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Applied and Environmental Microbiology, September 2007, p. 5775-5781, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00060-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Directed Evolution of Vibrio fischeri LuxR for Improved Response to Butanoyl-Homoserine Lactone{triangledown} ,{dagger}

Andrew C. Hawkins,1 Frances H. Arnold,2,3 Rainer Stuermer,4 Bernhard Hauer,4 and Jared R. Leadbetter1*

Environmental Science & Engineering,1 Biochemistry & Molecular Biophysics,2 Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California 91125,3 Fine Chemicals & Biocatalysis Research, BASF Aktiengesellschaft, GVF/E-B009, D67056 Ludwigshafen, Germany4

Received 10 January 2007/ Accepted 25 July 2007

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 the 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.

* Corresponding author. Mailing address: Environmental Science & Engineering, California Institute of Technology, 1200 E. California Blvd., Mail Code 138-78, Pasadena, CA 91125-7800. Phone: (626) 395-4182. Fax: (626) 395-2940. E-mail: jleadbetter{at}caltech.edu

{triangledown} Published ahead of print on 3 August 2007.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, September 2007, p. 5775-5781, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00060-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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