The structure of a pyoverdine from Pseudomonas sp. CFML 96.188 and its relation to other pyoverdines with a cyclic C-terminus

From Pseudomonas sp. CFML 96.188 a pyoverdine was isolated and its primary structure was elucidated by spectroscopic methods and degradation reactions. This strain is of interest as it accepts the structurally different pyoverdines from several other Pseudomonas strains. They all have in common as a specific structural feature a C-terminal cyclic substructure, the importance of which for the recognition of a pyoverdine at the cell surface of a given strain will be discussed.     

The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate

Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii. This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P. reactans, both belonging to the collective species P. fluorescens. A recent study has shown that a group of P. tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al. 2000). One group of strains is characterized by the pyoverdine described by Demange et al. (1990). A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P. aureofaciens. However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical. They are also distinguishable from the P. tolaasii type strain.     

Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations

The lungs of cystic fibrosis patients are frequently colonized by Pseudomonas aeruginosa, which produces high-affinity fluorescent peptidic siderophores, pyoverdines. Three pyoverdines which differ in their peptide chain and are easily differentiated by isoelectric focusing exist, only one being produced by a given strain. P. aeruginosa isolates from cystic fibrosis patients of a German hospital were analyzed by sequential, pulse-field gel electrophoresis (PFGE) and for pyoverdine production and type. Only producers of type I and type II pyoverdine were found. There was a perfect correlation between the type of pyoverdine produced and the clonality determined by PFGE. PFGE clone C, the most prevalent among cystic fibrosis patients, and found in an aquatic environment, produced type II pyoverdine. Pyoverdine-negative mutants seemed to increase as a function of the lung colonization time, but retained the capacity to take up pyoverdines. Most isolates that took up type II pyoverdine were also able to utilize type I pyoverdine as judged by growth stimulation experiments. No correlation was observed between the loss of pyoverdine production and mucoidy.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

Characterization of the pyoverdines ofAzotobacter vinelandii ATCC 12 837 with regard to heterogeneity

Summary Azotobacter vinelandii strain ATCC 12 837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows thatA. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to theAzotobacter-type family of pyoverdines.     

Tin-Carbon Cleavage of Organotin Compounds by Pyoverdine from Pseudomonas chlororaphis

The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu²⁺ and Sn⁴⁺, respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.     

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.     

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake

Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.     

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa

Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.     

Defined media for optimal pyoverdine production by Pseudomonas fluorescens 2-79

Pseudomonas fluorescens strain 2-79 (NRRL-15132) produces a fluorescent yellow-green pyoverdine when cultured on Fe(III)-poor medium. When cultured on Fe(III)-rich medium, strain 2-79 produces an antibiotic, phenazine 1-carboxylic acid, which is effective in suppressing plant fungal diseases such as take-all of wheat. A 2³ factorial design was used to examine pyoverdine production as a function of the presence or absence of Bacto casamino acids, purines-pyrimidines and vitamins in an iron-deficient medium. Amino acids were found to be an important factor (P=0.0002). A Plackett-Burman design was used to identity eight amino acids, out of the 19 present in casamino acids, that were responsible for the increased pyoverdine production: methionine, valine, isoleucine, tyrosine, proline, phenylalanine, glutamic acid, and glycine. Biomass was enhanced only by glutamic acid. Correspondence to: W. S. Kisaalita     

Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida

We investigated the regulation of the psbA and pvdA pyoverdine biosynthesis genes, which encode the L-ornithine N(5)-oxygenase homologues in Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1, respectively. We demonstrate that pyoverdine(B10), as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, pyoverdine(PAO1) has no apparent role in the positive regulation of the pvdA gene.     

Tin-carbon cleavage of organotin compounds by pyoverdine from Pseudomonas chlororaphis

The triphenyltin (TPT)-degrading bacterium Pseudomonas chlororaphis CNR15 produces extracellular yellow substances to degrade TPT. Three substances (F-I, F-IIa, and F-IIb) were purified, and their structural and catalytic properties were characterized. The primary structure of F-I was established using two-dimensional nuclear magnetic resonance techniques; the structure was identical to that of suc-pyoverdine from P. chlororaphis ATCC 9446, which is a peptide siderophore produced by fluorescent pseudomonads. Spectral and isoelectric-focusing analyses revealed that F-IIa and F-IIb were also pyoverdines, differing only in the acyl substituent attached to the chromophore part of F-I. Furthermore, we found that the fluorescent pseudomonads producing pyoverdines structurally different from F-I showed TPT degradation activity in the solid extracts of their culture supernatants. F-I and F-IIa degraded TPT to monophenyltin via diphenyltin (DPT) and degraded DPT and dibutyltin to monophenyltin and monobutyltin, respectively. The total amount of organotin metabolites produced by TPT degradation was nearly equivalent to that of the F-I added to the reaction mixture, whereas DPT degradation was not influenced by monophenyltin production. The TPT degradation activity of F-I was remarkably inhibited by the addition of metal ions chelated with pyoverdine. On the other hand, the activity of DPT was increased 13- and 8-fold by the addition of Cu(2+) and Sn(4+), respectively. These results suggest that metal-chelating ligands common to pyoverdines may play important roles in the Sn-C cleavage of organotin compounds in both the metal-free and metal-complexed states.     

Pyoverdine and histicorrugatin-mediated iron acquisition in <Emphasis Type="Italic">Pseudomonas thivervalensis</Emphasis>

The genome of Pseudomonas thivervalensis LMG 21626^T has been sequenced and a genomic, genetic and structural analysis of the siderophore mediated iron acquisition was undertaken. Pseudomonas thivervalensis produces two structurally new siderophores, pyoverdine PYO_thi which is typical for P. thivervalensis strains and a closely related strain, and the lipopeptidic siderophore histicorrugatin which is also detected in P. lini. Histicorrugatin consists out of an eight amino acid long peptide which is linked to octanoic acid. It is structurally related to the siderophores corrugatin and ornicorrugatin. Analysis of the proteome for TonB-dependent receptors identified 25 candidates. Comparison of the TonB-dependent receptors of P. thivervalensis with the 17 receptors of its phylogenetic neighbor, P. brassicacearum subsp. brassicacearum NFM 421, showed that NFM 421 shares the same set of receptors with LMG 21626^T, including the histicorrugatin receptor. An exception was found for their cognate pyoverdine receptor which can be explained by the observation that both strains produce structurally different pyoverdines. Mass analysis showed that NFM 421 did not produce histicorrugatin, but the analogue ornicorrugatin. Growth stimulation assays with a variety of structurally distinct pyoverdines produced by other Pseudomonas species demonstrated that LMG 21626^T and NFM 421 are able to utilize almost the same set of pyoverdines. Strain NFM 421 is able utilize two additional pyoverdines, pyoverdine of P. fluorescens Pf0–1 and P. citronellolis LMG 18378^T, these pyoverdines are probably taken up by the FpvA receptor of NFM 421.     

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine–Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric–pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor–ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric–pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric–pyoverdines, i.e. the chromophore, iron, and its chelating groups.     

Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation

Pyoverdines, the main siderophores produced by fluorescent Pseudomonads, comprise a fluorescent dihydroxyquinoline chromophore attached to a strain-specific peptide. These molecules are thought to be synthesized as non-fluorescent precursor peptides that are then modified to give functional pyoverdines. Using the fluorescent properties of PVDI, the pyoverdine produced by Pseudomonas aeruginosa PAO1, we were able to show that PVDI was not present in the cytoplasm of the bacteria, but large amounts of a fluorescent PVDI precursor PVDIp were stored in the periplasm. Like PVDI, PVDIp is able to transport iron into P. aeruginosa cells. Mutation of genes encoding the periplasmic PvdN, PvdO and PvdP proteins prevented accumulation of PVDIp in the periplasm and secretion of PVDI into the growth medium, indicating that these three enzymes are involved in PVDI synthesis. Mutation of the gene encoding PvdQ resulted in the presence of fluorescent PVDI precursor in the periplasm and secretion of a functional fluorescent siderophore that had different isoelectric properties to PVDI, suggesting a role for PvdQ in the periplasmic maturation of PVDI. Mutation of the gene encoding the export ABC transporter PvdE prevented PVDI production and accumulation of PVDIp in the periplasm. These data are consistent with a model in which a PVDI precursor peptide is synthesized in the cytoplasm and exported to the periplasm by PvdE where siderophore maturation, including formation of the chromophore moiety, occurs in a process involving the PvdN, PvdO, PvdP and PvdQ proteins.     

Pyoverdine biosynthesis and secretion in Pseudomonas aeruginosa: implications for metal homeostasis

Pyoverdines are siderophores produced by fluorescent Pseudomonads to acquire iron. At least 60 different pyoverdines produced by diverse strains have been chemically characterized. They all consist of a dihydroquinoline‐type chromophore linked to a peptide. These peptides are of various lengths and the sequences are strain specific. Pyoverdine biosynthesis in Pseudomonas aeruginosa and fluorescent Pseudomonads is a complex process involving at least 12 different proteins, starting in the cytoplasm and ending in the periplasm. The cellular localization of pyoverdine precursors was recently shown to be consistent with their biosynthetic enzymes. In the cytoplasm, pyoverdine appears to be assembled at the inner membrane and particularly at the old cell pole of the bacterium. Mature pyoverdine is uniformly distributed throughout the periplasm, like the periplasmic enzyme PvdQ. Secretion of pyoverdine involves a recently identified ATP‐dependent efflux pump, PvdRT‐OpmQ. This efflux system does not only secrete newly synthesized pyoverdine but also pyoverdine that already transported iron into the bacterial periplasm and any pyoverdine–metal complex other than ferri‐pyoverdine present in the periplasm. This review considers how these new insights into pyoverdine biosynthesis and secretion contribute to our understanding of the role of pyoverdine in iron and metal homeostasis in fluorescent Pseudomonads.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11

Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd^? and S11 PqqF^? by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd^? and S11 PqqF^? was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES–NaOH (pH?7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd^? and S11 PqqF^?. Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.     

绿针假单胞菌的研究进展及农业应用潜力

绿针假单胞菌(Pseudomonas chlororaphis)是目前研究较多的生防菌种之一.19世纪初被Miguela首次分离,将其鉴定为假单胞菌(Pseudomonas),并将机会性病原菌绿脓杆菌作为其模式菌株,而后Peix于2007年重新将其分类为绿针假单胞菌(P.chlororaphis).目前该菌种已报道有4...     

Phenazine-1-carboxylic acid biosynthesis in <Emphasis Type="Italic">Pseudomonas Chlororaphis</Emphasis> GP72 is positively regulated by the sigma factor RpoN

The production of phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) makes Pseudomonas chlororaphis GP72 an effective biocontrol agent. In order to understand how production of PCA is regulated by RpoN, an insertional mutation in rpoN has been made in P. chlororaphis GP72. Production of PCA in the rpoN mutant strain GP72N decreased both in King’s B medium and in Pigment Producing Medium. Moreover, the expression of the translational fusion phzA′–′lacZ was reduced about 2-fold in GP72N compared to wild type strain, whatever the growth medium is. Complementation of rpoN gene in mutant GP72N restored its motility and its PCA biosynthesis ability. However, overexpression of RpoN had no major effects on the expression of the RpoN-dependent phenotypes described in this study for P. chlororaphis GP72. These results suggest that RpoN is involved as a positive regulator in the regulation of PCA biosynthesis in P. chlororaphis GP72.