 (2006). https://doi.org/10.1038/nbt1209

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