Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains.

Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.     

Ferrisiderophore reductases of Pseudomonas. Purification, properties and cellular location of the Pseudomonas aeruginosa ferripyoverdine reductase.

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of M(r) 27,000-28,000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical M(r) as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas.     

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.     

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.     

Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida

Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa.To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined.The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.     

Uptake of Pyocin S3 Occurs through the Outer Membrane Ferripyoverdine Type II Receptor of Pseudomonas aeruginosa

Pyocin S3 was found to kill exclusively Pseudomonas aeruginosa isolates producing type II pyoverdine (exemplified by strain ATCC 27853). Killing was specifically inhibited by addition of type II ferripyoverdine. All Tn5 mutants resistant to pyocin S3 were defective for pyoverdine-mediated iron uptake and failed to produce an 85-kDa iron-repressed outer membrane protein. We conclude that this protein is probably the type II ferripyoverdine receptor that is used by pyocin S3 to gain entry into the cell.     

Fluorescent Pseudomonas mainly produce the dihydro form of pyoverdine at low specific growth rate

AIMS: To analyse the influence of cell growth rate and iron concentration on the production of pyoverdines (PVDs) and of their reduced dihydro forms by three fluorescent Pseudomonas strains (P. putida BTP16, P. fluorescens BTP7 and P. aeruginosa 7NSK2). METHODS: PVD and dihydropyoverdine (DHPVD) productions were determined by LC ESI-MS and spectrophotometry during batch and chemostat culture at different dilution rates. SIGNIFICANCE: The relatively high PVD-to-DHPVD ratio (0.57) observed in pH-controlled batch cultures suggested that a base-catalysed chemical oxidation of the dihydroform is not the prime mechanism involved in generating PVDs. Interestingly, in chemostat cultures the PVD-to-DHPVD ratio was significantly reduced at low specific growth rate. Our results suggest that the oxidation of DHPVD to PVD is catalysed by an iron-dependent enzymatic reaction rather than a chemical oxidation.     

Pyocin S2 (Sa) kills Pseudomonas aeruginosa strains via the FpvA type I ferripyoverdine receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.     

Siderophore typing,a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.     

Iron release from ferrisiderophores. A multi-step mechanism involving a NADH/FMN oxidoreductase and a chemical reduction by FMNH2.

Release of iron from various ferrisiderophores (ferripyoverdines, ferrioxamines B and E, ferricrocin, ferrichrome A, ferrienterobactin and its analog ferric N,N',N'-tri(1,3,5-Tris) 2,3-dihydroxybenzoylaminomethylbenzene) was obtained through an enzymic reduction of iron, involving NADH, FMN and the ferripyoverdine reductase of Pseudomonas aeruginosa PAO1. The iron released from the same complexes was also obtained through chemical reduction of iron involving FMNH2. Evidence is given that the enzymic process acts through a FMNH2 reduction; the P. aeruginosa enzyme, purified according to its ferripyoverdine-reductase activity [Hallé, F. & Meyer, J. M., Eur. J. Biochem. 209, 613-620], functions as a NADH:FMN oxidoreductase, the FMNH2 produced being able to chemically reduce the iron complexed by siderophores. The general occurrence of such a multi-step mechanism, which denies the existence of specific ferrisiderophore reductases, is discussed.     

Mutational analysis of a bifunctional ferrisiderophore receptor and signal-transducing protein from Pseudomonas aeruginosa

The FpvA protein of Pseudomonas aeruginosa strain PAO1 mediates uptake of a siderophore, ferripyoverdine. It is also a component of a signal transduction pathway that controls production of an exotoxin, a protease, pyoverdine, and FpvA itself. The purpose of the research described here was to dissect these different functions of FpvA. Signaling involves an N-terminal domain of FpvA, and it was shown that this domain is probably located in the periplasm, as expected. Short peptides were inserted at 36 sites within FpvA by linker insertion mutagenesis. The effects of these mutations on the presence of FpvA in the outer membrane, on FpvA-mediated uptake of ferripyoverdine, and on pyoverdine synthesis and gene expression were determined. Five of the mutations resulted in the absence of FpvA from the outer membrane of the bacteria. All of the remaining mutations eliminated either the transport or signaling function of FpvA and most affected both functions. Three mutations prevented transport of ferripyoverdine but had no effect on the signal transduction pathway showing that transport of ferripyoverdine is not required for the trans-membrane signaling process. Conversely, eight mutations affected pyoverdine-mediated signaling but had no effect on transport of ferripyoverdine. These data show that insertions throughout FpvA resulted in loss of function and that signaling and transport are separate and discrete functions of FpvA.     

Siderotyping of Antarctic fluorescent Pseudomonas strains.

Five fluorescent Pseudomonas strains isolated from Antarctica have been previously recognized as producing three structurally different pyoverdines. In the present work, siderotyping procedures have been used to classify these strains, together with 1282 isolates of different origins, into siderovars. The strain biodiversity encountered within each siderovar, as well as the potential taxonomic value of the siderovars, are described and discussed. It is concluded that a majority of antarctic strains are commonly distributed worldwide. One strain, however, presenting a particular pyoverdine structure found in a unique other isolate, was apparently much more specific to cold environment.     

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.     

Analysis of the draft genome of Pseudomonas fluorescens ATCC17400 indicates a capacity to take up iron from a wide range of sources,including different exogenous pyoverdines

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.     

Effectiveness of Acetic Acid, Betadine, Amphyll, Polymyxin B, Colistin, and Gentamicin Against Pseudomonas aeruginosa

The in vitro effectiveness of three germicides and three chemotherapeutic drugs against hospital-isolated strains of Pseudomonas aeruginosa was determined. Solutions of 1% acetic acid, 0.5% Amphyll, and 0.1% Betadine were bactericidal for six strains tested within 15 min at room temperature. Over a 3-year period at the Albany Medical Center Hospital, the percentages of strains of P. aeruginosa susceptible to polymyxin B, colistin, and gentamicin did not increase. During this 3-year period, polymyxin B and colistin were administered at the hospital but gentamicin was not.     

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.     

Distribution and evolution of ferripyoverdine receptors in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, which is also able to cause severe opportunistic infections in humans. The colonization of the host is importantly affected by the production of the high-affinity iron (III) scavenging peptidic siderophore pyoverdine. The species P. aeruginosa can be divided into three subgroups ('siderovars'), each characterized by the production of a specific pyoverdine and receptor (FpvA). We used a multiplex PCR to determine the FpvA siderovar on 345 P. aeruginosa strains from environmental or clinical origin. We found about the same proportion of each type in clinical strains, while FpvA type I was slightly over-represented (49%) in environmental strains. Our multiplex PCR also detected the presence or absence of an additional receptor for type I pyoverdine (FpvB). The fpvB gene was in fact present in the vast majority of P. aeruginosa strains (93%), regardless of their siderovar or their origin. Finally, molecular analyses of fpvA and fpvB genes highlighted a complex evolutionary history, probably linked to the central role of iron acquisition in the ecology and virulence of P. aeruginosa .