In vitro-binding of the natural siderophore enantiomers pyochelin and enantiopyochelin to their AraC-type regulators Pch
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In vitro-binding of the natural siderophore enantiomers pyochelin and enantiopyochelin to their AraC-type regulators PchR in Pseudomonas Po-Chi Lin • Zeb A. Youard • Cornelia Reimmann
Received: 19 August 2013 / Accepted: 8 September 2013 / Published online: 15 September 2013 Ó Springer Science+Business Media New York 2013
Abstract The enantiomeric siderophores pyochelin and enantiopyochelin of Pseudomonas aeruginosa and Pseudomonas protegens promote growth under iron limitation and activate transcription of their biosynthesis and uptake genes via the AraC-type regulator PchR. Here we investigated siderophore binding to PchR in vitro using fluorescence spectroscopy. A fusion of the N-terminal domain of P. aeruginosa PchR with maltose binding protein (MBP-PchR0 PAO) bound iron-loaded (ferri-) pyochelin with an affinity (Kd) of 41 ± 5 lM. By contrast, no binding occurred with ferri-enantiopyochelin. Stereospecificity of a similar fusion protein of the P. protegens PchR (MBPPchR0 CHA0) was less pronounced. The Kd’s of MBPPchR0 CHA0 for ferri-enantiopyochelin and ferri-pyochelin were 24 ± 5 and 40 ± 7 lM, respectively. None of the proteins interacted with the iron-free siderophore enantiomers, suggesting that transcriptional activation by PchR occurs only when the respective siderophore actively procures iron to the cell. Keywords Siderophore Pseudomonas Iron Pyochelin AraC-type regulator
P.-C. Lin Z. A. Youard C. Reimmann (&) De´partement de Microbiologie Fondamentale, Universite´ de Lausanne, Baˆtiment Biophore, Quartier UNIL-Sorge, 1015 Lausanne, Switzerland e-mail: [email protected]
Introduction As a cofactor of many redox-dependent enzymes, iron is essential for most organisms including bacteria. Despite its abundancy in nature, iron is not readily accessible for microorganisms as it forms poorly soluble ferric hydroxides in the oxic environment and is bound to iron transport and storage proteins in the mammalian host. To acquire iron, bacteria produce and release siderophores, which bind ferric iron avidly and transport it to the cytoplasm via specific outer membrane receptors and inner membrane permeases (Guerinot 1994; Wandersman and Delepelaire 2004). Siderophore biosynthesis and uptake is tightly regulated to guarantee a sufficient iron acquisition and to prevent an iron overload, which would generate toxic hydroxyl radicals through the Fenton chemistry (Andrews et al. 2003). Under iron-replete conditions, siderophore biosynthesis and uptake genes are therefore repressed. In many bacteria this occurs by the Fur repressor, which, when complexed with ferrous iron, binds to conserved sequences in target promoters and prevents transcription (Escolar et al. 1999). When iron becomes limiting, Fur loses its cofactor and dissociates from its binding sites, thus allowing gene expression to occur. However, full expression of siderophore biosynthesis and/or uptake genes often involves additional regulators whose activities are controlled by the siderophores themselves. This positive regulation, also
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