RESUMO
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-PchR'PAO) bound iron-loaded (ferri-) pyochelin with an affinity (Kd) of 41 ± 5 µM. By contrast, no binding occurred with ferri-enantiopyochelin. Stereospecificity of a similar fusion protein of the P. protegens PchR (MBP-PchR'CHA0) was less pronounced. The Kd's of MBP-PchR'CHA0 for ferri-enantiopyochelin and ferri-pyochelin were 24 ± 5 and 40 ± 7 µM, 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.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Fenóis/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas/metabolismo , Sideróforos/metabolismo , Tiazóis/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Fenóis/química , Ligação Proteica , Pseudomonas/genética , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sideróforos/química , Espectrometria de Fluorescência , Estereoisomerismo , Tiazóis/química , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
The bacterial siderophore pyochelin is composed of salicylate and two cysteine-derived heterocycles, the second of which is modified by reduction and N-methylation during biosynthesis. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation, whereas the second cysteine remains in its L-configuration. Stereochemistry is opposite in the Pseudomonas fluorescens siderophore enantio-pyochelin, in which the first ring originates from L-cysteine and the second ring from D-cysteine. Both siderophores promote growth of the producer organism during iron limitation and induce the expression of their biosynthesis genes by activating the transcriptional AraC-type regulator PchR. However, neither siderophore is functional as an iron carrier or as a transcriptional inducer in the other species, demonstrating that both processes are highly stereospecific. Stereospecificity of pyochelin/enantio-pyochelin-mediated iron uptake is ensured at two levels: (i) by the outer membrane siderophore receptors and (ii) by the cytosolic PchR regulators.
Assuntos
Ferro/química , Ferro/metabolismo , Fenóis/química , Pseudomonas/química , Pseudomonas/metabolismo , Sideróforos/química , Tiazóis/química , Sequência de Aminoácidos , Dados de Sequência Molecular , Estrutura Molecular , Oxirredução , Fenóis/metabolismo , Pseudomonas/genética , Alinhamento de Sequência , Sideróforos/genética , Sideróforos/metabolismo , Estereoisomerismo , Tiazóis/metabolismoRESUMO
The siderophore pyochelin of Pseudomonas aeruginosa promotes growth under iron limitation and induces the expression of its biosynthesis genes via the transcriptional AraC/XylS-type regulator PchR. Pseudomonas fluorescens strain CHA0 makes the optical antipode of pyochelin termed enantio-pyochelin, which also promotes growth and induces the expression of its biosynthesis genes when iron is scarce. Growth promotion and signalling by pyochelin and enantio-pyochelin are highly stereospecific and are known to involve the pyochelin and enantio-pyochelin outer-membrane receptors FptA and FetA, respectively. Here we show that stereospecificity in signalling is also based on the stereospecificity of the homologous PchR proteins of P. aeruginosa and P. fluorescens towards their respective siderophore effectors. We found that PchR functioned in the heterologous species only if supplied with its native ligand and that the FptA and FetA receptors enhanced the efficiency of signalling. By constructing and expressing hybrid and truncated PchR regulators we showed that the weakly conserved N-terminal domain of PchR is responsible for siderophore specificity. Thus, both uptake and transcriptional regulation confer stereospecificity to pyochelin and enantio-pyochelin biosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fenóis/química , Pseudomonas aeruginosa/metabolismo , Pseudomonas fluorescens/metabolismo , Tiazóis/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Ferro/metabolismo , Ligantes , Dados de Sequência Molecular , Pseudomonas aeruginosa/genética , Sideróforos/metabolismo , Fatores de Transcrição/químicaRESUMO
The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2''R, 4''R (pyochelin I) and 4'R, 2''S, 4''R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.