Development and Application of an Assay for Uranyl Complexation by Fungal Metabolites, Including Siderophores

An assay to detect UO₂²⁺ complexation was developed based on the chrome azurol S (CAS) assay for siderophores (B. Schwyn and J. B. Neilands, Anal. Biochem. 160:47-56, 1987) and was used to investigate the ability of fungal metabolites to complex actinides. In this assay the discoloration of two dyed agars (one containing a CAS-Fe³⁺ dye and the other containing a CAS-UO₂²⁺ dye) caused by ligands was quantified. The assay was tested by using the siderophore desferrioxamine B (DFO), and the results showed that there was a regular, reproducible relationship between discoloration and the amount of siderophore added. The ratio of the discoloration on the CAS-UO₂²⁺ agar to the discoloration on the CAS-Fe³⁺ agar was independent of the amount of siderophore added. A total of 113 fungi and yeasts were isolated from three soil samples taken from the Peak District National Park. The fungi were screened for the production of UO₂²⁺ chelators by using the CAS-based assay and were also tested specifically for hydroxamate siderophore production by using the hydroxamate siderophore auxotroph Aureobacterium flavescens JG-9. This organism is highly sensitive to the presence of hydroxamate siderophores. However, the CAS-based assay was found to be less sensitive than the A. flavescens JG-9 assay. No significant difference between the results for each site for the two tests was found. Three isolates were selected for further study and were identified as two Pencillium species and a Mucor species. Our results show that the new assay can be effectively used to screen fungi for the production of UO₂²⁺ chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Mycorrhizal Fungi: Siderophore Production

Abstract

Mycorrhizal fungi, which commonly occur in natural as well as agricultural soils, are known to enhance plant uptake of nutrients, including metal ions present as trace concentrations. As mycorrhizal infection is a widespread feature of plant communities, it seems appropriate to review the data on mycorrhizal fungi and their potential to produce siderophores.

Based on a bioassay with Aureobacteriumflavescens JG-9 it was shown that a number of ectomycorrhizal fungi (EM) produce hydroxamate siderophores. Also an arbuscular mycorrhizal (AM) grass species, which showed greater iron uptake than nonmycorrhizal controls, tested positively when bioassayed for hydroxamate siderophores. Encoid mycorrhizal fungi, too, have been demonstrated to be capable of producing hydroxamate-type siderophores. However, only in the case of the eridoid mycorrhizal fungi the main siderophores have been isolated and subsequently identified as ferricrocin and fusigen, respectively. The biotechnological and ecological significance of studies of the siderophore biosynthesis by mycorrhizal fungi is discussed.     

Siderophore-mediated iron uptake in different strains of Anabaena sp.

Anabaena sp. strain 6411, which produces the dihydroxamate siderophore schizokinen to facilitate iron uptake, is also capable of using the related siderophore aerobactin. The two siderophores compete for the same iron transport system, but there is a markedly higher affinity for ferric schizokinen than for ferric aerobactin. The trihydroxamate siderophore ferrioxamine B is far less effective as an iron donor in this organism. Anabaena sp. strain 7120 appears to be closely related to strain 6411. It synthesizes schizokinen as its major siderophore and shows rates of iron uptake from ferric schizokinen, ferric aerobactin, and ferrioxamine B which are similar to those observed with strain 6411. Anabaena cylindrica Lemm. 7122 and 1611, on the other hand, differ from strain 6411. In contrast to schizokinen, the hydroxamate which they produce in response to iron starvation cannot be extracted with water from the organic layer and does not support the growth of the siderophore auxotroph Arthrobacter flavescens JG-9. Strain 7122 can use its endogenous siderophore or schizokinen to promote iron uptake, but at 50-fold-lower rates than are observed with Anabaena sp. strain 6411 or 7120.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1

Siderophore production by the magnetic bacterium Magnetospirillum magneticum AMB-1 is elicited by sufficient iron rather than by iron starvation. In order to clarify this unusual pattern, siderophore production was monitored in parallel to iron assimilation using the chrome azurol sulfonate assay and the ferrozine method respectively. Iron concentration lowered approximately five times less than its initial concentration only within 4 h post-inoculation, rendering the medium iron deficient. A concentration of at least 6 microM Fe(3+) is required to initiate siderophore production. The propensity of M. magneticum AMB-1 for the assimilation of large amounts of iron accounts for the rapid depletion of iron in the medium, thereby triggering siderophore excretion. M. magneticum AMB-1 produces both hydroxamate and catechol siderophores.     

Chemical characterization and quantification of siderophores produced by marine and terrestrial aspergilli

Ten aspergilli (five each from marine and terrestrial habitats) were screened for siderophore production. All test isolates produced siderophores as indicated by a positive reaction in the FeCl(3) test, chrome azurol sulphonate assay, and chrome azurol sulphonate agar plate test. Further, the test isolates were compared for their siderophore production potential and chemical characteristics. Examination of the chemical nature of the siderophores revealed that all test isolates produced hydroxamate siderophores that were trihydroxamate hexadentates. Wide-spread occurrence of siderophores in marine isolates indicate their functional role in maintaining overall productivity of coastal waters. Among all test aspergilli, marine Aspergillus versicolor was found to be the largest siderophore producer (182.5 microg/mL desferrioxamine mesylate equivalent), least siderophore production was recorded in a marine strain of Aspergillus niger (3.5 microg/mL desferrioxamine mesylate equivalent).     

Growth stimulation of Brevibacterium sp. by siderophores

AIMS: To assess which types of siderophores are typically produced by Brevibacterium and how siderophore production and utilization traits are distributed within this genus. METHODS AND RESULTS: During co-cultivation experiments it was found that growth of B. linens Br5 was stimulated by B. linens NIZO B1410 by two orders of magnitude. The stimulation was caused by the production of hydroxamate siderophores by B. linens NIZO B1410 that enabled the siderophore-auxotrophic strain Br5 to grow faster under the applied iron-limited growth conditions. Different patterns of siderophore production and utilization were observed within the genus Brevibacterium. These patterns did not reflect the phylogenetic relations within the group as determined by partial 16S rDNA sequencing. Most Brevibacterium strains were found to utilize hydroxamate siderophores. CONCLUSIONS: Brevibacteria can produce and utilize siderophores although certain strains within this genus are siderophore-auxotrophic. SIGNIFICANCE AND IMPACT OF THE STUDY: It is reported for the first time that brevibacteria produce and utilize siderophores. This knowledge can be utilized to stimulate growth of auxotrophic strains under certain conditions. Enhancing the growth rate of Brevibacterium is of importance for the application of this species, for example, for cheese manufacturing or for industrial production of enzymes or metabolites.     

Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae.     

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Effect of pH,L-ornithine and L-proline on the hydroxamate siderophore production by Hymenoscyphus ericae,a typical ericoid mycorrhizal fungus

Since ericoid mycorrhizae become dominant in heathland plant communities on acid soils, we assessed the effect of pH on the hydroxamate siderophore production by a typical ericoid mycorrhizal fungus under pure culture conditions. In addition, we determined whether the supplementation of the nutrient medium with L-ornithine or L-proline as precursors for hydroxamate siderophores would enhance their biosynthesis. The results indicate that the hydroxamate siderophore production by Hymenoscyphus ericae has its optimum at pH 4.5 (between 3.5 and 5.5). L-ornithine rather than L-proline appears to favour the biosynthesis of hydroxamate siderophores.     

Chemical nature, ligand denticity and quantification of fungal siderophores

Thirtyfive siderophore producing fungi were categorized for their hydroxamate, catecholate or carboxylate nature by chemical and bioassays. Out of 35 fungi, 30 were hydroxamates and 5 showed carboxylate nature. However, none of the fungi produced catecholate type of siderophores. Eighteen out of 29 fungi were trihydroxamate and the rest 11 fungi were dihydroxamates. Twenty-three fungi were hexadentate and 6 were tetradentate in nature. Quantification of siderophores using standard compounds deferrioxamine mesylate and rhizoferrin revealed that Phanerochaete chrysosporium produced maximum among the hydroxamate producing fungi and Mycotypha africana resulted maximum among the carboxylate producing fungi.     

Siderophore-Mediated Aluminum Uptake by Bacillus megaterium ATCC 19213

The bacterium Bacillus megaterium ATCC 19213 is known to produce two hydroxamate siderophores, schizokinen and N-deoxyschizokinen, under iron-limited conditions. In addition to their high affinity for ferric ions, these siderophores chelate aluminum. Aluminum was absorbed by B. megaterium ATCC 19213 through the siderophore transport receptor, providing an extra pathway for aluminum accumulation into iron-deficient bacteria. At low concentrations of the metal, siderophore-mediated uptake was the dominant process for aluminum accumulation. At high concentrations of aluminum, passive transport dominated and siderophore production slowed the passive transport of aluminum into the cell. Siderophore production was affected by the aluminum content in the media. High concentrations of aluminum increased production of siderophores in iron-limited cultures, and this production continued into stationary phase. Aluminum did not stimulate siderophore production in iron-replete cultures. The production of siderophores markedly affected aluminum uptake. This has direct implications on the toxicity of heavy metals under iron-deficient conditions.     

Siderophores and related outer membrane proteins produced by pseudomonads isolated from eels and freshwater

A total of 46 environmental pseudomonads, together with six type strains, were examined for their siderophore-producing activity. All strains were able to grow under iron-limiting conditions, gave orange halos in the CAS agar assay, and produced hydroxamates, and some of them also produced phenolate-type compounds. Bioassays showed that all strains, except Pseudomonas aeruginosa, promoted growth of mutant strain Arthrobacter flavescens JG-9, deficient in hydroxamate production, and some of them promoted growth of Salmonella typhimurium enb-1, which requires enterobactin for growth. The presence of iron-regulated outer membrane proteins was observed, the molecular size of the main induced proteins ranged between 76 and 93 kDa.     

Relationship ofAzotobacter chrooccum siderophores with nitrogen fixation

In iron-limited medium, a siderophore producing soil isolate ofAzotobacter chroococcum showed a high level of hydroxamate with relatively low level of nitrogen fixation. Inclusion of iron in the medium resulted in increased nitrogen fixation with decreased hydroxamate production. Under shake culture conditions, the level of both hydroxamate and catechol type of siderophores decreased after 2 d of incubation in iron-deficient medium. However, under iron-sufficient conditions, both siderophore production and nitrogen fixation increased with time although the level of siderophore was quite low. A number of soil isolates and mutants ofA. chrococcum were tested for nitrogen fixation, hydroxamate and catechol type of siderophore production. Wide variation was observed in the siderophore level and nitrogen fixation in the cultures tested. Nitrogen fixation was higher in the iron-sufficient medium than in iron-limited one while hydroxamate yield was higher in iron-limited medium than in the iron-sufficient one in all the cultures. Inclusion of ammonium acetate in the medium induced catechol synthesis in more than 60% of the cultures.     

Bioassay for siderophore utilization by Candida albicans

A convenient plate assay which is sensitive to medium pH has been developed to evaluate potential siderophores of Candida albicans. Adding a siderophore to a filter paper disk on chemically defined Lee's agar (final pH 7.2) seeded with the test strain reversed the growth inhibitory effects of the supplemented (25-100 micrograms/ml) iron chelator ethylenediaminedi(o-hydroxyphenylacetic acid), to provide a zone of growth stimulation. This bioassay has been used to demonstrate the structure-activity relationships of ferrichrome and several water-soluble hydroxamate peptide building blocks of this natural siderophore. Of all compounds so evaluated, ferrichrome exhibited the best activity.     

假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

Physiological properties and production of siderophores detected in Yersinia enterocolitica strain 4-32

A hydrophilic compound with siderophore activity was isolated from a culture of Yersinia enterocolitica 4-32 grown in an iron-deficient medium. It was found that the siderophore secreted did not belong to the catecholamide and hydroxamate type of siderophores and not yersiniabactin. Supplementation of cultures of Y. enterocolitica 4-32 with sodium chloride (300 mM) resulted in a decrease in the production of siderophores.     

Chemical Characterization,Crossfeeding and Uptake Studies on Hydroxamate Siderophore of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ_max at 224 nm and minor fraction appeared as catecholate with a λ_max of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml⁻¹ respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.     

Siderophore production in relation to N2 fixation and iron uptake in pigeon pea-Rhizobium symbiosis

Thirty-one bradyrhizobial and rhizobial strains infecting pigeon pea were screened for siderophore production using Chrome Azurol S (CAS) agar plate as well as a CAS assay solution. Of a total of 31 strains only 23 showed siderophore production. Of the 23 siderophore-positive strains, 21 strains showed the production of hydroxamate while 6 strains showed the presence of catechol type of siderophore. A large variation in the quantity of hydroxamate and catechol produced by different rhizobial strains was observed (1.03–3.73 μg hydroxamate N per mg protein; 0.19–3.43 μmol/L of catechol per mg protein). Maximum nodule biomass was produced by strain PP-11 (CC-1020); strain G-14 formed minimum nodule biomass. Nitrogen contents of low, moderate and high siderophore-producing strains were 11.4, 15.4, 20.9 mg per plant, respectively, iron contents were 1445, 1768 and 2003 ppm, respectively. Siderophore production was related to N₂-fixing efficiency.     

Growth and siderophore production inBradyrhizobium (lupin) strains under iron limitation

SixBradyrhizobium (lupin) strains were evaluated for their ability to produce siderophores using four chemical assays. Two strains gave positive reactions with chrome azurol S assay (CAS) and produced hydroxamate-type siderophores. The other four strains gave negative results for siderophore production using the four assays. Generation time, growth yield and hydroxamate production of one strain (WPBS 3201 D) were affected by the iron concentration of the culture medium and the previous culture history of the cells. Resuspension of washed cells grown previously in media supplemented with 0 and 20 μmol/L Fe into differing iron regimes (0, 0.5, 1, 2, 4, 8, 10, 15 and 20 μmol/L Fe) suggest that the extent of hydroxamate production depended on the growth history of the cells. Cells pregrown in 20 μmol/L Fe produced a high amount of hydroxamates compared with cells pregrown in iron-free medium when resuspended in medium containing up to 4 μmol/L Fe. Cells pregrown in 20 μmol/L Fe were more sensitive to iron repression than those pregrown in 0.5 μmol/L Fe. Mannitol was the best carbon source for siderophore production. Siderophore synthesis was inhibited by 4-chloromercuribenzenesulfonic acid, 2,4-dinitrophenol, sodium azide and MgCl₂ suggesting that an energized membrane and a mercapto group are essential and required for hydroxamate synthesis in strain WPB5 3201 D.