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@example.com
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