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

Collins, Cynthia H. and Leadbetter, Jared R. and Arnold, Frances H. (2006) Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR. Nature Biotechnology, 24 (6). pp. 708-712. ISSN 1087-0156. https://resolver.caltech.edu/CaltechAUTHORS:20130502-083943317

[img] PDF (Supplementary Fig. 1 Activation of gfpuv transcription with 3OC6HSL and C6HSL by wild-type LuxR and LuxR-R67M) - Supplemental Material
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[img] PDF (Supplementary Fig. 2 Activation of gfpuv transcription with 3OC12HSL and C12HSL by wild-type LasR and LasR-R61M) - Supplemental Material
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[img] PDF (Supplementary Fig. 3 Protein accumulation of LuxR, LuxR-G2E and LuxR-G2E-R67M is dependent upon acyl-HSL binding) - Supplemental Material
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[img] PDF (Supplementary Figure 4 Maps of plasmids used in this study) - Supplemental Material
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[img] PDF (Supplementary Table 1 Nucleotide and amino acid changes in luxR/LuxR and lasR/LasR mutants) - Supplemental Material
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[img] PDF (Supplementary Table 2 Synthetic oligonucleotides used in this study) - Supplemental Material
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[img] PDF (Supplementary Note Characterization of the dual-selection system) - Supplemental Material
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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 respond. 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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ORCID:
AuthorORCID
Leadbetter, Jared R.0000-0002-7033-0844
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2006 Nature Publishing Group. Received 30 January; accepted 26 March; published online 21 May; corrected after print 25 July 2006; doi:10.1038/nbt1209. 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 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. Competing Interests Statement: The authors declare that they have no competing financial interests.
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Funding AgencyGrant Number
NSFCCF-0522831
NIH1 R01 GM074712-01A1
Issue or Number:6
Record Number:CaltechAUTHORS:20130502-083943317
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130502-083943317
Official Citation:Collins, C. H., J. R. Leadbetter, et al. (2006). "Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR." Nat Biotech 24(6): 708-712.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38226
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 May 2013 23:36
Last Modified:10 Jun 2020 23:08

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