Chemical characterization of fungal siderophores

Siderophores of twenty fungi belonging to Zygomycotina (5 Mucorales), Ascomycotina (7 aspergilli, 6 penicillia, Neurospora crassa) and Deuteromycotina (Fusarium dimerum) were examined for their chemical nature. Siderophores produced by fungi other than Mucorales were all hydroxamates. Mucorales produced carboxylate siderophores. Catecholate type of siderophores were not detectable. Hydroxamate siderophores were mostly (9 out of 15) trihydroxamates, while six were dihydroxamates. Monohydroxamate nature was not shown by any of the 15 test fungal siderophores. In ligand properties, 12 out of 15 hydroxamate siderophores formed hexadentate ligands, while two formed tetradentates and one bidentate. There was good correlation between number of hydroxamate groups and ligand property.     

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.     

Interactions between iron availability, aluminium toxicity and fungal siderophores

The influence of iron, aluminium and of the combined application of both metals on microbial biomass and production of siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and Hymenoscyphus ericae) were investigated. All three species showed a strong iron regulation and Al-sensitivity of siderophore biosynthesis although several differences remained species dependent. Inhibitory effects of Fe and Al on siderophore-production were additive and the higher binding capacity of siderophores towards iron could be compensated by a higher Al-availability. Although pH itself is also important for regulation of siderophore biosynthesis, an indirect effect of Al on siderophore production via an Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a reduced biomass production were compensated by high Fe-availability, whereas the addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.     

Isolation and identification of siderophores produced by cyanobacteria

Cyanobacteria are one of the most successful and oldest forms of life that are present on Earth. They are prokaryotic photoautotrophic microorganisms that colonize so diverse environments as soil, seawater, and freshwater, but also stones, plants, or extreme habitats such as snow and ice as well as hot springs. This diversity in the type of environment they live in requires a successful adaptation to completely different conditions. For this reason, cyanobacteria form a wide range of different secondary metabolites. In particular, the cyanobacteria living in both freshwater and sea produce many metabolites that have biological activity. In this review, we focus on metabolites called siderophores, which are low molecular weight chemical compounds specifically binding iron ions. They have a relatively low molecular weight and are produced by bacteria and also by fungi. The main role of siderophores is to obtain iron from the environment and to create a soluble complex available to microbial cells. Siderophores play an important role in microbial ecology; for example, in agriculture they support the growth of many plants and increase their production by increasing the availability of Fe in plants. The aim of this review is to demonstrate the modern use of physico-chemical methods for the detection of siderophores in cyanobacteria and the use of these methods for the detection and characterization of the siderophore-producing microorganisms. Using high-performance liquid chromatography-mass spectrometry (LC-MS), it is possible not only to discover new chemical structures but also to identify potential interactions between microorganisms. Based on tandem mass spectrometry (MS/MS) analyses, previous siderophore knowledge can be used to interpret MS/MS data to examine both known and new siderophores.     

Bacterial siderophores : structure and NMR assignment of pyoverdins Pa,siderophores ofPseudomonas aeruginosa ATCC 15692

Summary In iron-deficient conditions,Pseudomonas aeruginosa ATCC 15692 synthesizes two major siderophores, pyoverdins Pa and pyoverdin Pa B. Two other compounds, pyoverdin Pa A (occurring from hydrolysis of pyoverdin Pa during the culture) and pyoverdin Pa C (occurring artifactually during the purification procedure) were also isolated. All these compounds possess the same partly cyclic peptide chain wherel-Orn(OH · HCO) isN -formyl,N -hydroxy-l-ornithine. The chain is bound to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and having the (S) configuration. The four pyoverdins differ only in the acyl substituent bound to the nitrogen atom bound to carbon C3 of the chromophore. This is succinamide (pyoverdin Pa), succinic acid (pyoverdin Pa A), methyl succinate (pyoverdin Pa C) and 2-oxoglutaric acid (pyoverdin Pa B). The complete¹H- and¹³CNMR assignments, using two-dimensional total correlation NMR spectroscopy (TOCSY) and rotating-frame Overhauser enhancement spectroscopy (ROESY) procedures, as well as¹H-¹³C correlations, are reported. The complete sequence of the peptide using CH-NH correlations was achieved by NMR and confirmed the partly cyclic structure earlier reported using fast-atom-bombardment mass spectrometry (FAB-MS) on the siderophores and their dansylated fragments [Briskot G, Taraz K, Budzikiewicz H (1989)Liebigs Ann Chem: 375–384]. The use of these NMR procedures appears to be a tool of choice and a complementary approach to FAB-MS in the structure determination of some complex pyoverdins.Abbreviations Ser serine - Arg arginine - Thr ethreonine - Lys lysine - OHOrn N -hydroxyornithine - Chr chromophore     

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.     

Tris(trimethylsilyl)stannyl alkali derivatives: Syntheses and NMR spectroscopic properties

Starting from tetrakis(trimethylsilyl)stannane, the tris(trimethylsilyl)stannyl alkali derivatives (Me₃Si)₃SnM, (M = Li, Na, K, Rb, Cs) were prepared in excellent yields. Reaction with MgBr₂ · Et₂O afforded bis[tris(trimethylsilyl)stannyl]magnesium. Reaction products were investigated by means of multinuclear NMR spectroscopy. At low temperatures, coupling of ⁷Li and ¹¹⁹Sn between [(Me₃Si)₃Sn]⁻ and [Li · 3THF]⁺ (337 Hz) or [Li · 12Cr4]⁺ (275 Hz), was observed. NMR chemical shifts and coupling constants of the stannyl anions exhibit a strong dependency on the nature of the cation, solvent system, concentration and temperature. In addition, the molecular structure of tris(trimethylsilyl)stannyl sodium · 15Cr5 was determined by X-ray crystallography. The [Na · 15Cr5]⁺ and [(Me₃Si)₃Sn]⁻ units are joined by a direct Sn-Na contact, 3.0775(18) Å in length.     

高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究

采用改进的CAS检测平板从东湖中筛选得到了一株高产铁载体细菌sp-f,并用CAS检测液定量检测其分泌铁载体量,发现其As/Ar仅0.09(OD680),Su(Siderophore Unit)为90%,达到产铁载体菌最高级。用BIOLOG检测板,结合细菌生理生化反应、形态观察和16S rDNA序列比对分析等分类鉴定方法,确定sp-f为一株荧光假单胞菌。P.fluorescenssp-f生长过程中胞外铁载体的量在对数生长前期累积达到最高后有所减少,至稳定期时菌液中铁载体量达到稳定。在已知铁载体特异吸收峰波长下,用反向高效液相色谱检测无铁环境和高铁环境下培养液上清,比较发现sp-f上清含有3种含儿茶酚胺类基团铁载体,其中包括荧光和非荧光性的脓菌素,200μmol/L Fe2 可完全抑制荧光性质脓菌素的分泌,但非荧光脓菌素的分泌不受抑制,并且对非脓菌素的儿茶酚胺类铁载体的合成分泌反而具有一定的诱导作用。     

Effect of the lipid interface on the catalytic activity and spectroscopic properties of a fungal lipase

Lipase from the fungi Thermomyces (formerly Humicola) lanuginosa (TlL) is widely used in industry. This interfacial enzyme is inactive under aqueous conditions, but catalytic activation is induced on binding to a lipid-water interface. In order for protein engineering to design more efficient mutants of TlL for specific applications, it is important to characterize its interfacial catalysis. A complete analysis of steady-state kinetics for the hydrolysis of a soluble substrate by TlL has been developed using an interface different from the substrate. Small vesicles of 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or other anionic phospholipids are a neutral diluent interface for the partitioning of substrate and enzyme. TlL binds to these interfaces in an active or open form, thus implying a displacement of the helical lid away from the active site. A study of the influence of substrate and diluent concentration dependence of the rate of hydrolysis provides a basis for the determination of the primary interfacial catalytic parameters. The interfacial activation is not supported by zwitterionic vesicles or by large anionic vesicles of 100 nm diameter, although TlL binds to these interfaces. Using a combination of fluorescence-based techniques applied to several mutants of TlL with different tryptophan residues we have shown that TlL binds to phospholipid vesicles in different forms rendering different catalytic activities, and that the open lid conformation is achieved and stabilized by a combination of electrostatic and hydrophobic interactions between the enzyme's lipid-binding face and the interface.     

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).     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Development and application of an assay for uranyl complexation by fungal metabolites,including siderophores

An assay to detect UO(2)(2+) 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-Fe(3+) dye and the other containing a CAS-UO(2)(2+) 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-UO(2)(2+) agar to the discoloration on the CAS-Fe(3+) 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 UO(2)(2+) 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 UO(2)(2+) chelating ligands. We suggest that hydroxamate siderophores can be produced by mucoraceous fungi.     

Nature of rhizosphere fungal flora of certain plants

Summary An attempt has been made in this study to see whether there existed a specific rhizosphere microflora. A comparison of the fungi shows that fungal species derived from four different plants show more specificity in the rhizoplane regions than in the rhizosphere. It is the rhizoplane region which needs critical and thorough study to understand the exact nature of micro-organisms associated with particular plant roots.     

Oncomodulin. 1H NMR and optical stopped-flow spectroscopic studies of its solution conformation and metal-binding properties

As deduced from its 1H NMR spectrum, oncomodulin's solution conformation is very similar to the tertiary structure of other single domain 2-site calcium-binding proteins of the troponin C class. Despite its extensive amino acid sequence homology with parvalbumins, however, oncomodulin differs significantly from these proteins in its Ca(II)----Ln(III) exchange characteristics. Although the relative affinity of Lu(III) for the EF site of Ca2-oncomodulin was normal, beta Lu:EF/beta Ca:EF being 175 +/- 15, displacement of Ca(II) from the CD site was not favored, beta Lu:CD/beta Ca:CD being 1.2 +/- 0.1. Lineshape analyses of several 1H NMR resonances generated by the Lu(III) titration of Ca2-oncomodulin indicated that Ca(II)----Ln(III) exchange at the CD site was 15-20 s-1, approximately 100 times faster than exchange at the CD site of parvalbumins. Analyses of the distribution of metal-bound oncomodulin species showed that Ca(II)----Lu(III) exchange was cooperative, the coefficient of cooperativity being estimated as 5 +/- 1. The kinetics of the release of Yb(III) from oncomodulin as measured by optical stopped-flow techniques corroborated the observed cooperativity in metal binding; the off-rate constant of Yb(III) from the EF site of Yb2-oncomodulin was 0.0036 s-1, approximately 19 times slower than the release of Yb(III) from the EF site of Ca1Yb1-oncomodulin. We attribute part of the reduced preference of small Ln(III)s for the CD site of oncomodulin to a combination of this site's inherent incompressibility (Williams, T.C., Corson, D.C. & Sykes, B.D. (1984) J. Am. Chem. Soc. 106, 5698-5702) and the Glu----Asp substitution at sequence position 59, the residue which chelates metal at the -X coordination position. Like the CD site in oncomodulin, site III in troponin C has not only a lower affinity for calcium relative to the CD site of parvalbumins but also aspartic acid at its -X position; a water molecule bridges the gap between bound metal and the carboxyl group of the relatively short side chain of Asp-114 (Herzberg, O. & James, M. N. G. (1985) Biochemistry 24, 5298-5302). Hence, we suggest that Asp-59 in oncomodulin binds metal only indirectly through an intervening water molecule, a proposal which is consistent with the CD site's reduced affinity for ions the size of Ca(II) or smaller.     

(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties

Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).     

Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophores in iron uptake and storage

To acquire iron, all species have to overcome the problems of iron insolubility and toxicity. In response to low iron availability in the environment, most fungi excrete ferric iron-specific chelators--siderophores--to mobilize this metal. Siderophore-bound iron is subsequently utilized via the reductive iron assimilatory system or uptake of the siderophore-iron complex. Furthermore, most fungi possess intracellular siderophores as iron storage compounds. Molecular analysis of siderophore biosynthesis was initiated by pioneering studies on the basidiomycete Ustilago maydis, and has progressed recently by characterization of the relevant structural and regulatory genes in the ascomycetes Aspergillus nidulans and Neurospora crassa. In addition, significant advances in the understanding of utilization of siderophore-bound iron have been made recently in the yeasts Saccharomyces cerevisiae and Candida albicans as well as in the filamentous fungus A. nidulans. The present review summarizes molecular details of fungal siderophore biosynthesis and uptake, and the regulatory mechanisms involved in control of the corresponding genes.     

Crystallographic and NMR spectroscopic protein structures: Interresidue contacts

Interresidue pair contacts were analyzed in detail for four pairs of protein structures solved using X-ray analysis (X-ray) and nuclear magnetic resonance (NMR). In the four NMR structures, at distances of ≤4.0 Å, the total number of pair contacts was 4–9% lower and, in general, the pair contacts were 0.02–0.16 Å shorter compared to the X-ray structures. Each of the four structural pairs contained 83–94% common pair contacts (CPCs), which were formed by identical residues in both structures; the other 6–17% were longer intrinsic pair contacts (IPCs) formed by different residues in NMR and X-ray structures, while the latter contained more IPC. Every NMR structure contained three types of CPC that were shorter, longer, or equal to the identical contact pairs in the X-ray structure of this protein. Methodologically different short CPCs prevailed at a known distance dependence of the interresidue contact density in 60–61 pairs of NMR/X-ray structures. Among the analyzed four structural pairs, contact shortening appeared upon the energy minimization of the crambin NMR structure and upon solving the ubiquitin, hen lysozyme, and monomeric hemoglobin NMR structures using X-PLOR software with decreased van der Waals atomic radii. The degree of contact shortening in the NMR structures diminished with an increase in the NMR data used to solve these structures. Among the 60 pairs of NMR/X-ray structures, the major difference between α-helical and β-structural proteins in the dependences on interresidue distances of average contact density appeared due to strong α/β differences in the backbone local geometry.     

NMR spectroscopic filtration of polypeptides and proteins in complex mixtures

Due to the inherent complexity of the natural biological environment, most studies on polypeptides, proteins and nucleic acids have so far been performed in vitro, away from physiologically relevant conditions. Nuclear magnetic resonance is an ideal technique to extend the in vitro analysis of simple model systems to the more complex biological context. This work shows how diffusion-based spectroscopic selection can be combined with isotopic labeling to tackle and optimize the NMR analysis of specific macromolecules in multicomponent mixtures. Typical media include cell-free systems containing overexpressed proteins, lysates and proteolytic mixtures. We present a few variants of diffusion-edited HSQC pulse sequences for the selective spectroscopic detection of protein and polypeptide resonances within complex mixtures containing undesired species of smaller molecular weight. Due to diffusion-based filtering, peak intensities of fast diffusing small molecules are attenuated more than peaks due to large molecules. The basic sequence, denoted as PFGSTE-HSQC, combines translational diffusion-ordering with two dimensional heteronuclear single quantum correlation spectroscopy. The GCSTE-HSQC and BPPSTE-HSQC sequences include bipolar gradients and are therefore suitable for both diffusion-based filtering and determination of diffusion coefficients of individual mixture components. Practical applications range from protein stability/folding investigations in physiologically relevant contexts to prescreening of tertiary fold and resonance assignments in structural genomics studies. A few applications of diffusion-edited HSQC to an E. coli cell lysate containing the (15)N-labeled B domain of streptococcal protein G (GB1), and to a (15)N-labeled N-acetylglycine/apomyoglobin mixture are presented. In addition, we provide specific guidelines for experimental setup and parameter optimization.