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Originally published In Press as doi:10.1074/jbc.M609238200 on December 5, 2006

J. Biol. Chem., Vol. 282, Issue 5, 2987-2995, February 2, 2007
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Real Time Fluorescent Resonance Energy Transfer Visualization of Ferric Pyoverdine Uptake in Pseudomonas aeruginosa

A ROLE FOR FERROUS IRON*

Jason Greenwald{ddagger}12, Françoise Hoegy{ddagger}1, Mirella Nader{ddagger}, Laure Journet{ddagger}2, Gaëtan L. A. Mislin{ddagger}, Peter L. Graumann§, and Isabelle J. Schalk{ddagger}3

From the {ddagger}Métaux et Microorganismes: Chimie, Biologie, et Applications, UMR 7175-LC1 Institut Gilbert-Laustriat, CNRS-Université Louis Pasteur, ESBS, Boulevard Sébastien Brant, F-67413 Illkirch, Strasbourg, France and the §Institüt für Mikrobiologie, Stefan Meier Strasse 19, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.


Received for publication, September 29, 2006 , and in revised form, December 1, 2006.

* This work was supported in part by the Centre National de la Recherche Scientific, the Association Vaincre la Mucoviscidose (French Association against Cystic Fibrosis), Agence Nationale de Recherche Grant ANR-05-JCJC-0181-01 (to I. S.), and the Deutsche Forschungegemeinschaft (to P. G.). 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 These authors contributed equally to this work.

2 Supported by European Molecular Biology Organization postdoctoral fellowships.

3 To whom correspondence should be addressed: Métaux et Microorganismes: Chimie, Biologie, et Applications, UMR 7175-LC1 Institut Gilbert-Laustriat, ESBS, Blvd. Sébastien Brant, BP 10412, F-67413 Illkirch, Strasbourg, France. Tel.: 33-3-90-24-47-19; Fax: 33-3-90-24-48-29; E-mail: schalk{at}esbs.u-strasbg.fr.


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