Pyoverdine-mediated Iron Uptake Research Articles

Pseudomonas aeruginosa responds to altered membrane phospholipid composition by adjusting the production of two-component systems, proteases and iron uptake proteins

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. To achieve these, bacteria use adaptation mechanisms involving covalent modification and remodelling of the acyl chain length of PLs. However, little is known about bacterial pathways regulated by PLs. Here, we investigated proteomic changes in the biofilm of P. aeruginosa phospholipase mutant (∆plaF) with altered membrane PL composition. The results revealed profound alterations in the abundance of many biofilm-related two-component systems (TCSs), including accumulation of PprAB, a key regulator of the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of several proteases, in ∆plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional response. Moreover, proteomics and biochemical assays revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in ∆plaF, while proteins from alternative iron-uptake systems were accumulated. These suggest that PlaF may function as a switch between different iron-acquisition pathways. The observation that PL-acyl chain modifying and PL synthesis enzymes were overproduced in ∆plaF reveals the interconnection of degradation, synthesis and modification of PLs for proper membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that alteration of PL composition in ∆plaF plays a role for the global adaptive response in P. aeruginosa mediated by TCSs and proteases. Our study revealed the global regulation of virulence and biofilm by PlaF and suggests that targeting this enzyme may have therapeutic potential.

PvdN Enzyme Catalyzes a Periplasmic Pyoverdine Modification

Pyoverdines are high affinity siderophores produced by a broad range of pseudomonads to enhance growth under iron deficiency. They are especially relevant for pathogenic and mutualistic strains that inhabit iron-limited environments. Pyoverdines are generated from non-ribosomally synthesized highly modified peptides. They all contain an aromatic chromophore that is formed in the periplasm by intramolecular cyclization steps. Although the cytoplasmic peptide synthesis and side-chain modifications are well characterized, the periplasmic maturation steps are far from understood. Out of five periplasmic enzymes, PvdM, PvdN, PvdO, PvdP, and PvdQ, functions have been attributed only to PvdP and PvdQ. The other three enzymes are also regarded as essential for siderophore biosynthesis. The structure of PvdN has been solved recently, but no function could be assigned. Here we present the first in-frame deletion of the PvdN-encoding gene. Unexpectedly, PvdN turned out to be required for a specific modification of pyoverdine, whereas the overall amount of fluorescent pyoverdines was not altered by the mutation. The mutant strain grew normally under iron-limiting conditions. Mass spectrometry identified the PvdN-dependent modification as a transformation of the N-terminal glutamic acid to a succinamide. We postulate a pathway for this transformation catalyzed by the enzyme PvdN, which is most likely functional in the case of all pyoverdines.

The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance ofPseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment for such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here, we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, nonsiderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by subinhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development.

Influence of siderophore pyoverdine synthesis and iron-uptake on abiotic and biotic surface colonization of Pseudomonas putida S11

Fluorescent pseudomonads produce a characteristic fluorescent pigment, pyoverdines as their primary siderophore for iron acquisition under iron-limiting conditions. Here, we report the identification of a random transposon mutant IST3 of Pseudomonas putida S11 showing tolerance to iron starvation stress condition and increased pyoverdine production. The insertion of the Tn5 transposon was found to be in pstS gene of pstSR operon encoding sensor histidine kinase protein of the two-component signal transduction system. A pyoverdine negative derivative of IST3 mutant constructed was sensitive to iron stress condition. It indicated that increased survival of IST3 under iron-limiting condition was due to higher pyoverdine production. The iron starvation tolerant mutant (IST3) exhibited enhanced pyoverdine-mediated iron uptake in minimal medium which significantly improved its biofilm formation, seed adhesion and competitive root colonization.

Loss of the oxidative stress regulator OxyR inPseudomonas aeruginosaPAO1 impairs growth under iron-limited conditions

Pyoverdine is the main siderophore secreted by fluorescent pseudomonads to scavenge iron in the extracellular environment. Iron uptake, however, needs to be tightly regulated, because free iron stimulates the formation of highly toxic oxygen derivatives. In the opportunistic pathogen Pseudomonas aeruginosa, the transcriptional regulator OxyR plays a key role in the upregulation of defense mechanisms against oxidative stress as it stimulates the expression of the antioxidant genes katB, ahpB and ahpCF after contact with oxidative stress-generating agents. Inactivation of the oxyR gene in Pseudomonas fluorescens ATCC 17400 and in P. aeruginosa PAO1 impairs pyoverdine-mediated iron uptake. The pyoverdine utilization defect can be restored by complementation with the oxyR gene of P. aeruginosa, as well as by adding catalase. Growth of the oxyR mutant in low- or high-iron media is also impaired at a low, but not at a high inoculum density. Uptake of radioactive (59)Fe pyoverdine is, however, not affected by the oxyR mutation, nor is the transcription of the fpvA gene encoding the ferripyoverdine receptor, suggesting that the defect lies in the inability to remove iron from the ferrisiderophore.

Genomic, genetic and structural analysis of pyoverdine-mediated iron acquisition in the plant growth-promoting bacterium Pseudomonas fluorescens SBW25

BackgroundPyoverdines (PVDs) are high affinity siderophores, for which the molecular mechanisms of biosynthesis, uptake and regulation have been extensively studied in Pseudomonas aeruginosa PAO1. However, the extent to which this regulatory model applies to other pseudomonads is unknown. Here, we describe the results of a genomic, genetic and structural analysis of pyoverdine-mediated iron uptake by the plant growth-promoting bacterium P. fluorescens SBW25.ResultsIn silico analysis of the complete, but un-annotated, SBW25 genome sequence identified 31 genes putatively involved in PVD biosynthesis, transport or regulation, which are distributed across seven different regions of the genome. PVD gene iron-responsiveness was tested using 'lacZ fusions to five PVD loci, representative of structural and regulatory genes. Transcription of all fusions increased in response to iron starvation. In silico analyses suggested that regulation of fpvR (which is predicted to encode a cytoplasmic membrane-spanning anti-sigma factor) may be unique. Transcriptional assays using gene expression constructs showed that fpvR is positively regulated by FpvI (an extracytoplasmic family (ECF) sigma factor), and not directly by the ferric uptake regulator (Fur) as for PAO1. Deletion of pvdL, encoding a predicted non-ribosomal peptide synthetase (NRPS) involved in PVD chromophore biosynthesis confirmed the necessity of PvdL for PVD production and for normal growth in iron-limited media. Structural analysis of the SBW25 PVD shows a partly cyclic seven residue peptide backbone, identical to that of P. fluorescens ATCC13525. At least 24 putative siderophore receptor genes are present in the SBW25 genome enabling the bacterium to utilize 19 structurally distinct PVDs from 25 different Pseudomonas isolates.ConclusionThe genome of P. fluorescens SBW25 contains an extensively dispersed set of PVD genes in comparison to other sequenced Pseudomonas strains. The PAO1 PVD regulatory model, which involves a branched Fpv signaling pathway, is generally conserved in SBW25, however there is a significant difference in fpvR regulation. SBW25 produces PVD with a partly cyclic seven amino acid residue backbone, and is able to utilize a wide variety of exogenous PVDs.

Taxonomic heterogeneity, as shown by siderotyping, of strains primarily identified as Pseudomonas putida

One hundred and forty-four fluorescent pseudomonad strains isolated from various environments (soil, water, plant rhizosphere, hospital) and received as Pseudomonas putida (83 strains), P. putida biovar A (49 strains), P. putida biovar B (10 strains) and P. putida biovar C (2 strains), were analysed by the pyoverdine-isoelectrofocusing and pyoverdine-mediated iron uptake methods of siderotyping. Both methods demonstrated a great diversity among these strains, which could be subdivided into 35 siderovars. Some siderovars specifically included strains that have subsequently been transferred to well-defined Pseudomonas species, e.g. Pseudomonas monteilii or Pseudomonas mosselii, or which could be related by their siderotype to Pseudomonas jessenii or Pseudomonas mandelii. Other siderovars included strains sharing a high level of DNA-DNA relatedness (>70%), thus demonstrating that siderotyping could easily circumscribe strains at the species level. However, a group of seven strains, including the type strain, P. putida ATCC 12633T, were allocated into four siderovars, despite sharing DNA-DNA relatedness values of higher than 70 %. Interestingly, the strong genomic relationships between these seven strains were supported by the structural relationships among their pyoverdines, thus reflecting their phylogenetic affinities. These results strongly support the view that pyoverdine-based siderotyping could be used as a powerful tool in Pseudomonas taxonomy.

Pyoverdine-Mediated Iron Uptake in Pseudomonas aeruginosa : the Tat System Is Required for PvdN but Not for FpvA Transport

Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat-dependent export of proteins involved in Pvd biogenesis and ferric Pvd uptake (U. Ochsner, A. Snyder, A. I. Vasil, and M. L. Vasil, Proc. Natl. Acad. Sci. USA 99:8312-8317, 2002). Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.

Genomics of pyoverdine-mediated iron uptake in pseudomonads

Pyoverdines (PVDs) are complex siderophores produced by members of the fluorescent Pseudomonas. They comprise a dihydroxyquinoline fluorescent chromophore joined to a peptide of remarkably variable length and composition. In Pseudomonas aeruginosa, PVDs also function as signal molecules for the production of virulence factors. Genes responsible for the biosynthesis, excretion, uptake and regulation of these high-affinity siderophores are located either at a single locus or at up to three different loci in the genomes of the four pseudomonads analyzed. The peptide backbone of PVD is assembled by non-ribosomal peptide synthetases (NRPSs) and modified by accessory enzymes in the cytoplasm, and probably the periplasm. Regulation of PVD production and uptake depends on two extracytoplasmic sigma factors (ECF-σs), PvdS and FpvI, together with one anti-sigma, FpvR.

FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa.

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.

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.

Siderophore-Mediated Iron Uptake in Fluorescent Pseudomonas: Characterization of the Pyoverdine-Receptor Binding Site of Three Cross-Reacting Pyoverdines

Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine–peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.

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.

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.

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.

Siderotyping of fluorescent pseudomonads: characterization of pyoverdines of Pseudomonas fluorescens and Pseudomonas putida strains from Antarctica.

Five independent fluorescent pseudomonad isolates originating from Antarctica were analysed for their pyoverdine systems. A pyoverdine-related siderotyping, which involved pyoverdine-induced growth stimulation, pyoverdine-mediated iron uptake, pyoverdine analysis by electrophoresis and isoelectric focusing, revealed three different pyoverdine-related siderotypes among the five isolates. One siderotype, including Pseudomonas fluorescens 1W and P. fluorescens 10CW, was identical to that of P. fluorescens ATCC 13525. Two other strains, P. fluorescens 9AW and Pseudomonas putida 9BW, showed identical pyoverdine-related behaviour to each other, whereas the fifth strain, P. fluorescens 51W, had unique features compared to the other strains or to a set of 12 fluorescent Pseudomonas strains used as comparison material. Elucidation of the structure of the pyoverdines produced by the Antarctic strains supported the accuracy of the siderotyping methodology by confirming that pyoverdines from strains 1W and 10CW had the same structures as the P. fluorescens ATCC 13525 pyoverdine, whereas the 9AW and 9BW pyoverdines are probably identical with the pyoverdine of P. fluorescens strain 244. Pyoverdine from strain 51W appeared to be a novel pyoverdine since its structure was different from all previously established pyoverdine structures. Together with the conclusion that the Antarctic Pseudomonas strains have no special features at the level of their pyoverdines and pyoverdine-mediated iron metabolism compared to worldwide strains, the present work demonstrates that siderotyping provides a rapid means of screening for novel pyoverdines.

Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine

Pseudomonas aeruginosa strain K437 is defective in the production of a 90kDa ferripyoverdine receptor and is unable to grow in an iron-deficient medium in the presence of the non-metabolizable iron chelator 2,2'-dipyridyl (0.25 mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl-containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb xhoI-HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7-based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine-mediated iron-transport system are co-regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane-associated plasmid-encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA- and ORFB-deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron-deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine-mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA-ORFB-deficient mutants were not defective in septum formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains

Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.

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.