title: Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones.
abstract: 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.
authors.affiliation: Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Cynthia H
authors.initials: CH
authors.lastname: Collins
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
authors.forename: Jared R
authors.initials: JR
authors.lastname: Leadbetter
mesh_terms: D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism*; D048908:Aliivibrio fischeri / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019020:Directed Molecular Evolution; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins; C088386:homoserine lactone; D049452:Green Fluorescent Proteins; D015107:4-Butyrolactone