Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Utilization of exogenous siderophores by enterococci]

The study was undertaken to investigate the ability of enterococci to assimilate iron via siderophores of bacteria living in the same habitats in the human organism. The potential recipients of exogenous siderophores were six Enterococcus faecalis and six Enterococcus faecium strains, isolated from clinical materials of human origin. The donors of siderophores were Gram-negative rods (various species of the Enterobacteriaceae, Pseudomonas and Acinetobacter) and Gram-positive cocci (various species of Staphylococcus and Streptococcus). All of the investigated E. faecium and only two E. faecalis strains demonstrated the ability to utilize the siderophores of the aforementioned bacterial groups, predominantly the chelators of Gram-negative rods, those of Gram-positive cocci were utilized to a smaller extent. Four recipient strains from E. faecalis species did not demonstrate the ability to utilize siderophores synthesized by all of 40 investigated donor strains.     

Synthesis of siderophores by strains of Staphylococcus cohnii isolated from various environments

Siderophore activity as the feature of microorganisms enabling colonization of human body and the survival in inanimate environment was investigated in 108 strains of Staphylococcus cohnii; S. cohnii ssp. cohnii (50 strains) and S. cohnii ssp. urealyticus (58 strains). Strains were isolated from people, hospital and non-hospital environment. Highest siderophore activity was noted in strains S. cohnii ssp. urealyticus particularly from the inanimate environments origin. In 86% analyzed strains siderophores of hydroxamate class were detected. Larger amounts of these compounds were synthesized in strains S. cohnii ssp. urealyticus. Strains belonging to both subspecies from human origin showed lower activity of siderophores (total pool) and did not produce hydroxamate class chelators or produced very small amounts of these compounds.     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

Catecholates and mixed catecholate hydroxamates as artificial siderophores for mycobacteria

Different mono-, bis- or triscatecholates and mixed mono- or biscatecholate hydroxamates were synthesized as potential siderophores for mycobacteria. Siderophore activity was tested by growth promotion assays using wild type strains and iron transport mutants of mycobacteria as well as Gram-negative bacteria. Some triscatecholates and biscatecholate hydroxamates were active in mutants of Mycobacterium smegmatis deficient in mycobactin and exochelin biosynthesis or exochelin permease, respectively, indicating an uptake route independent of the exochelin/mycobactin pathway. Structure activity relationships were studied. Ampicillin conjugates of some of these compounds were inactive against mycobacteria but active against Gram-negative bacteria.     

Utilization of exogenous siderophores and natural catechols by Listeria monocytogenes.

Listeria monocytogenes does not produce siderophores for iron acquisition. We demonstrate that a number of microbial siderophores and natural iron-binding compounds are able to promote the growth of iron-starved L. monocytogenes. We suggest that the ability of L. monocytogenes to use a variety of exogenous siderophores and natural catechols accounts for its ubiquitous character.     

Utilization of glycerophosphodiesters by Staphylococcus aureus

The facultative pathogen Staphylococcus aureus colonizes the human anterior nares and causes infections of various organ systems. Which carbon, energy, and phosphate sources can be utilized by S. aureus in nutrient‐poor habitats has remained largely unknown. We describe that S. aureus secretes a glycerophosphodiesterase (glycerophosphodiester phosphodiesterase, EC 3.1.4.46), GlpQ, degrading the glycerophosphodiester (GPD) head groups of phospholipids such as human phosphatidylcholine (GroPC). Deletion of glpQ completely abolished the GroPC‐degrading activity in S. aureus culture supernatants. GroPC has been detected in human tissues and body fluids probably as a result of phospholipid remodelling and degradation. Notably, GroPC promoted S. aureus growth under carbon‐ and phosphate‐limiting conditions in a GlpQ‐dependent manner indicating that GlpQ permits S. aureus to utilize GPD‐derived glycerol‐3‐phosphate as a carbon and phosphate sources. Thus, S. aureus can use a broader spectrum of nutrients than previously thought which underscores its capacity to adapt to the highly variable and nutrient‐poor surroundings.     

Mechanisms of iron acquisition from siderophores by microorganisms and plants

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organism's expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.     

Reduction and transport of Fe from siderophores

Soils contain siderophores produced by bacteria and fungi; however, the role of siderophores in Fe nutrition of plants is uncertain. The Strategy I plant cucumber (Cucumis sativus L.) was used in an investigation of ferric chelate reduction activity and uptake and transport of Fe from ferric hydroxyethylethylenetriacetic acid (FeHEDTA) and ferric N,N–di–(2–hydroxybenzoyl)–ethylenediamine– N,N-diacetic acid (FeHBED) and the hydroxamate siderophores, ferric rhodotorulic acid (FeRA) and ferric ferrioxime B (FeFOB). Cucumber seedlings were grown in a hydroponic medium without Fe or supplied with 10 M FeHEDTA. Iron-deficient cucumber roots readily reduced FeHEDTA, while Fe-sufficient roots had low levels of ferric chelate reduction activity. The siderophore FeRA was reduced by Fe-deficient roots at 8% of the rate of FeHEDTA, while FeFOB was not reduced. The highly stable synthetic chelate FeHBED was reduced at 16% the rate of FeHEDTA. Fe transport to shoots by Fe-deficient seedlings from the slowly reducible complexes ⁵⁹FeRA and ⁵⁹FeHBED was, respectively, 74% and 73% of that transported from ⁵⁹FeHEDTA. The ferrous complexing agent, bathophenanthrolinedisulfonic acid (BPDS), had a strong inhibitory effect on uptake and transport of Fe from ⁵⁹FeHEDTA or ⁵⁹FeRA into shoots. An average of 11% as much Fe was transported to shoots of Fe-deficient seedlings from ⁵⁹FeFOB as from ⁵⁹FeHEDTA. Neither the Fe nutritional status of the seedlings nor the presence of BPDS influenced the uptake and transport of Fe from ⁵⁹FeFOB. It is concluded that cucumber roots may take up substantial amounts of Fe from FeRA and FeHBED following reduction, while small amounts of Fe may be taken up from FeFOB by a mechanism not involving reduction of the ferric siderophore at the root surface.     

Characterization of siderophores from Ustilago maydis

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10^–5 M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.     

Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans.

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 microM less than [Fe L-parabactin] less than 10 microM fit a regression line which suggests a low-affinity system (Km = 3.9 +/- 1.2 microM, Vmax = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 microM less than or equal to [Fe L-parabactin] less than or equal to 1 microM fit another line consistent with a high-affinity system (Km = 0.24 +/- 0.06 microM, Vmax = 108 pg-atoms of 55Fe min-1 mg of protein-1). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple Michaelis-Menten process with comparatively low affinity (Km = 3.1 +/- 0.9 microM, Vmax = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the lambda enantiomers. The A forms ferric parabactin (ferrin D- and L-parabactin A), in which the oxazoline ring is hydrolyzed to the open-chain threonyl structure, exhibit linear kinetics with a comparatively high Km (1.4 +/- 0.3 microM) and high Vmax (324 pg-atoms of 55Fe min-1 of protein-1). Furthermore, the marked stereospecificity seen between ferric D- and L-parabactins is absent; i.e., iron acquisition from ferric parabactin A is non stereospecific. The mechanistic implications of these findings in relation to a stereospecific high-affinity binding followed by a nonstereospecific postreceptor processing is discussed.     

Extracellular siderophores from Aspergillus ochraceous.

A large number of iron-chelating compounds (siderophores) were isolated from supernatants of iron-deficient cultures of a mold isolate, subsequently identified as Aspergillus ochraceous . Siderophores in their iron chelate form were purified to homogeneity by using Bio-Gel P2, silica gel, and C-18 bonded silica gel (reverse-phase) columns. Most of these compounds, as identified by 1H and 13C nuclear magnetic resonance spectroscopy and X-ray crystallography, belong to the ferrichrome family. The organism produces ferrirubin and ferrichrysin as the predominant and the second major compound (62 and 15% of the total siderophores), respectively. Ferrichrysin appears as the first siderophore in the medium on day 2 of growth. Several of the other siderophores are novel and ranged in quantities from 0.2 to 5% of the total. The trivial names asperchrome A, B1, B2, C, D1, D2, and D3 are proposed for these novel compounds, which are all members of the ferrichrome family, and all but the first one contain a common Orn1 - Orn2 - Orn3 - Ser1 -Ser2-Gly cyclic hexapeptide ring with three dissimilar ornithyl delta-N-acyl groups. Another compound which appeared late in the growth period was similar to fusarinine C ( fusigen ). All of these compounds showed growth factor activity to various extents in bioassays with Arthrobacter flavescens Jg-9. None of these compounds showed antibacterial activity against Escherichia coli or Bacillus megaterium.     

Synthesis of siderophores by pathogenicVibrio species

PathogenicVibrio species were assayed for the production and utilization of siderophores. Although some similarities were observed in the types of siderophores secreted, chemical and biological assays indicated that several different iron transport systems are expressed by members of this genus. Bioassays indicated that each species tested produced compounds that stimulated their growth in low-iron media. Phenolate compounds were detected by chemical assay of culture supernatants of iron-starvedV. cholerae, V. fluvialis, V. vulnificus, andV. anguillarum, but notV. parahaemolyticus orV. alginolyticus. Vibrio vulnificus synthesizes an additional, relatively species-specific compound. This unique siderophore has greater biological activity than theV. vulnificus phenolate.     

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.     

High-performance liquid chromatography of siderophores from fungi

Summary A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungiPenicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus andNeurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0–3) was possible.     

Exchange of iron by gallium in siderophores

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.