Metal uptake by pyochelin
Iron is an essential cofactor for almost all living organisms, including bacteria. However, the solubility of iron is very low under aerobic conditions at neutral pH, and in many environments bioavailable iron is not sufficient to sustain bacterial growth. Microbial pathogens encounter an iron-restricted environment also in the host where iron is bound to proteins (hemoglobin, myoglobin, cytochromes) or to high-affinity iron-carriers (transferrin, lactoferrin, ferritin). To acquire iron under these conditions, bacteria have evolved a number of different strategies, the most common is siderophore-mediated iron uptake. Siderophores are high-affinity iron chelators which are released to the extracellular environment where they complex iron and deliver it to the bacterial cell, via specific outer membrane receptors. Our lab is interested in pyochelin, a siderophore made by the opportunistic human pathogen Pseudomonas aeruginosa. Pyochelin biosynthesis occurs by a thiotemplate mechanism from salicylate and two molecules of cysteine. We have cloned the genes involved and have contributed to the elucidation of the biosynthetic pathway. In P. 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). We recently found that the Pseudomonas fluorescens strains Pf-5 and CHA0 make a different stereoisomeric mixture and we determined its structure to be enantio-pyochelin, the optical antipode of the P. aeruginosasiderophore. The biosynthesis of enantio-pyochelin is currently being investigated in our laboratory.
Biosynthesis of siderophores and their cognate uptake systems is tightly regulated to assure that they are produced only when needed and to avoid accumulation of iron which can be deleterious to the cell. Under iron-rich conditions, the Fur protein represses siderophore biosynthesis and uptake genes. Fur-mediated repression is alleviated when iron becomes limiting, allowing a basal level of gene expression to occur. Full expression often requires the presence of the siderophore. By a variety of different mechanisms, involving extracellular cytoplasmic function [ECF] sigma/anti-sigma factors, two-component regulatory systems, and AraC-type regulators, the siderophore induces the expression of genes necessary for its uptake and, in certain cases, also for its biosynthesis. We found that in P. aeruginosa the expression of the pyochelin biosynthetic and uptake genes requires the AraC-type transcriptional regulator PchR. In the presence of pyochelin, PchR binds to a conserved sequence (PchR-box) in the promoter region of these operons and regulates their transcription. A similar mechanism seems to occur in P. fluorescens as enantio-pyochelin and a PchR homolog were found to be required for the expression of enantio-pyochelin biosynthesis and uptake genes. We are in the process of investigating the interaction of the two PchR proteins with their corresponding effector molecule by fluorescence measurements and isothermal titration calorimetry.
Iron uptake with pyochelin and enantio-pyochelin is strictly stereospecific, meaning that neither siderophore is functional as an iron carrier or transcriptional inducer in the other species. Genetic, biochemical, and structural data have revealed that stereospecificity occurs (i) at the outer membrane, by the siderophore receptors FptA and FetA, and (ii) in the cytoplasm, by the two regulatory proteins PchR. In addition, we recently found that in P. fluorescens, the periplasmic binding protein-dependent ABC transporter FetCDE translocated enantio-pyochelin but not pyochelin across the inner membrane. We are currently studying the stereospecificity of the two PchR proteins in more detail.
1. L. Serino, C. Reimmann, H. Baur, M. Beyeler, P. Visca & D. Haas. 1995. Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa. Mol. Gen. Genet. 249, 217-228.
2. L. Serino, C. Reimmann, P. Visca, M. Beyeler, V. Della Chiesa & D. Haas. 1997. Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa. J. Bacteriol. 179, 248-257.
3. C. Reimmann, L. Serino, M. Beyeler & D. Haas. 1998. Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa. Microbiology 144, 3135-3148.
4. C. Reimmann, H.M. Patel, L. Serino, M. Barone, C.T. Walsh & D. Haas. 2001. Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa. J. Bacteriol. 183, 813-820.
5. C. Gaille, C. Reimmann & D. Haas. 2003. Isochorismate synthase (PchA), the first and rate-limiting enzyme in salicylate biosynthesis of Pseudomonas aeruginosa. J. Biol. Chem. 278, 16893-16898.
6. C. Reimmann, H. Patel, C.T. Walsh & D. Haas. 2004. PchC thioesterase optimizes the biosynthesis of the non-ribosomal peptide siderophore pyochelin in Pseudomonas aeruginosa. J. Bacteriol. 186, 6367-6373.
7. L. Michel, N. González, S. Jagdeep, T. Nguyen-Ngoc & C. Reimmann. 2005. PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector. Mol. Microbiol. 58, 495-509.
8. L. Michel, A. Bachelard & C. Reimmann. 2007. Ferripyochelin uptake genes are involved in pyochelin mediated signaling in Pseudomonas aeruginosa. Microbiology 153, 1508-1518.
9. Z.A. Youard, G.A. Mislin, P.A. Majcherczyk, I.J. Schalk & C. Reimmann. 2007. Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin. J. Biol. Chem. 282, 35546-35553.
10. F. Hoegy, X. Lee, S. Noel, D. Rognan, G.L.A. Mislin, C. Reimmann & I.J. Schalk. 2009. Stereospecificity of the siderophore pyochelin outer membrane transporters in fluorescent pseudomonads. J. Biol. Chem. 284, 14949-14957.
11. Z.A. Youard & C. Reimmann. 2010. Stereospecific recognition of pyochelin and enantio-pyochelin by the PchR proteins in fluorescent pseudomonads. Microbiology 156, 1772-1782.
12. Youard, N. Wenner & C. Reimmann. 2011. Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species.
Biometals 24, 513-522.
13. K. Brillet, C. Reimmann, G. Mislin, S. Noël, D. Rognan, I.J. Schalk & D. Cobessi. 2011. Pyochelin enantiomers and their outer-membrane siderophore transporters in fluorescent pseudomonads: structural bases for unique enantiospecific recognition. J. Am. Chem. Soc. 133, 16503-16509.
Swiss National Science Foundation for Scientific research (Project 3100A0-132998)