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Pyoverdines are important siderophores that guarantee iron supply to important pathogenic and non-pathogenic pseudomonads in host habitats. A key characteristic of all pyoverdines is the fluorescent dihydroxyquinoline group that contributes two ligands to the iron complexes. Pyoverdines are derived from the non-ribosomally synthesized peptide ferribactin, and their fluorophore is generated by periplasmic oxidation and cyclization reactions of d-tyrosine and l-diaminobutyric acid. The formation of the fluorophore is known to be driven by the periplasmic tyrosinase PvdP. Here we report that the putative periplasmic oxidoreductase PvdO of Pseudomonas fluorescens A506 is required for the final oxidation of dihydropyoverdine to pyoverdine, which completes the fluorophore. The pvdO deletion mutant accumulates dihydropyoverdine, and this phenotype is fully complemented by recombinant PvdO. The autoxidation of dihydropyoverdine at alkaline pH and the presence of high copper concentrations can mask this phenotype. Mutagenesis of conserved residues with potential catalytic function identified Glu-260 as an essential residue whose mutation abolished function without affecting stability or transport. Glu-260 of PvdO is at the exact position of the active-site cysteine in the structurally related formylglycine-generating enzyme. Evolution thus used the same protein fold for two distinct functionalities. As purified PvdO was inactive, additional factors are required for catalysis., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

From a pyoverdin-negative mutant of Pseudomonas fluorescens AF76 a new lipopeptidic siderophore (ornicorrugatin) could be isolated. It is structurally related to the siderophore of Pseudomonas corrugata differing in the replacement of one Dab unit by Orn.

Structure elucidation of naturally occurring linear and cyclic peptidic compounds can be complicated by rearrangement reactions induced upon collision activation (CA) when parts of the molecule migrate, suggesting incorrect substitution patterns. Such complex rearrangements are examined and discussed for two iron complexing compounds produced by the bacterial genus Pseudomonas (so-called pyoverdins). Various MS2- and MS3-product ion experiments were performed using a quadrupole-ion trap (QIT) at low resolution and a FT-ICR at high resolution allowing accurate mass determinations. The results of the multidimensional study confirm the proposed processes. On the basis of the series of tandem-MS experiments the structure of a new pyoverdin from a P. fluorescens strain [PVD(D47)] is deduced., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

The effect of a bacteriocin of Enterococcus on the oxidative metabolism of sensitive bacteria was investigated through the detection of oxidative stress by chemiluminescence (CL). The bacteriocin named EntB was purified to study the action on Staphylococcus aureus isolated from cosmetic. Chromatographic separation of EntB indicated different states of oligomerization with molecular weights multiple of 12,000Da monomeric form. The monomer purified by ion exchange was studied in its capacity to affect the oxidative metabolism of S. aureus, which showed increase of anion superoxide (O(2)(-)) when incubated with EntB. This effect was compared to the action of EntB on leukocytes as an assay of toxicity. EntB did not generate significant oxidative stress in leukocytes. Pyoverdin, a leukotoxic pigment of Pseudomonas fluorescens, was taken as reference, and it was found that this pigment caused similar oxidative stress to EntB in S. aureus; however, pyoverdin generated high production of anion superoxide (O(2)(-)) in leukocytes, while EntB did not increase the level of O(2)(-).

When a pyoverdin (PV), (a siderophore) from Pseudomonas fluorescens, binds aluminum 1:1, its natural fluorescence almost doubles, whereas PV-Fe is non-fluorescent. Complex formation allows [Al] determination down to 1 mug/l. Fe(III) in the sample interferes with [Al] determination, but added after PV, improves the assay's performance. Ascorbic acid does not eliminate Fe(III) interference. PV-Al fluorescence could have analytical and toxicological applications.

From Pseudomonas chlororaphis D-TR 133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions. Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate. This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules. An unexpected feature is that the main pyoverdine of P. chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly. So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.

The siderophores produced by Pseudomonas fluorescens G173 are unusual in several respects. So far all pyoverdins with a C-terminal cyclopeptidic substructure have in common that the epsilon-amino group of an in-chain Lys is bound amidically to the carboxyl group of a C-terminal Ser or Thr and that N5-formyl-N5-hydroxy Orn (FoOHOrn) is the next amino acid after Lys. FoOHOrn may (cyclotetrapeptidic structures) be or may not (cyclotripeptidic structures) be followed by a further amino acid. In the pyoverdin described here Orn instead of Lys is the amino acid forming the cycle, FoOHOrn is replaced by AcOHOrn which does not follow the branching Orn but is the penultimate amino acid and finally the last amino acid is Asp. The producing strain which had been classified as Pseudomonas fluorescens may well be a new species. Pyoverdins are frequently accompanied by ferribactins which are considered to be their biogenetic precursors. They always have the same amino acid chain as the co-occurring pyoverdins but the pyoverdin chromophore is replaced by a condensation product of L-Dab and D-Tyr with the amino group of Tyr bound to the gamma-carboxyl group of Glu. A ferribactin having these structural characteristics is produced by the investigated strain, but it is accompanied by derivatives where the alpha-amino group of Glu is partially or completely transformed into a hydroxamic acid by substitution with a hydroxyl and/or acetyl group.

From Pseudomonas fluorescens PL7 and PL8 structurally related pyoverdins were isolated and their primary structures were elucidated by spectroscopic methods and degradation reactions. Despite of some structural differences both Fe(III) complexes are taken up by either strain with a high rate. The implications regarding the recognition at the cell surface are discussed.

Pyoverdin was purified by solvent extraction, gel filtration, and ionic exchange chromatography. Assays of cytotoxic of pyoverdin were done with human leukocytes and macrophages from the peritoneum of mice. Both cell quantities showed a significant reduction. Death was followed by lysis in a dose-dependent form. The mechanism of action of pyoverdin involved the stimulation of reactive oxygen species (ROS) measured by Nitroblue Tetrazolium (NBT) reaction and chemiluminescence (CL). UV radiation at 368 nm increased the leukotoxicity; expositions of 5 min were enough to photostimulate the effect of pyoverdin on cellular oxydative metabolism, which increased between 35.4 and 53.2%. Genestein, an inhibitor of tyrosine kinases, counteracted the ROS stimuli of pyoverdin, suggesting endocytic mechanism of action for this pigment. The little chloroquine interference on oxydative stress indicated that intraphagosomal pH and the stimuli of reactive nitrogen intermediaries (RNI) seem to be of less importance than ROS in pyoverdin action on leukocytes., (Copyright 2001 Academic Press.)

By comparison of the NMR data of the ferribactins from Pseudomonas chlororaphis ATCC 9446 and of P. fluorescens 18.1 with those of their Ga3+-complexes as models for the Fe3+-complexes it will be shown that only two bidentate ligands are provided for complexation, both located in the peptide chain. The two remaining free sites of the octahedral metal ion are probably occupied by solvent molecules.

The structure of the pyoverdin siderophore of Pseudomonas fluorescens 18.1 was elucidated by spectroscopic methods and chemical degradation. By cross feeding studies structurally closely related pyoverdins containing a C-terminal cyclopeptidic substructure were tested regarding the mutual recognition by the producing strains. Partial recognition of foreign pyoverdins was observed.

Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, D- and L-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins.

The effect of three forms pyoverdin on mouse liver was studied. Significant increases of alanine amino transferase (ALT) and aspartate amino transferase (AST) were obtained in mice after ingestion of water with forms A and C. The effect on liver was more evident with A than with C. Pyoverdin was purified by means of salt saturation, solvent extractions and ion-exchange chromatography. Fluorescent peaks obtained in the presence of light were different from those eluted under dark conditions. The relative amounts of pyoverdin A, B and C varied when dark purification procedure was employed. Form A decreased while C increased in the absence of light. Optimum conditions for C were in the dark without iron. When C was exposed to light, it changed to form A. Fast Atom Bombardment (FAB) mass spectrometry of pyoverdin form C gave a form at M+ = 1324 m.u., which is 9 m.u. less than pyoverdin purified in the presence of light. The results suggest that light can influence pyoverdin stability and toxicity.

When Pseudomonas putida ATCC 39167 and plant-deleterious Pseudomonas fluorescens A225 were grown in an iron-deficient culture medium, they each produced two different novel yellow-green fluorescent pseudobactins: P39167-I, II and PA225-I, II. Pseudobactin P39167-I has a molecular formula of C46H65O23N13 and is monoanionic at neutral pH. P39167-II has the molecular formula of C46H63O22N13 and no charge at neutral pH. Pseudobactin PA225-I has a molecular formula of C46H65O24N13 and is monoanionic at neutral pH whereas pseudobactin PA225-II has the molecular formula of C46H63O23N13 and no charge at neutral pH. All four of the pseudobactins contain a dihydroxyquinoline-based chromophore. The amino acid sequence for the octapeptide in case of pseudobactins from P. putida ATCC 39167 is Chr-Ser(1)-Ala(1)-AcOHOrn-Gly-Ala(2)-OHAsp-Ser(2)-Thr. In case of pseudobactins from P. fluorescens A225, the octapeptide has the sequence Chr-Ser(1)-Ala-AcOHOrn-Gly-Ser(2)-OHAsp-Ser(3)-Thr. For all four pseudobactins (P39167-I, II and PA225-I, II), the serine(1) residue of the octapeptide is attached to the carboxylic acid group on the C-11 of the fluorescent quinoline via an amide bond. Additionally, for pseudobactin P39167-II and PA225-II, the hydroxyl group of the serine(1) residue is also attached to the carboxyl group of threonine residue at the carboxy terminus of the peptide via an ester bond, resulting in a cyclic depsipeptide in contrast to the linear peptide chain of P39167-I and PA225-I. For all four pseudobactins, a malamide group is attached to the C-3 of the quinoline derived chromophore. The three bidentate iron(III) chelating groups in all four pseudobactins consist of a 1,2-dihydroxy aromatic group of the fluorescent chromophore, a hydroxy acid group of beta-hydroxy aspartic acid, and a hydroxamate group from the acylated Ndelta-hydroxyornithine. The amino acid constituents of the pseudobactins P39167 I, II are the same as those in pseudobactin A214, whereas those in A225 I, II are the same as in 7SR1, but in both cases the sequences are different. The uptake results indicate a single outer membrane receptor protein for ferric-pseudobactins in both organisms. The receptor proteins in the two species are similar but not identical.

Pseudomonas fluorescens grows and produces pigment in refrigerated human blood at 4 degrees C. Saline precipitation of plasma showed that globulin and albumin fractions retained pyoverdin at different concentrations. By dialysis it was possible to determine that the pigment attached or aggregated to the protein in total plasma as well as in the fraction obtained by saline precipitation. A greater binding was observed at the globulin fraction. Gamma-globulin immunological activity was reduced due to the interaction with pyoverdin, as much in agglutination reaction (VDRL) as in neutralization (Streptolysin O).

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C18 reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mM NH4HCO3/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P.fluorescens B10 (JL-3133).

From a strain of Pseudomonas fluorescens pseudobactin and several related compounds were isolated and their structures were elucidated. In this way a reference compound (5) could be obtained for the unambiguous determination of the absolute configuration of C-1 of the pyoverdin chromophore in newly isolated representatives of this class.