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.     

Specific inactivation of an antifungal bacterial siderophore by a fungal plant pathogen

Bacteria and fungi secrete many natural products that inhibit each other’s growth and development. The dynamic changes in secreted metabolites that occur during interactions between bacteria and fungi are complicated. Pyochelin is a siderophore produced by many Pseudomonas and Burkholderia species that induces systemic resistance in plants and has been identified as an antifungal agent. Through imaging mass spectrometry and metabolomics analysis, we found that Phellinus noxius, a plant pathogen, can modify pyochelin and ent-pyochelin to an esterification product, resulting in reduced iron-chelation and loss of antifungal activity. We also observed that dehydroergosterol peroxide, the fungal metabolite, is only accumulated in the presence of pyochelin produced through bacteria–fungi interactions. For the first time, we show the fungal transformation of pyochelin in the microbial interaction. Our findings highlight the importance of understanding the dynamic changes of metabolites in microbial interactions and their influences on microbial communities.Subject terms: Microbial ecology, Metabolomics

Microorganisms use various strategies to establish themselves within an ecological niche while facing keen competition in the environment. Natural products such as antibiotics, quorum sensing molecules, and siderophores are crucial in microbial interactions [1–3]. Certain microorganisms are equipped with uptake systems that enable them to acquire siderophores, even by those that may not produce them [4]. For example, pyochelin is a siderophore produced by many Pseudomonas and Burkholderia strains. Such bacterial strains are commonly found in soils, as endophytes, and from the rhizosphere where they may inhibit plant pathogens [5, 6].Burkholderia cenocepacia 869T2 was isolated as an endophyte and showed beneficial abilities to control banana Fusarium wilt [7]. It harbors many biosynthetic gene clusters of secondary metabolites, such as pyochelin, pyrrolnitrin, and pyrroloquinoline quinone [8]. Recently, we found that this strain could temporarily inhibit the growth of P. noxius, a fungal pathogen of brown root rot disease, which is prevalent in tropical and subtropical regions and has a wide host range covering over 200 plant species [9]. However, in the competition between fungi and bacteria, P. noxius can resist this inhibition and overwhelm bacterial colonies after 1–2 weeks under dual-culture conditions (Fig. S1). These results imply that fungi might have resistance responses and undergo metabolic changes in bacteria–fungi interactions [10]. Here we unveiled metabolic changes in the competitive interaction between B. cenocepacia 869T2 and P. noxius 2252 using the matrix-assisted laser desorption ionization-time of flight imaging mass spectrometry (MALDI-TOF IMS) [11, 12].We specifically monitored the metabolites in the inhibition region of B. cenocepacia 869T2 and P. noxius 2252 dual-culture using MALDI-TOF IMS. Several induced or enzymatically modified metabolites were detected, including m/z 275, 362, 383, and 427 (Fig. 1A). In particular, pyochelin (m/z 325), surrounding the B. cenocepacia 869T2 colony, showed asymmetric distribution in dual-culture samples. Near the P. noxius 2252 mycelia, a new metabolite with m/z 383 was detected with a complementary distribution to pyochelin (Fig. 1A). In LC-MS/MS-based molecular networking analysis [13], we found that this new metabolite structure is an esterification product of pyochelin and glycolic acid, which we named pyochelin-GA (Fig. 1B). We then constructed a pchF-null mutant strain, ΔpchF, which cannot produce pyochelin, and then dual cultured it with P. noxius. Pyochelin and pyochelin-GA were not observed in the MALDI-TOF IMS and LC-MS analysis of dual-culture samples (Fig. 1A and Fig. S2). We further inoculated P. noxius 2252 with pyochelin-GA-free extract harvested from B. cenocepacia 869T2 single culture, and the complementary distribution of pyochelin and pyochelin-GA was observed by MALDI-TOF IMS again (Fig. S3). These results demonstrated that pyochelin-GA was transformed from pyochelin by P. noxius 2252, rather than produced by B. cenocepacia 869T2 under dual-culture conditions.Open in a separate windowFig. 1Metabolic changes in the bacteria–fungi interaction.A Spatial distribution of selected mass signals (m/z) in MALDI-TOF IMS analysis of Phellinus noxius 2252 (Pn2252) dual-cultured with Burkholderia cenocepacia 869T2 (869T2) and a pchF-null mutant strain (Δ pchF). B Molecular networking analysis of pyochelin and analogs from the dual-culture sample. The red node is pyochelin, and the green node is pyochelin-GA. The structures of pyochelin, pyochelin-GA, and dehydroergosterol peroxide (DHEP), together with their mass signals in MALDI-TOF IMS, are shown. C Iron-chelating abilities of pyochelin and pyochelin-GA were evaluated by Chrome Azurol S liquid assay using different concentrations (2.5, 1.25, 0.63, 0.31, and 0.16 mM, n = 3). Proportions of siderophore units are shown in Fig. S14. D Fungal transformation of pyochelin and ent-pyochelin by treating P. noxius 2252 with ethyl acetate crude extracts of B. cenocepacia 869T2, Pseudomonas aeruginosa PAO1, and P. protegens Pf-5 for 8 h. LC-MS was used to monitor the signals of pyochelin (red), ent-pyochelin (blue), and transformation product 383 (black).The chemical structure of pyochelin-GA was further confirmed via total synthesis, NMR, and LC-MS/MS analysis (Supplementary Material and Methods, and Figs. S4–7). The purified pyochelin and pyochelin-GA were also evaluated for their iron-chelating ability. Chrome Azurol S assay indicated that pyochelin had the dose-dependent iron-chelating ability, but pyochelin-GA had lower iron-binding efficiency (Fig. 1C, Fig. S8). Pyochelin chelates iron in the extracellular medium and transports it into cells via the specific outer membrane transporter FptA. The X-ray structure of FptA-pyochelin-Fe indicated that the terminal carboxylic acid of pyochelin plays an essential role in the iron uptake ability [14, 15]. Our docking analysis suggested that the glycolic ester moiety of pyochelin-GA would affect the binding pocket shape of FptA and result in different binding properties compared to FptA-pyochelin (Fig. S9).Pyochelin and ent-pyochelin are produced independently by different biosynthetic gene clusters in Pseudomonas species [16]. To determine whether P. noxius 2252 can transform both enantiomers via this esterification process, we treated P. noxius 2252 with the extracts of pyochelin producers (P. aeruginosa PAO1 and B. cenocepacia 869T2) and an ent-pyochelin producer (P. protegens Pf-5). After 8 h of treatment, both pyochelin and ent-pyochelin were converted to pyochelin-GA (or ent-pyochelin-GA) (Fig. 1D), demonstrating this is a non-stereospecific transformation.To better understand the iron-chelating ability of pyochelin, we used pyochelin and pyochelin-GA to treat P. noxius 2252 under iron-deficiency conditions, by adding the iron chelator deferoxamine, and iron-rich conditions by adding FeCl₃ (Fig. 2). Pyochelin-GA did not affect the growth of P. noxius 2252 under all conditions. However, P. noxius 2252 was more sensitive to pyochelin in iron-deficient conditions and more resistant to pyochelin in iron-rich conditions, demonstrating that iron availability directly affected the tolerance of P. noxius 2252 to pyochelin. A similar phenomenon was reported previously for Aspergillus fumigatus [17].Open in a separate windowFig. 2Pyochelin inhibition of mycelial growth of Phellinus noxius 2252 is inversely associated with iron concentration.Pyochelin-GA did not have an inhibition effect on P. noxius 2252. Potato dextrose agar (PDA) with deferoxamine (DFO; 200 and 400 µM) was used to mimic iron-deficiency conditions. Iron-rich conditions was prepared by adding FeCl₃ (200 and 400 µM) in PDA. P. noxius 2252 was treated with 0.03, 0.06, 0.12, and 0.24 µmol of pyochelin or pyochelin-GA at 30 °C for 24 h. The antifungal assay was performed in two biological replicates.Using MALDI-TOF IMS analysis of the dual-culture of B. cenocepacia 869T2 and P. noxius 2252, we observed that several metabolites (e.g., m/z 275, 362, and 427) were only observed in the boundary of fungal mycelia (Fig. 1A). Although those metabolites were not detected in the dual-culture of ΔpchF and P. noxius 2252 (Fig. 1A), they were present when we treated P. noxius 2252 with pyochelin (Fig. S10). We identified the metabolite associated with m/z 427 as dehydroergosterol peroxide (DHEP) (Fig. S11), which was initially oxidized from ergosterol and dehydroergosterol [18]. Pyochelin can enhance intercellular reactive oxygen species (ROS) and ultimately disrupts membrane integrity, leading to cell death [17, 19, 20]. To clarify whether ROS induced the accumulation of DHEP, we treated P. noxius 2252 with pyochelin, pyochelin-GA, and 2,2′-bipyridyl (an iron chelator). Pyochelin and 2,2′-bipyridyl showed antifungal effects on P. noxius 2252 and induced ROS production (Fig. S12). However, the accumulation of DHEP in P. noxius 2252 was only associated with pyochelin treatment (Fig. S13). The induction of ROS in P. noxius 2252 by pyochelin and pyochelin-GA was not significantly different (Fig. S14). Therefore, we predict that pyochelin-induced accumulation of DHEP in P. noxius 2252 is independent of ROS production and iron-deficiency.Overall, we demonstrate that pyochelin transformation by fungi, in the interaction between pyochelin-producing bacteria and the plant pathogen P. noxius transforms pyochelin and ent-pyochelin into pyochelin-GA (and ent-pyochelin-GA). This product no longer functions as an iron chelator and no longer shows antifungal activity. The production of a fungal metabolite, dehydroergosterol peroxide, was induced explicitly by pyochelin through an unknown mechanism. These results highlight the importance of monitoring dynamic changes of metabolites in situ to better understand the functions and influences of metabolites on microbial community interactions.     

Synthesis and biological properties of thiazole-analogues of pyochelin,a siderophore of Pseudomonas aeruginosa

Pyochelin is a siderophore common to all strains of Pseudomonas aeruginosa utilized by this Gram-negative bacterium to acquire iron(III). FptA is the outer membrane transporter responsible of ferric-pyochelin uptake in P. aeruginosa. We describe in this Letter the synthesis and the biological properties (⁵⁵Fe uptake, binding to FptA) of several thiazole analogues of pyochelin. Among them we report in this Letter the two first pyochelin analogues able to bind FptA without promoting any iron uptake in P. aeruginosa.     

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.     

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa’s ability to colonize the GI tract but does decrease P. aeruginosa’s cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.     

Switching between apparently redundant iron-uptake mechanisms benefits bacteria in changeable environments

Bacteria often possess multiple siderophore-based iron uptake systems for scavenging this vital resource from their environment. However, some siderophores seem redundant, because they have limited iron-binding efficiency and are seldom expressed under iron limitation. Here, we investigate the conundrum of why selection does not eliminate this apparent redundancy. We focus on Pseudomonas aeruginosa, a bacterium that can produce two siderophores—the highly efficient but metabolically expensive pyoverdine, and the inefficient but metabolically cheap pyochelin. We found that the bacteria possess molecular mechanisms to phenotypically switch from mainly producing pyoverdine under severe iron limitation to mainly producing pyochelin when iron is only moderately limited. We further show that strains exclusively producing pyochelin grew significantly better than strains exclusively producing pyoverdine under moderate iron limitation, whereas the inverse was seen under severe iron limitation. This suggests that pyochelin is not redundant, but that switching between siderophore strategies might be beneficial to trade off efficiencies versus costs of siderophores. Indeed, simulations parameterized from our data confirmed that strains retaining the capacity to switch between siderophores significantly outcompeted strains defective for one or the other siderophore under fluctuating iron availabilities. Finally, we discuss how siderophore switching can be viewed as a form of collective decision-making, whereby a coordinated shift in behaviour at the group level emerges as a result of positive and negative feedback loops operating among individuals at the local scale.     

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 muM 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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.