Metal trafficking via siderophores in Gram-negative bacteria: specificities and characteristics of the pyoverdine pathway

Under iron-limiting conditions, fluorescent pseudomonads secrete fluorescent siderophores called pyoverdines (Pvd), which form complexes with iron that are then taken up by the bacteria. Pvds consist of a fluorescent chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and containing one of the bidentate groups involved in iron chelation, linked to a peptide moiety containing the other two bidentate groups required for binding to Fe³⁺. More than 100 different Pvds have been identified, with different peptide sequences, forming a wide family of siderophores. In the human opportunistic pathogen Pseudomonas aeruginosa, Pvd is necessary for infection and is considered to be a virulence factor. This review focuses on the mechanisms underlying iron uptake by the Pvd pathway in pseudomonads, taking into account recent biochemical and biophysical studies and recently solved 3D-structures of the Pvd outer membrane transporter FpvA in four different loading states. These data are discussed and compared with the mechanisms of siderophore–Fe uptake reported for other Gram-negative bacteria.     

Iron uptake and metabolism in pseudomonads

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.     

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.     

Diversity of root-associated fluorescent pseudomonads as affected by ferritin overexpression in tobacco

A transgenic tobacco overexpressing ferritin (P6) was recently shown to accumulate more iron than the wild type (WT), leading to a reduced availability of iron in the rhizosphere and shifts in the pseudomonad community. The impact of the transgenic line on the community of fluorescent pseudomonads was assessed. The diversity of 635 isolates from rhizosphere soils, rhizoplane + root tissues, and root tissues of WT and P6, and that of 98 isolates from uncultivated soil was characterized. Their ability to grow under iron stress conditions was assessed by identifying their minimal inhibitory concentrations of 8-hydroxyquinoline for each isolate, pyoverdine diversity by isoelectrofocusing and genotypic diversity by random amplified polymorphism DNA. The antagonistic activity of representative isolates and of some purified pyoverdines against a plant pathogen (Pythium aphanidermatum Op4) was tested in vitro. In overall, isolates taken from P6 tobacco showed a greater ability to grow in iron stress conditions than WT isolates. The antagonism by some of the representative isolates was only expressed under iron stress conditions promoting siderophore synthesis and their pyoverdines appeared to have a specific structure as assessed by mass spectrometry. For other isolates, antagonism was still expressed in the presence of iron, suggesting the involvement of metabolites other than siderophores. Altogether, these data indicate that the transgenic tobacco that over-accumulates iron selected fluorescent pseudomonads, less susceptible to iron depletion and more antagonistic to the tested plant pathogen than those selected by the tobacco WT.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Detection and differentiation of microbial siderophores by isoelectric focusing and chrome azurol S overlay

Siderophores are microbial, low molecular weight iron-chelating compounds. Fluorescent Pseudomonads produce different, strain-specific fluorescent siderophores (pyoverdines) as well as non-fluorescent siderophores in response to low iron conditions. We present an isoelectric focusing method applicable to unpurified as well as to purified pyoverdine samples where the fluorescent siderophores are visualized under UV illumination. Siderophores from different Pseudomonas sp., amongst which are P. aeruginosa, P. fluorescens and P. putida, including egg yolk, rhizospheric and clinical isolates as well as some derived Tn5 mutants were separated by this technique. Different patterns could be observed for strains known to produce different siderophores. The application of the chrome azurol S assay as a gel overlay further allows immediate detection of non-fluorescent siderophores or possibly degradation products with residual siderophore activity. The method was also applied to other microbial siderophores such as deferrioxamine B.     

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.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

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.     

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.     

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.     

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.     

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.     

Siderophores drive invasion dynamics in bacterial communities through their dual role as public good versus public bad

Microbial invasions can compromise ecosystem services and spur dysbiosis and disease in hosts. Nevertheless, the mechanisms determining invasion outcomes often remain unclear. Here, we examine the role of iron-scavenging siderophores in driving invasions of Pseudomonas aeruginosa into resident communities of environmental pseudomonads. Siderophores can be ‘public goods’ by delivering iron to individuals possessing matching receptors; but they can also be ‘public bads’ by withholding iron from competitors lacking these receptors. Accordingly, siderophores should either promote or impede invasion, depending on their effects on invader and resident growth. Using supernatant feeding and invasion assays, we show that invasion success indeed increased when the invader could use its siderophores to inhibit (public bad) rather than stimulate (public good) resident growth. Conversely, invasion success decreased the more the invader was inhibited by the residents’ siderophores. Our findings identify siderophores as a major driver of invasion dynamics in bacterial communities under iron-limited conditions.     

Siderotyping of fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis>: molecular mass determination by mass spectrometry as a powerful pyoverdine siderotyping method

The numerous pyoverdines so far characterized as siderophores of fluorescent Pseudomonas could be usually differentiated one from each others by the two physico-chemical and physiological methods of siderotyping, i.e., siderophore-isoelectrofocusing and siderophore-mediated iron uptake. As shown in the present paper, the structural diversity of the peptide chain characterizing these molecules results in a very large panel of molecular masses representing 64 different values ranging from 889 to 1,764 Da for the 68 compounds included in the study, with only a few structurally different compounds presenting an identical molecular mass. Thus, the molecular mass determination of pyoverdines through mass spectrometry could be used as a powerful siderotyping method.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.