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Indian Journal of Experimental Biology Vol. 43. October 2005, pp. 880-886 Chemical properties and NMR spectroscopic identification of certain fungal siderophores Arefa Baakza, B P Dave* & H C Dube Department of Life Scicnces, Bhavnagar University, Bhavnagar 364002. India Received 19 January 2005; rev ised 25 April 2005 Siderophores of six fungi viz. Aspergillus sp. ABp4, AllreobacidiulIl pullufwlS, Penicillil/l11 oxalic/III! , P. chrysosporiunz, My colypha ajricana and SYllcephalasln.t1ll racemosulIl were examined for their (I) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di-, or trihydroxamate; (3) amino acid composition; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicunz, P. chrysosporium, and M. ajricalla) exhibited net basic charge. siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racelllosum) were acidic and I fungus (A. pI/III/fans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporil/Ill was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, hi stidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporil/l1l, and valine in M. ajricana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. puill/ial/s) as asperchrome Fl. and AB-513 (M. ajricana) as rhizoferrin. The peak obtained for siderophore AB-5 (Aspergilil/s sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception. Keywords : Amino acids. Fungi, NMR. Siderophores Siderophores are low molecular weight « WOOD) virtually Fe (III) specific ligands produced by microorganisms to combat low iron stress 1.2. They facilitate the solubilization and transport of iron into the cell by a cognate transport system. No system analogous to siderophores has been known for any metal ion, thus, making iron unique in requiring such specific ligands. Regulation of iron uptake became a necessity when reducing atmosphere switched to oxidizing with the emergence of oxygenic photosynthesis by cyanobacteria 3 . Thus, siderophores are viewed as an evolutionary response to appearance of oxygen with concomitant oxidation of Fe (II) to Fe (Ill). Waring and Werkman 4 have documented the requirement of iron by microbes hence, emphasis has been laid on the role of iron plays in microbial life ranging from respiration to nucleic acid synthesis. Despite the fact that iron is the fourth most abundant element in the earth's crust, it is unavailable to and plants, as it forms insoluble oxyhydroxide polymers of FeOOH (e.g.geothite, haematite) at neutral to alkaline pH of the soil in which the earth abounds). Since, iron oxides are *Curres ponJing author: E-mail: [email protected] highly insoluble and also highly stable, free iron in an aerobic, aqueous environment is limited to an equilibrium concentration of 10- 17 M, a value far below that required for the optimum growth of microbes (l0- 8 to 10- 6 M). This accounts for the general occurrence of siderophores in all aerobic and facultative anaerobic microorganisms. Based on the chemical nature of their coordination sites, microbial siderophores are classified as hydroxamates, catecholates, carboxylates and mixed type. Hydroxamates are produced both by bacteria and fungi; catecholates are produced only by bacteria, whereas carboxylates are produced exclusively by mucoraceous fungi and a few bacteria (Rhizobium meliloti and Staphylococcus hyicus). Mixed types are produced by Pseudomonas fluorescens. In fungi, the hydroxamate siderophores are mostly orinithine- based, while bacterial hydroxamates are made of hydroxylated and acylated alkyl amines 6 - s _ While N°- hydroxyomithine is a characteristic building block in all fungal hydroxamate siderophores, N°-acyl residues may vary within the different fungal hydroxamate siderophores. This forms the basis for the recognition of three main series of fungal hydroxamate siderophores viz., Ferrichrome series, Fusarinines and

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  • Indian Journal of Experimental Biology Vol. 43 . October 2005 , pp. 880-886

    Chemical properties and NMR spectroscopic identification of certain fungal siderophores

    Arefa Baakza, B P Dave* & H C Dube

    Department of Life Scicnces, Bhavnagar University, Bhavnagar 364002. India

    Received 19 January 2005; revised 25 April 2005

    Siderophores of six fungi viz. Aspergillus sp. ABp4, AllreobacidiulIl pullufwlS, Penicillil/l11 oxalic/III! , P. chrysosporiunz , Mycolypha ajricana and SYllcephalasln.t1ll racemosulIl were examined for their (I) electrophoretic mobilities to determine the acidic, basic or neutral charge; (2) Fe (III) binding nature viz., mono-, di- , or trihydroxamate; (3) amino acid composition ; and (4) NMR (nuclear magnetic resonance) spectroscopy to determine their structure. Electrophoretic mobilities of siderophores of 3 fungi (P. oxalicunz, P. chrysosporium, and M. ajricalla) exhibited net basic charge. siderophores of 2 fungi (Aspergillus sp. ABp4 and S. racelllosum) were acidic and I fun gus (A. pI/III/fans) was neutral. Electrophoresis of ferrated siderophore at pH 2 and colour of the spots indicated that siderophores of Aspergillus sp. ABp4 and P. oxalicum and A. pullulans were trihydroxamates, whereas siderophore of P. chrysosporil/Ill was dihydroxamate. Amino acid composition of siderophores purified by XAD-2 column chromatography, revealed the presence of asparagine, histidine, and proline in Aspergillus sp. ABp4, serine and alanine in P. chrysosporil/l1l , and valine in M. ajricana. The structure of purified siderophores as revealed by NMR spectroscopy identified siderophore of AB - 2670 (A. puill/ial/s) as asperchrome Fl. and AB-513 (M. ajricana) as rhizoferrin. The peak obtained for siderophore AB-5 (Asperg ilil/s sp. ABp4) did not show resemblance to any known siderophore, therefore may be an exception .

    Keywords: Amino acids. Fungi , NMR. Siderophores

    Siderophores are low molecular weight WOOD) virtually Fe (III) specific ligands produced by microorganisms to combat low iron stress 1.2. They facilitate the solubilization and transport of iron into the cell by a cognate transport system. No system analogous to siderophores has been known for any metal ion, thus, making iron unique in requiring such specific ligands. Regulation of iron uptake became a necessity when reducing atmosphere switched to oxidizing with the emergence of oxygenic photosynthesis by cyanobacteria3. Thus, siderophores are viewed as an evolutionary response to appearance of oxygen with concomitant oxidation of Fe (II) to Fe (Ill). Waring and Werkman4 have documented the requirement of iron by microbes hence, emphasis has been laid on the role of iron plays in microbial life ranging from respiration to nucleic acid synthesis. Despite the fact that iron is the fourth most abundant element in the earth ' s crust, it is unavailable to microorgani~ms and plants, as it forms insoluble oxyhydroxide polymers of FeOOH (e.g.geothite, haematite) at neutral to alkaline pH of the soil in which the earth abounds). Since, iron oxides are

    *CurresponJing author: E-mail: [email protected]

    highly insoluble and also highly stable, free iron in an aerobic, aqueous environment is limited to an equilibrium concentration of 10-17 M, a value far below that required for the optimum growth of microbes (l0-8 to 10-6 M). This accounts for the general occurrence of siderophores in all aerobic and facultative anaerobic microorganisms .

    Based on the chemical nature of their coordination sites, microbial siderophores are classified as hydroxamates, catecholates, carboxylates and mixed type. Hydroxamates are produced both by bacteria and fungi; catecholates are produced only by bacteria, whereas carboxylates are produced exclusively by mucoraceous fungi and a few bacteria (Rhizobium meliloti and Staphylococcus hyicus). Mixed types are produced by Pseudomonas fluorescens. In fungi, the hydroxamate siderophores are mostly orinithine-based, while bacterial hydroxamates are made of hydroxylated and acylated alkyl amines6-s_ While N-hydroxyomithine is a characteristic building block in all fungal hydroxamate siderophores, N-acyl residues may vary within the different fungal hydroxamate siderophores. This forms the basis for the recognition of three main series of fungal hydroxamate siderophores viz., Ferrichrome series, Fusarinines and

  • BAAKZA e/ at. : PROPERTIES & NMR OF FUNGAL SIDEROPHORES 881

    Coprogens9 (Rhodotorulic acid is often regarded as a subclass of coprogens). In this study we have examined the siderophores for their (i) electrophoretic mobilities; (ii) Fe; (III) binding nature; (iii) amino acid composition; and (iv) structure elucidation by NMR spectroscopy.

    Materials and Methods Organisnzs----!Six test fungi belonging to

    Ascomycota (Aspergillus sp. ABp4, Penicillium oxalicum), Basidiomycota (,t\u reobasidium pullulans, Phanerochaete chrysosporium) and Zygomycota (Mycotypha africana. Syncephalastrum racemosum) were used for this study. The organisms were maintained on potato dextrose agar slants (PDA)IO and stored at 4C until used. These fungi produced siderophores as evidenced by the positi ve FeCI, test II, Chrome Azurol S (CAS) test 12 and CAS agar plate tese 2,13.

    Siderophore production medium - The fungi belonging to Ascomycota and BasidiG'mycota were grown in Grimm-Allen mediuml4 containing (per liter of distilled water) K2S0 4, 1 g; ammonium acetate, 3g; KZHP04. 3g; citric acid, Ig; sucrose, 20g; and adjusted to pH 6.8 with ammonia. The medium was then supplemented with thiamine, 2mg; CuS04.5H20, 0.005mg; MnS04. H20, 0.035mg; ZnS04.7H20, 2mg; and MgS04.7HzO, 80mg. Modified M9 medium

    l5 was used for fungi belonging to Zygomycota that contained (per liter of distilled water) glucose, 10 g; NazHP04, 7g; KH2P04,3g; NaCI, 0.5g; NH4CI, Ig; MgS04, 0.25g; CaCh, 0.015g; and pH adjusted to 7.2. The medium was then supplemented with thiamine, 0.005g; and ZnCI 2, 0.015g. The above media were decontaminated of iron by adding 8-hydroxyquinoline di ssolved in chloroforml6. Medium (50ml) was dispensed in a flask (500 ml) . All glass wares were soaked overnight in 6M. HCI and rinsed with distilled water several times to remove traces of iron prior to

    17 use .

    After 15 days of growth at their optimum temperatures, the mycelia were removed by filtration through Whatman No, 42 filter paper and the culture filtrates were used for further study.

    Electrophoretic mobilities of the siderophores -Paper electrophoresis is an important tool to determine the nature of charge present on a ferric siderophore . The act~,!1 rate of mobili ty of a ferric siderophore at a certain pH depends 01). the number of factors including the net charge on the molecule and

    the molecular weight of the compound. The compounds separate from one another during electrophoresis at pH 5 18 The information provided by electrophoresis helps in planning the purification of the siderophore extract l9 .

    Culture filtrate (0.1 ml with pH adjusted to 5.6) was spotted on Whatman No. 3 paper. The electrophoresis was run at approximately 30Y/cm for 1-2 hr in a flat bed device using a volatile buffer (5.7 ml glacial acetic acid, 24.3 ml pyridine per liter) at pH 5,612 The paper was dried carefully to remove all traces of pyridine and acetic acid. It was then sprayed on both sides with CAS assay solution. After a few minutes, pink spots appeared on a light blue background.

    Binding properties of siderophores-Electro-phoresis at pH 2 provides additional information on Fe (III) binding nature of the siderophore. The ferric monohydroxamate and the dihydroxamate complexes usually form deep purple and pink-purple colors respectively, while the ferric trihydroxamates remain red 19. The filtrates were ferrated with 2% aqueous FeCI3 solution. These filtrates (PH 2) were then spotted on Whatman No.3 filter paper strips and run at 30 V/cm for 1-2 hr in 4% formic acid. The color of the spot indicated the binding nature viz., mono-, di-and trihydroxamate of the siderophores .

    Extraction from culture broth I 9--S iderophores are produced inside the fungal cells under iron-deficient condition, and then excreted into the medium. Efficient extraction, and separation methods are needed to obtain the compounds in pure form, which is an essential pre-requisite for the elucidation of its structure. After removal of mycelia by filtrati on. the first step in the extraction process was the formation of ferric complexes of siderophores by addition of 2% aqueous FeCI] solution. The clear red supernatant was subjected to XAD-2 extraction.

    XAD-2 purification of siderophores - A column was prepared with XAD-2 as suggested by the manufacturer (Supelco, Bellfonte, PA). The dry resin was transferred to a beaker (500ml). Methanol ~as added to cover the resin bed by 2.5 - 5 cm. After complete mixing, the material was allowed to stand for 15 min. This was displaced by distilled water and allowed to stand for 15 min after stirring. Deionized water (approx. 20 - 25 ml) was added to the empty column before adding the resin slurry. Resin was slowly poured into the column and excess water was drained through the bottom. The column was then

  • 882 INDIAN J EXP BIOL, OCTOBER 2005

    back-washed for 30 min. After this the column was ready for use. The column was pre-washed with water, methanol and water again. The aqueous culture supernatant containing the ferric siderophores was slowly passed through the column. The ferric siderophores bound to the top of the column and pale-colour supernatant eluted out. The column was then washed with 5-10 bed volumes of water to remove all unbound components of the medium. Changing the solvent to methanol, ferric siderophores were eluted out as a single band. The column was left to equilibrate with methanol so that those siderophores which had adsorbed strongly to the resin, dissolved and resulted in a complete recovery of siderophores.

    Chemical degradation of siderophore for amino acid analysis--Chemical degradation should preferably be done on deferrisiderophores, as Fe (III) atom in the molecule may interfere with the reaction process l9. The XAD-2 extracted siderophores were deferrated. Deferration of ferric siderophores may be carried out by extraction of iron with 8-hydroxyquinoline20-22. The ferric siderophores were dissolved in a small volume of water, to which excess recrystallized 8-hydroxyquinoline was added. The suspension was slowly shaken overnight at room temperature. The greenish-black iron complex of 8-hydroxyquinoline as well as, unused reagent was removed by chloroform extraction (4-5 times). Addition of methanol or other organic solvents in the incubation mixture often causes insufficient deferration 19.

    Partial chemical degradation of deferrated siderophores (Aspergillus sp. ABp4, Phanerochaete chrysosporium, Mycotypha africana) was performed by adding 2 ml of 12N HCI to 2 ml of deferrated aqueous solution23-25 . The tube was sealed and heated at 110C for 16 hr. The excess acid was evaporated on a water bath at 50C. To the dried residue 1 ml of distilled water (containing 10% n-propanol) was added and 50 III of this was spotted on a silica gel GF254 plate. The plate was dried and developed in a saturated chamber containing butanol:acetic acid: water (4: 1: 1). The plates were sprayed with ninhydrin (0.2% in acetone) and the amino acids identified by comparing the Rf values to that of standard amino acids26. The amino acids were further confirmed by co-chromatography.

    NMR spectroscopr-Ferric iron present in the siderophore exists in a paramagnetic high spind5

    state, which causes severe line broadening of the

    NMR signals. For this reason, iron was removed from the ferric siderophore (XAD-2 extracted) prior to NMR spectroscopy. Deferration of ferric siderophores was carried out as mentioned in the previous section (chemical degradation).

    Deferrisiderophores tend to retain moisture in the samples, which interfere with IH NMR spectroscopy. Hence, samples were lyophilized and thoroughly dried prior to dissolving in a suitable deuteriated solvent. D20 is not regarded as a suitable solvent for deferrisiderophores27. CD30D is a good solvent used in the NMR spectroscopy of siderophores . Full and detailed spectra were obtained with (CD3h SO (d6-DMSO) and the information provided by the spectra in this solvent was useful in the complete identification of the siderophore.

    XAD-2 extracted siderophores of three fungi viz. , Aspergillus sp. ABp4 (AB-5), Aureobasidium pullulans (AB-2670), Mycotypha africana (AB-513) were deferrated and deferrisiderophores were lyophilized. These were thoroughly dried and then dissolved in (CD3)2 SO and subjected to NMR. All IH spectra were taken on Bruker 200 MHz with super conducting magnet in DMSO-d6, chemical shifts are in Sppm.

    Results and Discussion The results suggested that out of the six test fungi,

    siderophores of 3 fungi were basic (incidentally from each class of fungi), 2 acidic and 1 neutral (Table 1). Schwyn and Neilands l3 have observed electrophoretic mobilities and the net charge on siderophores of E.co/i, Rhizobium meliloti and Rhodotorula piliminae.

    Table 1 - Electrophoretic migration of siderophores

    Test fungi Electro2horetic mi![ation Net charge

    Anode Cathode No mobility

    Aspergillus sp. + Acidic ABp4 Penicillium + Basic oxalicum Aureobasidium Neutral pullulans Phanerochaete + Basic chrysosporium Mycotypha + Basic africana Syncephalastrnm + Acidic racemosum + indicates migration towards cathode or anode; - indicates no mobilit~

  • BAAKZA et al.: PROPERTIES & NMR OF FUNGAL SIDEROPHORES 883

    Budde and Leong28 have noted two types of compounds, neutral and acidic. Dave29 has suggested that the net charge on siderophores of 20 fungi belonging Zygomycota, Ascomycota and mitosporic fungi fell into five classes (i) acidic, (ii) basic, (iii) acidic and basic, (iv) acidic and neutral, (v) basic and neutral. The present result on M. africana belonging to Zygomycota were in accordance to those of Dave29

    who has also suggested siderophores of Mucorales to

    Table 2 - Fe (III) binding nature of hydroxamate siderophore in electrophoresis at pH 2 .

    Test fungi

    Aspergillus sp. ABp4 Penicillium oxalicum Aureobasidium pullulans Phanerochaele chrysosporium

    E 0. 0.

    Color of the Fe (III) binding spot nature

    Brownish red Trihydroxamate Brownish red Trihydroxamate

    Brown Trihydroxamate

    Pinkish purple Dihydroxamate

    01 ""II"NCONO'VMO'l 0 0'1 _W.nIJ'l\ClNClO"l N

    cD air.:,...:,...:,...:,...:....:w cO

    I \~V/f I

    48-2670 in .OMSO Recorded at CSHCRI

    be basic in nature. Koraeo has reported the electrophoretic mobilities of eleven fungi, which fell in the following groups- acidic, basic, acidic and basic, basic and neutral. Siderophores having basic charge are at an advantage because it increases affinity towards ferric iron 31 . Hence, basic siderophores having higher formation constants are more stable in the soil: The ferric siderophores of Mucorales are anionic at the pH range prevailing in the soil and are therefore expected to be mobile in the SOil32.

    The results of Fe (III) binding nature of ferrated hydroxamates by electrophoresis at pH 2 (Table 2) indicated that siderophores of 3 fungi (Aspergillus sp. ABp4, Penicillium oxalicum, Aureobasidium pullulans) were trihydroxamates as they produced reddish brown spot and one, Phanerochaete chrysosporium, was dihydroxamate that formed pinkish-purple spot. Monohydroxamates could not be

    W_~~O_N~'q'_m~_~~O'IN~O _m""ll"_m~~NN(\I_OOmm~~oo

    ~M~MNNNNNNNNN~~~~~O

    [

    . L .. C

    o -~"'~"'~

  • 884 INDIAN J EXP BIOL, OCTOBER 2005

    detected. The earlier results on Fe (III) binding nature of ferrated hydroxamate siderophores of 15 fungi have suggested that 11 are trihydroxamates and 4 are dihydroxamates29 . Monohydroxamates could not be detected. Earlier, Winkelmann 3} has suggested that trihydroxamates predominate in nature. which due to their chelating capacity form hexadentate trihydroxamates having a higher formation constant

    Table 3 - Type of amino acids present in the siderophore

    Test fungi

    Aspergillus sp. ABp4

    Phanerochaele

    chrysosporiulll

    Myco/ypha africana

    E a a

    Number

    of spots

    2

    2

    Amino acid present

    Histidine

    Asparagine/proline

    Serine

    Valine

    Cystine

    AB-513 in OMSC Recorded at CSMCIH

    than that of the corresponding hexadentate dihydroxamate or hexadentate monohydroxamates.

    The present results showed the presence of histidine, asparagine, proline in Aspergillus sp. ABp4, serine and valine in Phanerochaete chrysosporiulIl and only one amino acid i.e. cystine in Mycotyp/w africana (Table 3). Dave2,} has also reported the presence of valine in 5 out of 6 fungi studied and serine in 2 fungi.

    The peak position for AB-2670 (Fig. 1) matched with asperchrome FI, however the spectrum contains peaks 8.32 and 1.25 oppm for alanyl NH and CH3 fil l unique to asperchrome A along with a peak at 8.5 and 8.6 oppm for glycyl NH. Peaks between 3.13 and 4 .0 oppm merged with DMSO solvent peak. Further, sample purification and structural determination on high resolution NMR (>400 MHz instrument) would be able to give exact structure information. The peak position for AB-SI3 (Fig. 2) matched with rhizoferrin

    on on

    '" I

    o o C>

    rrrrrt i' iii iii iii, , i i II , Ii Ii' i I. t iii i II ,. Ii Ii" i "Ii Ii iii I i 1111111, iii I"'" i Ii I'''''' Ii iii" i i Ii Ii,., iii . i Ii Ii. iii i. Ii i iii" I II,,' i, I. ppm 12 10 B 6 4 2 0

    Fig. 2-IH nuclear magnetic resonance of siderophore of Mycotypha africana (AB 513) in DMSO.

  • BAAKZA et at.: PROPERTIES & NMR OF FUNGAL SIDEROPHORES 885

    .. -OOWMfI1U)""'CDlDfDPl-NN-ocnmco ..... r..c.Dtnlln('lt'll-o "OU)I'lQItl,...", .., ~~ -.;r. "Q". -...:"! ~~ 0\10'" 000 .. ......... - . ..

    mmmCD" ............................................. ..... ... ~""MC"1t"l"'('lC'U o

    ".ii.,."" .. """ . " .. , . ".""""""""iiiii"""""""""""""""""""""""'" ",i Ii", " . """, ,."" , PPM 12 10 B 6 4 2 0

    Fig 3---'H Nuclear magnetic resonance of siderophore of Aspergillus sp. ABp4 (AB5) in DMSO.

    along with unidentified peak. The peak of AB-5 (Fig. 3) did not match with the known siderophores and thus remained unidentified.

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  • 886 INDIAN J EXP BIOL, OCTOBER 2005

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