PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector

Under iron limitation, the opportunistic human pathogen Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted into the extracellular environment, pyochelin complexes ferric ions and delivers them, via the outer membrane receptor FptA, to the bacterial cytoplasm. Extracellular pyochelin also acts as a signalling molecule, inducing the expression of pyochelin biosynthesis and uptake genes by a mechanism involving the AraC-type regulator PchR. We have identified a 32 bp conserved sequence element (PchR-box) in promoter regions of pyochelin-controlled genes and we show that the PchR-box in the pchR-pchDCBA intergenic region is essential for the induction of the pyochelin biosynthetic operon pchDCBA and the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin and iron. PchR-box mutations that interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. We conclude that pyochelin, probably in its iron-loaded state, is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation suggests that the siderophore can enter the cytoplasm.     

Organotin Decomposition by Pyochelin, Secreted by Pseudomonas aeruginosa Even in an Iron-Sufficient Environment

A triphenyltin (TPT)-decomposing strain, Pseudomonas aeruginosa CGMCC 1.860, was screened out. It secreted an unknown TPT-decomposing factor into the medium, later shown to be pyochelin, even in the presence of 100 μM iron. To our knowledge, this is the first report of organotin decomposition by pyochelin.     

Involvement of Pyochelin and Pyoverdin in Suppression of Pythium-Induced Damping-Off of Tomato by Pseudomonas aeruginosa 7NSK2

The plant growth-promoting rhizobacterium Pseudomonas aeruginosa 7NSK2 produces three siderophores when iron is limited: the yellow-green fluorescent pyoverdin, the salicylate derivative pyochelin, and salicylic acid. This Pseudomonas strain was shown to be an efficient antagonist of Pythium-induced damping-off. The role of pyoverdin and pyochelin in the suppression of Pythium splendens was investigated by using various siderophore-deficient mutants derived from P. aeruginosa 7NSK2 in a bioassay with tomato (Lycopersicon esculentum). To provide more insight into the role of pyochelin in antagonism, mutant KMPCH, deficient in the production of pyoverdin and pyochelin, was complemented for pyochelin production. The complementing clone was further characterized by subcloning and transposon mutagenesis and used to generate a pyochelin-negative, pyoverdin-positive mutant by marker exchange. All mutants were able to reduce Pythium-induced preemergence damping-off to some extent. Production of either pyoverdin or pyochelin proved to be necessary to achieve wild-type levels of protection against Pythium-induced postemergence damping-off. Mutant KMPCH inhibited P. splendens but was less active than the parental strain. This residual protection could be due to the production of salicylic acid. Since pyoverdin and pyochelin are both siderophores, siderophore-mediated iron competition could explain the observed antagonism and the apparent interchangeability of the two compounds. We cannot, however, exclude the possibility that both siderophores act in an indirect way.     

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin

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.     

Isolation and characterization of Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis.

Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis were isolated after chemical mutagenesis by plating on a siderophore detection medium. Like the wild type, these mutants incorporated 7-[14C]salicylic acid into pyochelin, demonstrating that salicylic acid is an intermediate in the biosynthesis pathway of pyochelin.     

Isolation and characterization of Burkholderia cenocepacia mutants deficient in pyochelin production: pyochelin biosynthesis is sensitive to sulfur availability

The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.     

Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.     

Siderophore synthesis by mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients.

Nonmucoid Pseudomonas aeruginosa responds to iron deprivation by synthesizing the siderophores pyochelin and pyoverdine. When grown in iron-deficient medium, six mucoid P. aeruginosa strains isolated from cystic fibrosis patients synthesized copious amounts of the exopolysaccharide alginate. A procedure that eliminated the interference of alginate was developed so that siderophores could be extracted from the growth medium. All six isolates were then noted to produce both pyoverdine and pyochelin. This report thus confirms that mucoid P. aeruginosa, like its nonmucoid counterparts, elicits the siderophores commonly cited as those of the microbe.     

Postweaning diarrhoea in pigs: an interaction between change to dry food and colibacillosis

Abstract Production of pyochelin by Pseudomonas species was measured in ethyl acetate extracts of culture supernatants. Pyochelin was purified by paper chromatography, followed by two-dimensional, thin-layer chromatography. Strains of P. cepacia , P. multivorans , and P. fluorescens produced pyochelin, while P. stutzeri , P. putida and P. maltophila were negative for pyochelin production. Outer membranes prepared from these strains had a protein of M _r 14000 which was shown to bind [⁵⁹Fe]pyochelin.     

Synthesis and biological activity of pyochelin, a siderophore of Pseudomonas aeruginosa.

Pyochelin, a phenolic siderophore of Pseudomonas aeruginosa, was synthesized in three steps from salicylonitrile, L-cysteine, and L-N-methylcysteine. The synthetic product was determined to be identical to natural pyochelin by 1H nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, chromatographic analysis, and chemical reactivity with FeCl3 and ammoniacal silver nitrate reagent. Synthetic and natural pyochelin promoted bacterial growth in iron-depleted medium and were also found to mediate iron transport by P. aeruginosa to the same levels. Neopyochelin, a stereoisomeric by-product of the synthesis, showed less biological activity than did pyochelin in iron transport assays.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes.

Pseudomonas aeruginosa synthesizes two siderophores, pyochelin and pyoverdin, characterized by widely different structures, physicochemical properties, and affinities for Fe(III). Titration experiments showed that pyochelin, which is endowed with a relatively low affinity for Fe(III), binds other transition metals, such as Cu(II), Co(II), Mo(VI), and Ni(II), with appreciable affinity. In line with these observations, Fe(III) and Co(II) at 10 microM or Mo(VI), Ni(II), and Cu(II) at 100 microM repressed pyochelin synthesis and reduced expression of iron-regulated outer membrane proteins of 75, 68, and 14 kDa. In contrast, pyoverdin synthesis and expression of the 80-kDa receptor protein were affected only by Fe(III). All of the metals tested, except Mo(VI), significantly promoted P. aeruginosa growth in metal-poor medium; Mo(VI), Ni(II), and Co(II) were more efficient as pyochelin complexes than the free metal ions and the siderophore. The observed correlation between the affinity of pyochelin for Fe(III), Co(II), and Mo(VI) and the functional effects of these metals indicates that pyochelin may play a role in their delivery to P. aeruginosa.     

Crystal structure at high resolution of ferric-pyochelin and its membrane receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Bacterial siderophores: synthesis and biological activities of novel pyochelin analogues

The synthesis and biological activities of four pyochelin analogues substituted in different parts of the molecule are reported: 5-NHBoc-pyochelin, 3"N-Boc-pyochelin, 3"-nor-NH-pyochelin and neopyochelin II, the enantiomer of natural pyochelin. All these compounds complex iron(III) and transport it at different rates into the cells of Pseudomonas aeruginosa.     

Evolutionary dynamics of interlinked public goods traits: an experimental study of siderophore production in Pseudomonas aeruginosa

Public goods cooperation is common in microbes, and there is much interest in understanding how such traits evolve. Research in recent years has identified several important factors that shape the evolutionary dynamics of such systems, yet few studies have investigated scenarios involving interactions between multiple public goods. Here, we offer general predictions about the evolutionary trajectories of two public goods traits having positive, negative or neutral regulatory influence on one another's expression, and we report on a test of some of our predictions in the context of Pseudomonas aeruginosa's production of two interlinked iron‐scavenging siderophores. First, we confirmed that both pyoverdine and pyochelin siderophores do operate as public goods under appropriate environmental conditions. We then tracked their production in lines experimentally evolved under different iron‐limitation regimes known to favour different siderophore expression profiles. Under strong iron limitation, where pyoverdine represses pyochelin, we saw a decline in pyoverdine and a concomitant increase in pyochelin – consistent with expansion of pyoverdine‐defective cheats derepressed for pyochelin. Under moderate iron limitation, pyochelin declined – again consistent with an expected cheat invasion scenario – but there was no concomitant shift in pyoverdine because cross‐suppression between the traits is unidirectional only. Alternating exposure to strong and moderate iron limitation caused qualitatively similar though lesser shifts compared to the constant‐environment regimes. Our results confirm that the regulatory interconnections between public goods traits can significantly modulate the course of evolution, yet also suggest how we can start to predict the impacts such complexities will have on phenotypic divergence and community stability.     

Effect of pH on production of virulence factors by biofilm cells ofPseudomonas aeruginosa

Effect of pH on production of extracellular virulence factors of Pseudomonas aeruginosa grown on catheter in biofilm was determined. Alginate and proteinase production was higher at pH 8; in contrast, siderophores (pyochelin and pyoverdin) were synthesized more intensively at pH 5.     

Assessment of structural features of the pseudomonas siderophore pyochelin required for its ability to promote oxidant-mediated endothelial cell injury

We previously showed that iron chelated to the Pseudomonas aeruginosa siderophore pyochelin enhances oxidant-mediated injury to pulmonary artery endothelial cells by catalyzing hydroxyl radical (HO(*)) formation. Therefore, we examined pyochelin structural/chemical features that may be important in this process. Five pyochelin analogues were examined for (i) capacity to accentuate oxidant-mediated endothelial cell injury, (ii) HO(*) catalytic ability, (iii) iron transfer to endothelial cells, and (iv) hydrophobicity. All compounds catalyzed similar HO(*) production, but only the hydrophobic ones containing a thiazolidine ring enhanced cell injury. Transfer of iron to endothelial cells did not correlate with cytotoxicity. Finally, binding of Fe(3+) by pyochelin led to Fe(2+) formation, perhaps explaining how Fe(3+)-pyochelin augments H(2)O(2)-mediated cell injury via HO(*) formation. The ability to bind iron in a catalytic form and the molecule's thiazolidine ring, which increases its hydrophobicity, are key to pyochelin's cytotoxicity. Reduction of Fe(3+) to Fe(2+) may also be important.     

PchC thioesterase optimizes nonribosomal biosynthesis of the peptide siderophore pyochelin in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the antibiotic dihydroaeruginoate (Dha) and the siderophore pyochelin are produced from salicylate and cysteine by a thiotemplate mechanism involving the peptide synthetases PchE and PchF. A thioesterase encoded by the pchC gene was found to be necessary for maximal production of both Dha and pyochelin, but it was not required for Dha release from PchE and could not replace the thioesterase function specified by the C-terminal domain of PchF. In vitro, 2-aminobutyrate, a cysteine analog, was adenylated by purified PchE and PchF proteins. In vivo, this analog strongly interfered with Dha and pyochelin formation in a pchC deletion mutant but affected production of these metabolites only slightly in the wild type. Exogenously supplied cysteine overcame the negative effect of a pchC mutation to a large extent, whereas addition of salicylate did not. These data are in agreement with a role for PchC as an editing enzyme that removes wrongly charged molecules from the peptidyl carrier protein domains of PchE and PchF.