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J. Biol. Chem., Vol. 281, Issue 40, 29972-29987, October 6, 2006

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Direct Evidence for the Modulation of the Activity of the Erwinia chrysanthemi Quorum-sensing Regulator ExpR by Acylhomoserine Lactone Pheromone^*

Sandra Castang¹, Sylvie Reverchon, Patrice Gouet, and William Nasser²

From the Unité de Microbiologie et Génétique, Unité Mixte de Recherche CNRS-Université Lyon 1-Institut National des Sciences Appliquées de Lyon 5122, Domaine Scientifique de la Doua, bâtiment André Lwoff 10 rue Raphaël Dubois, 69622 Villeurbanne Cedex, France and the Laboratoire de BioCristallographie, Institut de Biochimie et Chimie des Protéines, UMR CNRS-UCBL 5086, 7 Passage du Vercors, 69367 Lyon Cedex 07, France

In Erwinia chrysanthemi production of pectic enzymes is controlled by a complex network involving several regulators. Among them is ExpR, the quorum-sensing regulatory protein. ExpR is a member of the LuxR family of transcriptional regulators, the activity of which is modulated by the binding of diffusible N-acylhomoserine lactone pheromones to the N-terminal receptor site of the proteins. Previous in vitro DNA-ExpR binding studies suggested that ExpR might activate pectic enzyme production and repress its cognate gene expression. This report presents genetic evidence that ExpR represses its own gene expression in the absence of pheromone and that the addition of pheromone promotes concentration-dependent de-repression. In vitro experiments show that (i) ExpR binds target DNA in the absence of pheromone and that the pheromone dissociates ExpR-DNA complexes, (ii) ExpR binds target DNA in a non-cooperative fashion, and (iii) two molecules of pheromone are bound per molecule of ExpR dimer. In the absence of N-(3-oxo-hexanoyl)-homoserine lactone, ExpR prevents RNA polymerase access to the expR promoter, thereby directly repressing transcription initiation. The presence of pheromone renders the expR promoter accessible to RNA polymerase and results in the de-repression of transcription initiation. Overall we have established that there is a direct modulation of the repressive activity of a LuxR family regulator by a pheromone. Furthermore, site-directed mutagenesis experiments strongly suggest that the ExpR residues Leu-19, Tyr-31, and Ser-125 are involved in the transduction of conformational changes induced by ligand binding, and this provides new insights into the structure-function relationship of this bacterial regulator family.

Received for publication, February 22, 2006 , and in revised form, July 5, 2006.

^* The work was supported in part from the Région Rhône-Alpes (Programme Emergence 2002), the CNRS, and the Ministère de l'Education Nationale de la Recherche et de la Technologie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Region Rhône-Alpes Emergence fellowship.

² To whom correspondence should be addressed. Tel.: 33-4-72432695; Fax: 33-4-72431584; E-mail: william.nasser{at}insa-lyon.fr.

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