Quantification of Hydroxamate Siderophores in Soil Solutions of Podzolic Soil Profiles in Sweden

Concentrations up to 2 and 12 nM of the hydroxamate siderophores ferrichrome and ferricrocin, respectively, were identified in soil solutions of podzolic forest soils at four sites in both northern and southern Sweden. No ferrichrysin was detected. As with the dissolved organic carbon and low molecular mass organic acids, the highest concentrations of the siderophores were found in the upper layers i.e. the mor layer, the eluvial and upper illuvial horizons. At the southern sites, the concentrations of ferrichrome and ferricrocin were both of similar magnitude and did not differ between the two sites. In contrast, soil solutions at the two northern sites contained more ferricrocin than ferrichrome; the ferricrocin concentrations were also higher at the northern sites than at the southern sites. Analyses were performed by high performance liquid chromatography with a porous graphitic carbon column on which ferrichrome, ferricrocin and ferrichrysin were separated. Detection by electrospray ionization mass spectrometry (ESI-MS) combined with on-line sample pre-concentration, by means of column-switching, enabled detection limits of 0.1–0.2 nM for ferrichrome, ferrichrysin and ferricrocin. The structural identities of the siderophores were further verified by MS/MS fragmentation. Fragmentation of ferrichrome, ferricrocin and ferrichrysin occurred mainly via peptide cleavage. The most intense fragments were typified by the loss of one of the three iron(III) chelating hydroxamate residues, i.e N⁵-acyl-N⁵-hydroxy ornithine.     

Role of siderophores in iron storage in spores of Neurospora crassa and Aspergillus ochraceus.

Spores of Neurospora crassa 74A are lacking in ferritinlike iron pools, as demonstrated by M?ssbauer spectroscopic analysis. The cyclic hexapeptide siderophore ferricrocin constituted 47% of the total iron content in spores. After germination and growth, the ferricrocin iron pool disappeared, indicating that the metal was utilized. In spores of Aspergillus ochraceus, 74% of the total iron content was bound by ferrichrome-type siderophores. Siderophores may function as iron storage forms in fungal systems.     

The relation between spruce monoculture and incipient podzolisation in ochreous brown earths of the Belgian Ardennes

Summary The effects of coniferous monoculture on the distribution of C, Fe and Al in the upper horizons of brown ochreous earths of the Belgian Ardennes were investigated by comparing 5 soil profiles developed under an 80 year old spruce stand with another 5 profiles developed under a climactic broad-leaved forest (beechwood).Organic carbon, Fe and Al were extracted with 0.1N NaOH/Na-tetraborate solution buffered at pH 9.7: recent studies have shown that this extraction is particularly appropriate for the detection of incipient podzolisation in brown earths-brown podzolic soils intergrades.Even if most of the classic podzolisation indexes fail to illustrate differences, nevertheless our results show that fulvic acids and organo-ferric complexes are present in significantly greater amounts in the upper part of the cambic (B)₁ horizon of the soils developed under conifers. Moreover, this podzolic tendancy is confirmed by the weathering patterns of the clay minerals in the A₁(B) horizons developed under spruces, i.e. a more pronouced weathering of chloritic layers than those observed in the beechwood soil, with a correlative genesis of more abundant smectite-like minerals. One may therefore conclude that the change in the humus type (moder to mor) after the planting of spruce trees, has been sufficient, within the local climatic and edaphic context, to promote incipient podzolisation.     

Soil microfungi in three Swedish coniferous forests

Microfungi were isolated with the soil washing technique from five different soil horizons in three podzolic coniferous forests in Sweden. Totally 126 species were identified of which 45 were found at two and 24 were found at three sites. The most common genera were Mortierella and Penicillium . Other genera isolated in high frequences were Oidiodendron, Tolypocladium, Trichoderma and Verticillium . Pronounced differences in species composition were found between the soil layers, while great similarities were shown in identical soil layers at different sites.     

Isolation and identification of hydroxamate siderophores of ericoid mycorrhizal fungi

Three ericoid mycorrhizal fungi were grown in pure culture under iron deprivation: (i) the ascomyceteHymenoscyphus ericae, a characteristic endophyte of ericaceous plants on acid soils; (ii) the hyphomyceteOidiodendron griseum, an ericoid mycorrhizal fungus which is also a soil-borne fungus able to colonize wood; and (iii) an endophyte of the calciculous ericaceous plantRhodothamnus chamaecistus. All three fungi produced several hydroxamate siderophores which were isolated in the ferric form by adsorption to Amberlite XAD-2, gel chromatography on Sephadex LH20 and by HPLC on a C₁₈ reversed-phase column. Siderophores were identified by (i) co-chromatography with known fungal siderophores, (ii) ion spray mass spectrometry after semi-preparative HPLC and (iii) analyzing their electrophoretic behavior. WhileH. ericae andO. griseum were similar in producing ferricrocin as their principal siderophore, the endophyte ofR. chamaecistus produced mainly fusigen.     

Siderophores in environmental research: roles and applications

Siderophores are organic compounds with low molecular masses that are produced by microorganisms and plants growing under low iron conditions. The primary function of these compounds is to chelate the ferric iron [Fe(III)] from different terrestrial and aquatic habitats and thereby make it available for microbial and plant cells. Siderophores have received much attention in recent years because of their potential roles and applications in various areas of environmental research. Their significance in these applications is because siderophores have the ability to bind a variety of metals in addition to iron, and they have a wide range of chemical structures and specific properties. For instance, siderophores function as biocontrols, biosensors, and bioremediation and chelation agents, in addition to their important role in weathering soil minerals and enhancing plant growth. The aim of this literature review is to outline and discuss the important roles and functions of siderophores in different environmental habitats and emphasize the significant roles that these small organic molecules could play in applied environmental processes.     

Current and historical patterns of heavy metals pollution in Estonia as reflected in natural media of different ages: ICP Vegetation,ICP Forests and ICP Integrated Monitoring data

In order to characterise current and historical pattern of heavy metal (HM) pollution in Estonia, this article will compare the concentrations and stocks of Cd, Cr, Cu, Ni, Pb and Zn represented in current deposition (data from 18 local precipitation stations) with natural media of three different ages: 1–3-year-old moss carpet (ICP Vegetation moss survey data from 99 open area plots), 3–5-year-old litter layer, and several-decades-old organic layer (mor humus) of coniferous forest, in mostly podzolic soils (ICP Forest soil survey data, 75 stands).Objectives of this study are (1) to assess differences in HM retention and accumulation in various aged media of coniferous stands (2) to estimate territorial differences in current HM distribution and previously accumulated concentrations and stores of HM (3) to compare territorial distribution of HM concentration in Estonia between five different regions: N-W; N-E; S-W; S-E and Western insular region, whereas the local oil shale industry in N-E part of Estonia has been the main source of HM pollution over a long period of time and therefore may have an effect on HM regional distribution.Comparing the studied media, three types of HM retention patterns were detected: (1) for Cu, Ni, Cr (2) for Cd, Pb, (3) for Zn. The mean current level of HM deposition in Estonia is low comparison to previous decades, especially the 1980s. The effect of the previously significantly higher exposure of HM emissions and deposition is preserved in older part of soil organics (OF), where the highest stocks and concentrations of HMs (with the exception of Zn) are currently found. The HM proportions in fly ash of oil shale and in OF layer of soil were very similar with regards to Ni and Cr—indicating their origin from the oil shale industry in the N-E region. According to spatial distribution analysis, the greatest accumulated storages of Ni and Cr in OF layer of coniferous forest soils are characteristic to S-W Estonia.     

Column Leaching Using Dry Soil to Estimate Solid-Solution Partitioning Observed in Zero-Tension Lysimeters. 1. Method Development

In order to understand the reactions taking place between the soil solid phase and the soil solution, we require knowledge of the chemistry of the soil solution as it occurs in the field. This knowledge allows us to conduct experiments with environmentally relevant concentrations of macro and microelements in solution. Zero-tension lysimeters directly sample the mobile fraction of soil solutions. Unfortunately, they are expensive to sample and require long equilibration periods. Other solution extraction methods do not provide solutions similar in concentration to lysimeters, either because they sample a different fraction of the soil solution or due to the impacts of the sampling process. The processes that produce lysimeter solutions cannot be emulated; however, to estimate lysimeter solution chemistry, we developed a standard protocol to produce solutions that resemble lysimeter solutions from podzolic soils using air-dried samples. We washed air-dried soil columns sequentially with de-ionized water until the electrical conductivity (EC) of the leachates stabilized and then leached the columns using an environmentally relevant concentration of a weak salt solution. We hypothesize that the stabilization point of the EC of the soil solution is indicative of the point at which soluble salts and organic material precipitated during sampling and storage are removed from the soil surface. Solutions produced by leaching, once the EC of wash solutions had stabilized, were comparable to lysimeter solutions from the area where samples were collected with respect to the concentrations of divalent cations, pH, EC and DOC.     

Siderophore sorption to clays

Siderophores are low molecular weight organic ligands exuded by some aerobic organisms and plants to acquire Fe under Fe-limited conditions. The hydroxamate siderophores may sorb to aluminosilicate clays through a variety of mechanisms depending upon the nature of the clay and of the siderophore along with solution conditions such as pH, ionic strength, and presence of metal cations. They may also affect metal binding to clays. Here, we review previous studies of siderophore sorption to aluminosilicate clays; briefly discuss how the techniques of X-ray diffractometry, Fourier-transform infrared spectroscopy, and X-ray absorption spectroscopy may be applied to such studies; review effects of siderophores on metal sorption to clays; and highlight some areas for future research.     

Siderophore production by actinobacteria

Produced by bacteria, fungi and plants, siderophores are low-molecular-weight chelating agents (200–2,000 Da) to facilitate uptake of iron (Fe). They play an important role in extracellular Fe solubilization from minerals to make it available to microorganisms. Siderophores have various chemical structures and form a family of at least 500 different compounds. Some antibiotics (i.e., albomycins, ferrimycins, danomycins, salmycins, and tetracyclines) can bind Fe and some siderophores showed diverse biological activities. Functions and applications of siderophores derived from actinobacteria were reviewed to better understand the diverse metabolites.     

Increasing Feldspar Tunneling by Fungi across a North Sweden Podzol Chronosequence

Tunnels in feldspar grains, assumed to be created by fungal hyphae, were first discovered in a boreal podzol. In this paper, we further describe the phenomenon of mineral tunneling by determining the rate of feldspar tunneling across a north Sweden podzol chronosequence. The chronosequence is a result of ongoing land uplift, which started after the retreat of glaciers about 9000 years ago. The sequence comprises a series of soils that began developing on glacial tills 190–7800 years ago. Feldspar tunneling was concentrated in the uppermost 2 cm of the E horizon, and its frequency increased significantly with soil age. Although no tunnels were found in feldspar grains from the youngest soil (190 years), they were seen more frequently in soils aged 2000 years and older. This lag phase in tunnel formation of about 2000 years coincided with the disappearance of the easily weatherable potassium-(K) and calcium (Ca)-containing minerals biotite and hornblende and with the appearance of etch pits on feldspar grains. In the oldest soil (7800 y), about 25% of the feldspar grains in the upper 2 cm of the E horizon were tunneled. Within site variation in tunnel frequency was high, and we were able to exclude spatial variations in mineralogy and texture as a possible explanation. The shape of the tunnels, their depth distribution, and the fungal hyphae found inside them all offer support for the previous assumption that their formation is mediated by biological activity involving fungi. The results of this investigation also indicate that the bioavailability of Ca and K may be a factor in tunnel formation. Received 12 March 2001; accepted 9 July 2001.     

水拉恩氏菌JZ-GX1产嗜铁素特性及其对林木病原菌的拮抗作用

【背景】嗜铁素被认为是一种具有开发和利用价值的新型生物活性物质,已被逐渐应用到植物病原菌的防治中。【目的】明确根际促生菌水拉恩氏菌(Rahnella aquatilis) JZ-GX1产嗜铁素最佳发酵培养条件,进一步探讨嗜铁素在防治植物根部病害中的潜在作用。【方法】采用摇瓶发酵法,通过铬天青(Chrome azurol S,CAS)检测分析,对影响JZ-GX1菌株嗜铁素分泌的几种发酵因子进行研究,并通过菌丝生长抑制速率法测定嗜铁素对两种林木病原菌的拮抗效果。【结果】以KMB为基础培养基,初始pH 8.0,装液量25 mL/50 mL,按1%接种量接种,在28°C下培养36 h可获得该菌株较高产量的嗜铁素;鉴定其嗜铁素类型为羧酸盐和异羟肟酸型的复合型铁载体;在最适条件下测得其发酵原液对樟疫霉(Phytophthoracinnamomi)和立枯丝核菌(Rhizoctoniasolani)的抑制率均达到100%。【结论】水拉恩氏菌JZ-GX1对碱性土壤上林木根部病害的防治具有较好的潜力。     

Ferricrocin - an ectomycorrhizal siderophore of Cenococcum geophilum

The ectomycorrhizal fungus Cenococcum geophilum was grown in low-iron medium and the excreted siderophores were extracted, purified and analyzed by HPLC. The principal hydroxamate siderophore produced, was identified as ferricrocin as confirmed by analytical HPLC, FAB-mass spectrometry and ¹H- and ¹³C-NMR spectra. Although the occurrence of ferricrocin has been shown earlier to occur in the ericoid mycorrhizal fungi, this is the first report of ferricrocin in a true ectomycorrhizal fungus which is taxonomically related to the ascomycetes.     

Estimating the Mean Residence Time of Lead in the Organic Horizon of Boreal Forest Soils using 210-lead, Stable Lead and a Soil Chronosequence

Knowledge about the residence time of lead in the organic horizon (mor layer; O-horizon) overlaying forest mineral soils is important for the prediction of past and future lead levels in the boreal environment. To estimate the mean residence time (MRT) of lead in the mor layer, we use in this study from Northern Sweden three different approaches: (1) lead-210 is applied as a tracer of lead migration; (2) estimations of loss rates of stable lead (concentrations and ²⁰⁶Pb/²⁰⁷Pb ratios) from the mor layer at an undisturbed forest setting, and (3) a study of lead in a soil series with sites of different age (a chronosequence of 20–220 years). In the last two approaches we compared measured inventories in the soil with estimated inventories derived using analyses of lake sediments. The results suggested a MRT of about 250 years in the mor layer in the mature forest and in the older parts of the chronosequence it was at least >170 years. The agreement between the three different approaches gives good credibility to this estimate. It is also supported by a modeling of trends in the ²⁰⁶Pb/²⁰⁷Pb ratio both between single cores and with depth in the mor layer. Our results suggest that it will take centuries for the deeper parts of the mor layer of undisturbed boreal forest soils to fully respond to decreased atmospheric lead pollution. However, data from the chronosequence indicate that the response could be much faster (MRT<50 years) in the mor layer at early stages of forest succession where graminoid and broadleaved litter fall dominates over conifer litter.     

The siderophore ferricrocin produced by specific foliar endophytic fungi in vitro

Production of extracellular siderophores is typical for many plant-associated microbes, both mutualistic and antagonistic. Various strains of mycorrhizal fungi produce siderophores, and siderophore production by pathogenic fungi is typically associated with virulence. We analyzed extracellular siderophore production along with production of antibacterial and antioxidant compounds in foliar endophytic fungi of Scots pine (Pinus sylvestris L.) and Labrador tea (Rhododendron tomentosum Harmaja). The siderophore produced in vitro was ferricrocin, quantities ranging between 7.9 and 17.6 μg/l. Only the fungi with antibacterial activity produced ferricrocin and any well-known siderophores were not detected in the broths of antioxidant-producing fungi. Therefore, production of ferricrocin is typical for some, but not all foliar endophytic fungi. Ferricrocin was detected in the leaves of Labrador tea, which suggests that ferricrocin may play a role in vivo in the interaction between the endophyte and plant host.     

Bacterial siderophores promote plant growth: Screening of catechol and hydroxamate siderophores

The aim of the study was to determine the quality and quantity of siderophores produced by bacteria isolated from plants' roots. The second aim was to determine the effect of siderophores on plants growth (Festuca rubra L. and Brassica napus L.). The study was carried out using bacteria isolated from roots of: Arabidopsis thaliana L., F. rubra, and Agrostis capillaris L., growing on the heavy metals contaminated area. The chrome azurol sulfonate (CAS) test, Arnow's test for catechol siderophores, and Csaksy's test for hydroxamate siderophores were performed. Among the bacteria, 42 isolates (39%) had a positive result in the CAS. Endophytic bacteria were mostly producing the catechol siderophores. It was found that F. rubra is the plant which is linked with the highest number of siderophores producing bacteria. The highest concentration of siderophores was noted for ectorhizospheric bacteria associated with A. thaliana, hyperaccumulating plant. It was found that hydroxamate siderophores are mainly produced by ectorhizosphere and rhizoplane bacteria. The siderophores producing bacteria reduced the toxicity of metals and improved the phytoremediation. Siderophores treatment increased the growth of plants in the biological assay, growing on two different soils: one highly contaminated with heavy metals and the second strongly alkaline soil.     

Site-specific rate constants for iron acquisition from transferrin by the Aspergillus fumigatus siderophores N′,N′′,N′′′-triacetylfusarinine C and ferricrocin

Aspergillus fumigatus is an opportunistic fungal pathogen that causes life-threatening infections in immunocompromised patients. Despite low levels of free iron, A. fumigatus grows in the presence of human serum in part because it produces high concentrations of siderophores. The most abundant siderophores produced by A. fumigatus are N',N',N'-triacetylfusarinine C (TAF) and ferricrocin, both of which have thermodynamic iron binding constants that theoretically allow them to remove transferrin (Tf)-bound iron. Urea-polyacrylamide gel electrophoresis was used to measure the change in concentration of Tf species incubated with TAF or ferricrocin. The rate of removal of iron from diferric Tf by both siderophores was measured, as were the individual microscopic rates of iron removal from each Tf species (diferric Tf, N-terminal monoferric Tf and C-terminal monoferric Tf). TAF removed iron from all Tf species at a faster rate than ferricrocin. Both siderophores showed a preference for removing C-terminal iron, evidenced by the fact that k(1C) and k(2C) were much larger than k(1N) and k(2N). Cooperativity in iron binding was observed with TAF, as the C-terminal iron was removed by TAF much faster from monoferric than from diferric Tf. With both siderophores, C-terminal monoferric Tf concentrations remained below measurable levels during incubations. This indicates that k(2C) and k(1C) are much larger than k(1N). TAF and ferricrocin both removed Tf-bound iron with second-order rate constants that were comparable to those of the siderophores of several bacterial pathogens, indicating they may play a role in iron uptake in vivo and thereby contribute to the virulence of A. fumigatus.     

Column Leaching using Dry Soil to Estimate Solid-Solution Partitioning Observed in Zero-Tension Lysimeters. 2. Trace Metals

The fundamental questions revolving around research into trace metals in soils are how much, and in what form, do metals exist in soil solutions. The mobile phase of soil solutions can be sampled by lysimeters, but cannot be consistently and accurately reproduced in laboratory extractions. We used a column leaching method developed specifically to produce solutions that were similar to those of lysimeters from northern forest podzolic soils. We hoped to yield reasonable estimates of the partitioning of Cd, Cu, Ni, Pb and Zn between the solid and solution phases observed in the field. The column leaching method produced solutions that were similar to lysimeter solutions in the concentrations of metals in solution. Partitioning coefficients (log K_d) calculated from average lysimeters solution concentrations ranged from 2.8 to 3.9 for Cd, 3.5 to 4.2 for Cu, 3.1 to 4.3 for Ni, 3.9 to 5.1 for Pb and 2.8 to 3.6 for Zn. Laboratory extractions produced very similar log K_d values ranging from 3.4 to 3.9 for Cd, 3.4 to 3.9 for Cu, 3.4 to 4.1 for Ni, 4.1 to 5.2 for Pb and 3.2 to 3.5 for Zn. According to a semi-mechanistic regression model based on observed lysimeter concentrations, the metal concentrations in solution were appropriate relative to known factors that influence metal partitioning in soils: pH and the concentrations of total metals and dissolved organic carbon. Partitioning coefficients based on laboratory extractions in the literature were on average an order of magnitude greater than those observed in lysimeters. When compared to the results of other laboratory extractions, the proposed extraction procedure appeared to be an effective method to estimate the chemistry of soil solutions in the field.     

The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC)

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.     

Ferricrocin synthesis in Magnaporthe grisea and its role in pathogenicity in rice

Iron is an essential element for the growth of nearly all organisms. In order to overcome the problem of its low bioavailability, microorganisms (including fungi) secrete siderophores, high-affinity iron chelators. As the acquisition of iron is also a key step in infection processes, siderophores have been considered as potential virulence factors in several host–pathogen interactions. Most fungi produce siderophores of the hydroxamate-type, which are synthesized by non-ribosomal peptide synthetases (NRPSs). Magnaporthe grisea , the causal agent of rice blast disease, produces ferricrocin as intracellular storage siderophore and excretes coprogens. In the M. grisea genome we identified SSM1 , an NRPS gene, and a gene encoding an l -ornithine N5-monooxygenase ( OMO1 ) that is clustered with SSM1 and responsible for catalysing the first step in siderophore biosynthesis, the N⁵ hydroxylation of ornithine. Disruption of SSM1 confirmed that the gene encodes ferricrocin synthetase. Pathogenicity of these mutants towards rice was reduced, suggesting a role of this siderophore in pathogenicity of M. grisea .