RESUMO
OBJECTIVES: To alter DNA binding specificity of Vibrio fischeri LuxR and to expand the toolbox for constructing synthetic networks. RESULTS: A mutation library (about 10,000 individuals) of the DNA binding domain of LuxR were generated. A genetic selection was performed to obtain LuxR mutants that recognize three lux box DNA variants that are not recognized by wild-type LuxR. Six LuxR mutants were identified. The evolved LuxR mutants were further characterized by measuring the transcriptional activities of different combinations of LuxR mutants and lux box variants. Varied transcriptional activities were found in these LuxR-lux box pairs. The background expressions of the evolved LuxR-lux box systems are more tightly regulated than the wild-type LuxR-lux box system. CONCLUSION: The LuxR transcriptional system was evolved to recognize three lux box DNAs which are not recognized by wild-type LuxR.
Assuntos
Aliivibrio fischeri/metabolismo , Aliivibrio fischeri/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Biologia Sintética/métodos , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/genética , Ativação Transcricional/fisiologiaRESUMO
In the phylum of Proteobacteria, quorum sensing (QS) system is widely driven by synthesis and response of N-acyl homoserine lactone (AHL) signalling molecules. AHL is synthesized by LuxI homologue and sensed by LuxR homologue. Once the AHL concentration achieves a threshold level, it triggers the regulation of target genes. In this study, QS activity of Citrobacter amalonaticus strain YG6 which was isolated from clams was investigated. In order to characterise luxI/R homologues, the genome of C. amalonaticus strain YG6 (4.95 Mbp in size) was sequenced using Illumina MiSeq sequencer. Through in silico analysis, a pair of canonical luxI/R homologues and an orphan luxR homologue were identified and designated as camI, camR, and camR2, respectively. A putative lux box was identified at the upstream of camI. The camI gene was cloned and overexpressed in E. coli BL21 (DE3)pLysS. High-resolution triple quadrupole liquid chromatography mass spectrometry (LC-MS/MS) analysis verified that the CamI is a functional AHL synthase which produced multiple AHL species, namely Nbutyryllhomoserine lactone (C4-HSL), Nhexanoyllhomoserine lactone (C6-HSL), Noctanoyllhomoserine lactone (C8-HSL), Ntetradecanoyllhomoserine lactone (C14-HSL) and Nhexadecanoyllhomoserine lactone (C16-HSL) in C. amalonaticus strain YG6 and camI gene in recombinant E. coli BL21(DE3)pLysS. To our best knowledge, this is the first functional study report of camI as well as the first report describing the production of C14-HSL by C. amalonaticus.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Percepção de Quorum/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Acil-Butirolactonas , Proteínas de Bactérias/genética , Sequência de Bases , Hidrolases de Éster Carboxílico/genética , Citrobacter/genética , Citrobacter/metabolismo , DNA Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos/genética , Homosserina/análogos & derivados , LactonasRESUMO
A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.