Thu, 21 Aug 2008 17:25:59 BST CiteULike: macowell Leadbetter http://www.citeulike.org/user/macowell/author/Leadbetter CiteULike.org en-gb Copyright © 2004-2008 citeulike.org http://www.citeulike.org/user/macowell/article/1570059 <i>Mol Microbiol, Vol. 55, No. 3. (Feb 2005), pp. 712-23.</i><br /><br />LuxR-type transcriptional regulators play key roles in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. These proteins mediate quorum control by changing their interactions with RNA polymerase and DNA in response to binding their cognate acyl-HSL. The evolutionarily related LuxR-type proteins exhibit considerable diversity in primary sequence and in their response to acyl-HSLs having acyl groups of differing length and composition. Little is known about which residues determine acyl-HSL specificity, and less about the evolutionary time scales required to forge new ones. To begin to examine such issues, we have focused on the LuxR protein from Vibrio fischeri, which activates gene transcription in response to binding its cognate quorum signal, 3-oxohexanoyl-homoserine lactone (3OC6HSL). Libraries of luxR mutants were screened for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL), with which wild-type LuxR interacts only weakly. Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL; these variants also displayed increased sensitivities to pentanoyl-HSL and tetradecanoyl-HSL, while maintaining a wild-type or greater response to 3OC6HSL. The most sensitive variants activated gene transcription as strongly with C8HSL as the wild type did with 3OC6HSL. With one exception, the amino acid residues involved were restricted to the N-terminal, 'signal-binding' domain of LuxR. These residue positions differed from critical positions previously identified via 'loss-of-function' mutagenesis. We have demonstrated that acyl-HSL-dependent quorum-sensing systems can evolve rapidly to respond to new acyl-HSLs, suggesting that there may be an evolutionary advantage to maintaining such plasticity. Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones Cynthia Collins Frances Arnold Jared Leadbetter Mol Microbiol, Vol. 55, No. 3. (Feb 2005), pp. 712-23. 2007-08-16T20:20:58-00:00 2005 Mol Microbiol 55 3 712 23 4-butyrolactone bacterial directed evolution expression fischeri fluorescent foundationalsb gene genetic green models molecular mutagenesis mutation proteins regulation repressor signal site-directed syntheticbiology trans-activators transcription transduction vibrio http://www.citeulike.org/user/macowell/article/1570032 <i>Nat Biotechnol, Vol. 24, No. 6. (Jun 2006), pp. 708-12.</i><br /><br />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. Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR Cynthia Collins Jared Leadbetter Frances Arnold Nat Biotechnol, Vol. 24, No. 6. (Jun 2006), pp. 708-12. 2007-08-16T20:20:57-00:00 2006 Nat Biotechnol 24 6 708 12 cell coli communication engineering enhancement escherichia foundationalsb genetic protein proteins recombinant repressor syntheticbiology trans-activators