Siderophore-mediated Iron Transport Research Articles

The dual role of TonB genes in turnerbactin uptake and carbohydrate utilization in the shipworm symbiont Teredinibacter turnerae.

This study highlights diversity in iron acquisition and regulation in bacteria. The mechanisms of iron acquisition and its regulation in Teredinibacter turnerae, as well as its connection to cellulose utilization, a hallmark phenotype of T. turnerae, expand the paradigm of bacterial iron acquisition. Two of the four TonB genes identified in T. turnerae exhibit functional redundancy and play a crucial role in siderophore-mediated iron transport. Unlike typical TonB genes in bacteria, none of the TonB genes in T. turnerae are clearly iron regulated. This unusual regulation could be explained by another important finding in this study, namely, that the two TonB genes involved in iron transport are also essential for cellulose utilization as a carbon source, leading to the expression of TonB genes even under iron-rich conditions.

LCN2-Fungal siderophore-iron binding and uptake leads to oxidative stress and cell death in hepatocellular carcinoma cell line HepG2

Microorganisms produce non-ribosomal peptides called siderophores for the purpose of iron acquisition. Mammalian immune system is well-known for producing small secretory proteins called lipocalins upon bacterial infection. These proteins sequester siderophores produced by invading bacterial pathogens rendering them unable to acquire iron from the host. However, this is not their sole function. In addition to transferrin and lactoferrin, lipocalins are also known to transport siderophore-bound iron to the host cells. While binding of bacterial siderophores with human lipocalin is well studied, binding of the fungal counterpart is still not confirmed and fully understood. Apart from pathogen-affected cells, developing cancerous cells also show varying expression level of different proteins including those involved in iron transport. The possibility of exogenous fungal siderophore-mediated iron transport via lipocalin and its receptor in mammalian cells has not yet been explored much. In present investigation we have checked differential expression of human lipocalin, LCN2 in hepatocellular carcinoma cell lines HepG2 as well as its normal counterpart WRL-68 and computationally determined the feasibility of LCN2 binding with fungal siderophore. Further in case of a stable complex being formed, whether this complex has the ability to transport iron through its specific receptor was assessed. Also, we have tried to explore possible mechanism of fungal-siderophore mediated oxidative stress leading to significant cell death in cancerous cells. This study will thus be useful towards finding a new way of treating hepatocellular carcinoma via inducing siderophore-mediated cell death in cancerous cells. Communicated by Ramaswamy H. Sarma

Recent Advances in the Siderophore Biology of Shewanella.

Despite the abundance of iron in nature, iron acquisition is a challenge for life in general because the element mostly exists in the extremely insoluble ferric (Fe3+) form in oxic environments. To overcome this, microbes have evolved multiple iron uptake strategies, a common one of which is through the secretion of siderophores, which are iron-chelating metabolites generated endogenously. Siderophore-mediated iron transport, a standby when default iron transport routes are abolished under iron rich conditions, is essential under iron starvation conditions. While there has been a wealth of knowledge about the molecular basis of siderophore synthesis, uptake and regulation in model bacteria, we still know surprisingly little about siderophore biology in diverse environmental microbes. Shewanella represent a group of γ-proteobacteria capable of respiring a variety of organic and inorganic substrates, including iron ores. This respiratory process relies on a large number of iron proteins, c-type cytochromes in particular. Thus, iron plays an essential and special role in physiology of Shewanella. In addition, these bacteria use a single siderophore biosynthetic system to produce an array of macrocyclic dihydroxamate siderophores, some of which show particular biological activities. In this review, we first outline current understanding of siderophore synthesis, uptake and regulation in model bacteria, and subsequently discuss the siderophore biology in Shewanella.

Iron chelating bacteria: a carrier for biofortification and plant growth promotion

Biofortification is the process by which the nutritional quality of food crops is improved through agronomic practices, conventional plant breeding, or modern biotechnology to focus on malnourishment in developing countries. Under iron restricted environment certain bacteria (iron chelating bacteria) produced iron chelating molecules called as siderophore. This review gives an overview of Need for biofortification, Plant growth promoting rhizobacteria, Plant growth promoting consortia, importance of iron for human health, uptake of iron in plants, iron chelating (siderophore producing) bacteria as plant growth promotor, siderophore, generalized mechanism for siderophore-mediated iron transport in bacteria and the possible approaches to enhancing iron content in plants by implementing iron chelating bacteria as biotechnological carrier for increasing plant nutrition, yield and quality.
 
 
 

Uninterrupted Expression of CmSIT1 in a Sclerotial Parasite Coniothyrium minitans Leads to Reduced Growth and Enhanced Antifungal Ability.

Coniothyrium minitans is an important mycoparasite of Sclerotinia sclerotiorum. In addition, it also produces small amounts of antifungal substances. ZS-1TN1812, an abnormal mutant, was originally screened from a T-DNA insertional library. This mutant showed abnormal growth phenotype and could significantly inhibit the growth of S. sclerotiorum when dual-cultured on a PDA plate. When spraying the filtrate of ZS-1TN1812 on the leaves of rapeseed, S. sclerotiorum infection was significantly inhibited, suggesting that the antifungal substances produced by this mutant were effective on rapeseed leaves. The thermo-tolerant antifungal substances could specifically suppress the growth of S. sclerotiorum, but could not significantly suppress the growth of another fungus, Colletotrichum higginsianum. However, C. higginsianum was more sensitive to proteinous antibiotics than S. sclerotiorum. The T-DNA insertion in ZS-1TN1812 activated the expression of CmSIT1, a gene involved in siderophore-mediated iron transport. It was also determined that mutant ZS-1TN1812 produced hypha with high iron levels. In the wild-type strain ZS-1, CmSIT1 was expressed only when in contact with S. sclerotiorum, and consistent overexpression of CmSIT1 showed similar phenotypes as ZS-1TN1812. Therefore, activated expression of CmSIT1 leads to the enhanced antifungal ability, and CmSIT1 is a potential gene for improving the control ability of C. minitans.

Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17

The neutrophil gelatinase-associated lipocalin (NGAL, also known as LCN2) and its cellular receptor (LCN2-R, SLC22A17) are involved in many physiological and pathological processes such as cell differentiation, apoptosis, and inflammation. These pleiotropic functions mainly rely on NGAL's siderophore-mediated iron transport properties. However, the molecular determinants underlying the interaction between NGAL and its cellular receptor remain largely unknown. Here, using solution-state biomolecular NMR in conjunction with other biophysical methods, we show that the N-terminal domain of LCN2-R is a soluble extracellular domain that is intrinsically disordered and interacts with NGAL preferentially in its apo state to form a fuzzy complex. The relatively weak affinity (≈10 μm) between human LCN2-R-NTD and apoNGAL suggests that the N terminus on its own cannot account for the internalization of NGAL by LCN2-R. However, human LCN2-R-NTD could be involved in the fine-tuning of the interaction between NGAL and its cellular receptor or in a biochemical mechanism allowing the receptor to discriminate between apo- and holo-NGAL.

Lifestyle evolution in cyanobacterial symbionts of sponges.

ABSTRACTThe “Candidatus Synechococcus spongiarum” group includes different clades of cyanobacteria with high 16S rRNA sequence identity (~99%) and is the most abundant and widespread cyanobacterial symbiont of marine sponges. The first draft genome of a “Ca. Synechococcus spongiarum” group member was recently published, providing evidence of genome reduction by loss of genes involved in several nonessential functions. However, “Ca. Synechococcus spongiarum” includes a variety of clades that may differ widely in genomic repertoire and consequently in physiology and symbiotic function. Here, we present three additional draft genomes of “Ca. Synechococcus spongiarum,” each from a different clade. By comparing all four symbiont genomes to those of free-living cyanobacteria, we revealed general adaptations to life inside sponges and specific adaptations of each phylotype. Symbiont genomes shared about half of their total number of coding genes. Common traits of “Ca. Synechococcus spongiarum” members were a high abundance of DNA modification and recombination genes and a reduction in genes involved in inorganic ion transport and metabolism, cell wall biogenesis, and signal transduction mechanisms. Moreover, these symbionts were characterized by a reduced number of antioxidant enzymes and low-weight peptides of photosystem II compared to their free-living relatives. Variability within the “Ca. Synechococcus spongiarum” group was mostly related to immune system features, potential for siderophore-mediated iron transport, and dependency on methionine from external sources. The common absence of genes involved in synthesis of residues, typical of the O antigen of free-living Synechococcus species, suggests a novel mechanism utilized by these symbionts to avoid sponge predation and phage attack.

An anti-Aspergillusprotein fromEscherichia coliDH5α: Putative inhibitor of siderophore biosynthesis inAspergillus fumigatus

An antifungal protein designated as anti-Aspergillus protein (AAP), produced by Escherichia coli DH5α, was purified and characterised. It exhibited a molecular weight of 60kDa on Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis and depicted 99% purity on ultra performance liquid chromatography. The purified protein manifested antimycotic potential against pathogenic isolates of Aspergillus spp., depicting a minimum inhibitory concentration in the range 15.62-31.25μgml(-1) and 5.0-10.0μg per disc, using microbroth dilution, spore germination inhibition and disc diffusion assays respectively. In vitro toxicity tests demonstrated that it showed no toxicity against human erythrocytes at doses up to 1000μgml(-1) . Matrix-assisted laser desorption ionisation-Time-of-flight analysis of trypsin-digested peptides of purified protein and subsequent Mascot search revealed that several peptides of AAP have identity with bacterial siderophore biosynthetic protein, i.e. non-ribosomal peptide synthetase enzyme, involved in critical step of fungal siderophore biosynthesis. Siderophore-based inhibition was further corroborated by Chrome azurol S assay. Hence, the antagonistic effect might be the result of impediment in siderophore-mediated iron uptake and transport process which may cause critical consequences on Aspergillus growth and virulence.

Photostability of photosynthetic complexes coupled to silver nanowires

We investigate the influence of plasmonic excitations in silver nanowires upon the photostability of photosynthetic complex placed at their vicinity. Strong interaction between the two nanostructures is evident through increase of the fluorescence intensity of the photosynthetic complexes close to the nanowires as compared to uncoupled ones and shortening of the fluorescence lifetime. Analysis of movies collected using a wide-field fluorescence microscope with respect to temporal intensity variations for photosynthetic complexes located along and at the ends of the nanowires, indicate that the photostability is unaffected by the interaction with plasmonic excitations in metallic nanoparticles. Therefore, we conclude that in the case of studied hybrid nanostructures coupling with metallic nanoparticles results in higher number of photons emitted from chlorophyll molecules. Full Text: PDF References R.E. Blankenship, Wiley-Blackwell, ISBN: 978-0-632-04321-7 (2002). Ł. Bujak, N. Czechowski, D. Piatkowski, R. Litvin, S. Mackowski, T.H.P. Brotosudarmo, R.J. Cogdell, S. Pichler, W. Heiss, "Fluorescence enhancement of light-harvesting complex 2 from purple bacteria coupled to spherical gold nanoparticles", Appl. Phys. Lett. 99, 173701 (2011). CrossRef N. Czechowski, P. Nyga, M.K. Schmidt, T.H.P. Brotosudarmo, H. Scheer, D. Piatkowski, S. Mackowski, "Absorption Enhancement in Peridinin–Chlorophyll–Protein Light-Harvesting Complexes Coupled to Semicontinuous Silver Film", Plasmonics 7, 115 (2012). CrossRef J.B. Nieder, R. Bittl, M. Brecht, "Fluorescence Studies into the Effect of Plasmonic Interactions on Protein Function", Angew. Chem. Int. Ed. 49, 10217 (2010). CrossRef A. O. Govorov, I. Carmeli, "Hybrid Structures Composed of Photosynthetic System and Metal Nanoparticles:  Plasmon Enhancement Effect", Nano Letters 7, 620 (2007). CrossRef M. Olejnik, B. Krajnik, D. Kowalska, M. Twardowska, N. Czechowski, E. Hofmann, S. Mackowski, "Imaging of fluorescence enhancement in photosynthetic complexes coupled to silver nanowires", Appl. Phys. Lett. 102, 083703/1 (2013). CrossRef P. Anger, P. Bharadwaj, L. Novotny, "Enhancement and Quenching of Single-Molecule Fluorescence", Phys. Rev. Lett. 96, 113002 (2006). CrossRef J.R. Lakowicz, "Radiative Decay Engineering: Biophysical and Biomedical Applications", Analytical Biochemistry 298(1), 1 (2001). CrossRef K. Ray, R. Badugu, J.R. Lakowicz, "Metal-Enhanced Fluorescence from CdTe Nanocrystals:  A Single-Molecule Fluorescence Study", J. Am. Chem. Soc. 128, 8998 (2006). CrossRef S. Mackowski, S. Wörmke, A.J. Maier, T.H.P. Brotosudarmo, H. Harutyunyan, A. Hartschuh, A.O. Govorov, H. Scheer, C. Bräuchle, "Metal-Enhanced Fluorescence of Chlorophylls in Single Light-Harvesting Complexes", Nano Letters 8, 558 (2008). CrossRef A.D. Ferguson, E. Hofmann, J. W. Coulton, K. Diederichs, W. Welte, "Siderophore-Mediated Iron Transport: Crystal Structure of FhuA with Bound Lipopolysaccharide", Science 282, 2215 (1998). CrossRef K.E. Korte, S.E. Skrabalak, Y. Xia, "Rapid synthesis of silver nanowires through a CuCl- or CuCl 2 -mediated polyol process", J. Mater. Chem. 18, 437 (2008). CrossRef T.H.P. Brotosudarmo, E. Hofmann, R.G. Hiller, S. Wörmke, S. Mackowski, A. Zumbusch, C. Bräuchle, H. Scheer, "Peridinin–chlorophyll–protein reconstituted with chlorophyll mixtures: Preparation, bulk and single molecule spectroscopy", FEBS Letters 580, 5257 (2006). CrossRef

Hal Is a Bacillus anthracis Heme Acquisition Protein

The metal iron is a limiting nutrient for bacteria during infection. Bacillus anthracis, the causative agent of anthrax and a potential weapon of bioterrorism, grows rapidly in mammalian hosts, which suggests that it efficiently attains iron during infection. Recent studies have uncovered both heme (isd) and siderophore-mediated (asb) iron transport pathways in this pathogen. Whereas deletion of the asb genes results in reduced virulence, the loss of three surface components from isd had no effect, thereby leaving open the question of what additional factors in B. anthracis are responsible for iron uptake from the most abundant iron source for mammals, heme. Here, we describe the first functional characterization of bas0520, a gene recently implicated in anthrax disease progression. bas0520 encodes a single near-iron transporter (NEAT) domain and several leucine-rich repeats. The NEAT domain binds heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of hemoproteins. The NEAT domain also binds hemoglobin and can acquire heme from hemoglobin in solution. Finally, deletion of bas0520 resulted in bacilli unable to grow efficiently on heme or hemoglobin as an iron source and yielded the most significant phenotype relative to that for other putative heme uptake systems, a result that suggests that this protein plays a prominent role in the replication of B. anthracis in hematogenous environments. Thus, we have assigned the name of Hal (heme-acquisition leucine-rich repeat protein) to BAS0520. These studies advance our understanding of heme acquisition by this dangerous pathogen and justify efforts to determine the mechanistic function of this novel protein for vaccine or inhibitor development.

Sorption of ferric iron from ferrioxamine B to synthetic and biogenic layer type manganese oxides

Siderophores are biogenic chelating agents produced in terrestrial and marine environments that increase the bioavailability of ferric iron. Recent work has suggested that both aqueous and solid-phase Mn(III) may affect siderophore-mediated iron transport, but scant information appears to be available about the potential roles of layer type manganese oxides, which are relatively abundant in soils and the oligotrophic marine water column. To probe the effects of layer type manganese oxides on the stability of aqueous Fe–siderophore complexes, we studied the sorption of ferrioxamine B [Fe(III)HDFOB +, an Fe(III) chelate of the trihydroxamate siderophore desferrioxamine B (DFOB)] to two synthetic birnessites [layer type Mn(III,IV) oxides] and a biogenic birnessite produced by Pseudomonas putida GB-1. We found that all of these predominantly Mn(IV) oxides greatly reduced the aqueous concentration of Fe(III)HDFOB + at pH 8. Analysis of Fe K-edge EXAFS spectra indicated that a dominant fraction of Fe(III) associated with the Mn(IV) oxides is not complexed by DFOB as in solution, but instead Fe(III) is specifically adsorbed to the mineral structure at multiple sites, thus indicating that the Mn(IV) oxides displaced Fe(III) from the siderophore complex. These results indicate that layer type manganese oxides, including biogenic minerals, may sequester iron from soluble ferric complexes. We conclude that the sorption of iron–siderophore complexes may play a significant role in the bioavailability and biogeochemical cycling of iron in marine and terrestrial environments.

Siderophore-promoted dissolution of synthetic and biogenic layer-type Mn oxides

Siderophores are biogenic chelating agents exuded in terrestrial and marine environments to increase the bioavailablity of ferric iron. Recent work suggests that both solid and aqueous manganese may affect the aqueous speciation of siderophores and thus siderophore-mediated iron transport. Although the interaction of the trihydroxamate siderophore desferrioxamine B (DFOB) with several lower-valence manganese oxides has been studied, the effects of siderophores on Mn(III,IV) oxide dissolution are unknown. To remedy this situation, we measured the dissolution rates of two synthetic layer-type Mn(IV) oxides and a biogenic oxide produced by a model organism, Pseudomonas putida GB-1. For pH 5–7, we find that all minerals studied dissolve by traditional reductive (R1) dissolution, yielding Mn(II); for pH 7–9, dissolution yields Mn(III)–siderophore complexes, either by selective ligand-promoted dissolution of structural Mn(III) or by reduction of >Mn(IV) to >Mn(III) followed by complexation and solubilization of Mn(III) by DFOB. Because reductive dissolution results in siderophore oxidation, manganese oxide dissolution at acidic pH may provide a significant abiotic sink for siderophores in natural waters. At alkaline pH, Mn(III)–siderophore complexes produced may profoundly affect the aqueous speciation of siderophores as well as provide a source of reactive Mn(III) complexes.

Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be 'schizokinen', a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to 'rhizobactin 1021' produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed 'schizokinen' and not a degradation product of 'rhizobactin 1021'. Since ferric-siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for 'ferric-schizokinen'. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.

Coordination Chemistry and Redox Processes in Siderophore-Mediated Iron Transport

In this mini-review we present an environmental iron mobility/transport scheme consisting of inter-related controls, whereby the first coordination shell of iron modulates the iron redox potential (E1/2), and the oxidation state of iron controls the chemistry of the first coordination sphere and therefore the immediate chemical environment of the iron. Siderophores (microbially generated iron specific chelators) may be viewed as iron redox mediators. Siderophore chelation of environmental iron in a reduced (Fe(II)) oxidation state results in facile air oxidation of iron due to the negative redox potentials observed for Fe-siderophore complexes. This solubilizes the iron and locks it into a specific coordination environment, thereby preventing hydrolysis and precipitation. The high-spin Fe3+ → Fe+ electron transfer process may be viewed as a switch that controls the thermodynamic stability and kinetic lability of the first coordination shell. Reduction of iron(III)-siderophore complexes to iron(II)-siderophore complexes decreases thermodynamic stability, increases the rate of siderophore ligand exchange, and increases the ease of siderophore donor atom protonation, thus facilitating a rapid turnover of the first coordination shell. Results are presented for iron-siderophore pH and oxidation state dependent speciation studies that are relevant to environmental and microbial iron mobility and transport.

Importance of the ornibactin and pyochelin siderophore transport systems in Burkholderia cenocepacia lung infections.

Previously, orbA, the gene encoding the outer membrane receptor for ferric-ornibactin, was identified in Burkholderia cenocepacia K56-2, a strain which produces ornibactin, salicylic acid, and negligible amounts of pyochelin. A K56-2 orbA mutant was less virulent than the parent strain in a rat agar bead infection model. In this study, an orbA mutant of B. cenocepacia Pc715j which produces pyochelin in addition to ornibactin and salicylic acid was constructed. The gene encoding the outer membrane receptor for ferric-pyochelin (fptA) was also identified. An fptA mutant was constructed in Pc715j and shown to be deficient in [(59)Fe]pyochelin uptake. A 75-kDa iron-regulated protein was identified in outer membrane preparations of Pc715j that was absent in outer membrane preparations of Pc715jfptA::tp. Pc715jfptA::tp and Pc715jorbA::tp produced smaller amounts of their corresponding siderophores. Both Pc715jorbA::tp and Pc715jfptA::tp were able to grow in iron starvation conditions in vitro. In the agar bead model, the Pc715jorbA::tp mutant was cleared from the lung, indicating that the pyochelin uptake system does not compensate for the absence of a functional ornibactin system. Pc715jfptA::tp persisted in rat lung infections in numbers similar to those of the parent strain, indicating that the ferric-ornibactin uptake system could compensate for the defect in ferric-pyochelin uptake in vivo. These studies suggest that the ornibactin uptake system is the most important siderophore-mediated iron transport system in B. cenocepacia lung infections.

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sitB and sitD genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti.

Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources. Tagged loci of these mutants were identified as sit B and sit D genes. These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria. In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants. The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S. meliloti.

Utilization and cell-surface binding of hemin by Histoplasma capsulatum.

Histoplasma capsulatum, a dimorphic fungus capable of causing severe respiratory illness in immunocompromised individuals, resides in macrophages during mammalian infection. Previous studies suggest that siderophore-mediated iron transport may be important for the acquisition of iron from transferrin while the organism resides in macrophages. However, iron is also present as hemin in the intracellular environment of the macrophage and may serve as a major source of iron during infection. Thus the ability of H. capsulatum to use hemin and heme-containing compounds was examined. Histoplasma capsulatum G217B was iron-starved by adding the iron chelator deferoxamine mesylate to the culture. The addition of 10 microM hemin in the presence of deferoxamine mesylate restored growth to the levels seen in the absence of the chelator. Histoplasma capsulatum was also cultivated in an iron-limited, chemically defined medium without the addition of chelators and it was determined that the organism could also use hemoglobin as a sole source of iron. The method of iron internalization from heme was examined by measuring hemin binding to the yeast-cell surface. The ability of H. capsulatum to bind hemin was related to the nutritional status of the cells. Cells grown under iron-limited conditions bound more heme to the cell surface than did cells grown in medium without chelator. Pretreatment of iron-starved cells with proteinase K eliminated the ability of the organism to bind hemin. Additionally, the pre-incubation of iron-starved H. capsulatum with hemin eliminated the ability of these cells to remove hemin from the solution, although pre-incubation of cells with the iron-free form of hemin, protoporphyrin IX, only modestly affected the ability of the organism to bind hemin. These results suggest that H. capsulatum uses hemin as a sole source of iron and that one mechanism of iron acquisition involves a cell-surface receptor for hemin.

Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport.

The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.