title: Purification and characterization of Pseudomonas aeruginosa LasR expressed in acyl-homoserine lactone free Escherichia coli cultures.
abstract: Quorum sensing systems are essential for bacterial communication. We report here the purification and characterization of the Pseudomonas aeruginosa LasR quorum sensing regulator purified from lysates of E. coli cultures grown in the absence of added acyl-homoserine lactones (AHL). We show by isothermal titration calorimetry that LasR recognizes different AHLs with an affinity of approximately 1 μM. The affinity of LasR for its cognate 3-Oxo-C12-AHL was similar to that of other AHLs, indicating that this regulator has not evolved to preferentially recognize its cognate AHL. The α-helical content as determined by CD spectroscopy was found to be in agreement with the corresponding value derived from the homology model. Analytical ultracentrifugation studies show that LasR is a mixture of monomers and dimers and that AHL binding does not alter its oligomeric state. Thermal unfolding studies indicate that LasR has a significant thermal stability and that AHL binding does not significantly alter the unfolding temperature. Two LasR-DNA complexes were observed in electrophoretic mobility shift assays using the hcnABC promoter that has two lux boxes. Taken together, data indicate that the presence of AHLs is not a requisite for correct LasR protein folding. The protein is able to bind AHL ligands in a reversible manner, revising initial concepts of this regulator. The availability of AHL-free protein will permit further studies to determine more precisely its mode of action.
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Andrés
authors.initials: A
authors.lastname: Corral Lugo
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Abdelali
authors.initials: A
authors.lastname: Daddaoua
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Ortega
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Morel
authors.affiliation: Department of Physical Chemistry, Faculty of Chemistry, Regional Campus of International Excellence "Campus Mare Nostrum", University of Murcia, 30071 Murcia, Spain.
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Díez Peña
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Espinosa-Urgel
authors.affiliation: Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/ Prof. Albareda, 1, 18008 Granada, Spain. Electronic address: tino.krell@eez.csic.es.
authors.forename: Tino
authors.initials: T
authors.lastname: Krell
mesh_terms: D054742:Acyl-Butyrolactones / Q000737:chemistry*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015534:Trans-Activators / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D054742:Acyl-Butyrolactones; D001426:Bacterial Proteins; C080365:LasR protein, Pseudomonas aeruginosa; D011994:Recombinant Proteins; D015534:Trans-Activators
keywords: Acyl-homoserine lactone; Quorum sensing; Transcriptional regulator