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Directed evolution of Vibrio fischeri LuxR for improved response to butanoyl-homoserine lactone

Hawkins, Andrew C. and Arnold, Frances H. and Stuermer, Rainer and Hauer, Bernhard and Leadbetter, Jared R. (2007) Directed evolution of Vibrio fischeri LuxR for improved response to butanoyl-homoserine lactone. Applied and Environmental Microbiology, 73 (18). pp. 5775-5781. ISSN 0099-2240. PMCID PMC2074898.

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[img] Archive (ZIP) (Data on third-generation (Table S1) and fourth-generation (Table S2) directed-evolution mutant sensitivities; third-generation (Fig. S1) and fourth-generation (Fig. S2) directed-evolution mutant AHL dose-response curves for C4HSL, C5HSL, 3OC6HSL) - Supplemental Material
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LuxR is the 3-oxohexanoyl-homoserine lactone (3OC6HSL) dependent transcriptional activator of the prototypical acyl-homoserine lactone (AHL) quorum sensing system of Vibrio fischeri. Wild-type LuxR exhibits no response to butanoyl-HSL (C4HSL) in quantitative bioassays at concentrations of up to 1 µM; a previously described LuxR variant (LuxR-G2E) exhibits a broadened response to diverse AHLs, including pentanoyl-HSL (C5HSL), but not to C4HSL. Here, two rounds of directed evolution of LuxR-G2E generated variants of LuxR that responded to C4HSL at concentrations as low as 10 nM. One variant, LuxR-G4E, had only one change, I45F, relative to the parent LuxR-G2E, which itself differs from wild-type at three residues. Dissection of the four mutations within LuxR-G4E demonstrated that at least three of these changes were simultaneously required to achieve any measurable C4HSL response. The four changes improved both sensitivity and specificity towards C4HSL relative to any of the other 14 possible combinations of those residues. These data confirm that LuxR is evolutionarily pliable and suggest that LuxR is not intrinsically asymmetric in its response to quorum sensing signals with different acyl-side chain lengths.

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Arnold, Frances H.0000-0002-4027-364X
Leadbetter, Jared R.0000-0002-7033-0844
Additional Information:© 2007, American Society for Microbiology. Received 10 January 2007/ Accepted 25 July 2007. AEM Accepts, published online ahead of print on 3 August 2007 We thank Amy Schaefer and members of our laboratories for their many helpful discussions and critical reading of the manuscript. This research was supported by the Defense Advanced Research Projects Agency (DARPA) Biological Input-Output Systems program under grant N66001-02-8929. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the DARPA. Supplemental material for this article may be found at
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)N66001-02-8929
Issue or Number:18
PubMed Central ID:PMC2074898
Record Number:CaltechAUTHORS:HAWaem07
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8600
Deposited By: Archive Administrator
Deposited On:22 Aug 2007
Last Modified:27 Apr 2020 22:32

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