title: Dual selection enhances the signaling specificity of a variant of the quorum-sensing transcriptional activator LuxR.
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.
authors.affiliation: Biochemistry & Molecular Biophysics, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Cynthia H
authors.initials: CH
authors.lastname: Collins
authors.forename: Jared R
authors.initials: JR
authors.lastname: Leadbetter
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D002450:Cell Communication / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C064778:LuxR autoinducer binding proteins