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

ABSTRACT: 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 microM; 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.

SUBMITTER: Hawkins AC

PROVIDER: S-EPMC2074898 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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

Hawkins Andrew C AC Arnold Frances H FH Stuermer Rainer R Hauer Bernhard B Leadbetter Jared R JR

Applied and environmental microbiology 20070803 18

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 microM; 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-G2 ...[more]

PMID: 17675429

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Project description:UnlabelledBurkholderia thailandensis has three acyl-homoserine lactone (AHL) LuxR-LuxI quorum-sensing circuits and two orphan LuxR homologs. Orphans are LuxR-type transcription factors that do not have cognate LuxI-type AHL synthases. One of the orphans, MalR, is genetically linked to the mal gene cluster, which encodes enzymes required for production of the cytotoxic polyketide malleilactone. Under normal laboratory conditions the mal gene cluster is silent; however, antibiotics like trimethoprim induce mal transcription. We show that trimethoprim-dependent induction of the mal genes requires MalR. MalR has all of the conserved amino acid residues characteristic of AHL-responsive LuxR homologs, but in B. thailandensis, MalR activation of malleilactone synthesis genes is not responsive to AHLs. MalR can activate transcription from the mal promoter in E. coli without addition of AHLs or trimethoprim. Expression of malR in B. thailandensis is induced by trimethoprim. Our data indicate that MalR binds to a lux box-like element in the mal promoter and activates transcription of the mal genes in an AHL-independent manner. Antibiotics like trimethoprim appear to activate mal gene expression indirectly by somehow activating malR expression. MalR activation of the mal genes represents an example of a LuxR homolog that is not a receptor for an AHL quorum-sensing signal. Our evidence is consistent with the idea that mal gene activation depends solely on sufficient transcription of the malR gene.ImportanceLuxR proteins are transcription factors that are typically activated by acyl-homoserine lactone (AHL) signals. We demonstrate that a conserved LuxR family protein, MalR, activates genes independently of AHLs. MalR is required for transcription of genes coding for synthesis of the cytotoxic polyketide malleilactone. These genes are not expressed when cells are grown under normal laboratory conditions. In laboratory culture, MalR induction of malleilactone requires certain antibiotics, such as trimethoprim, which increase malR expression by an unknown mechanism. At sufficient levels of malR expression, MalR functions independently of any external signal. Our findings show that MalR is an activator of the silent malleilactone biosynthesis genes and that MalR functions independently of AHLs.

Dataset Information

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

Publications

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

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