N α -Dimethylcoprogens Three novel trihydroxamate siderophores from pathogenic fungi

Summary Three novel siderophores have been isolated from a highly pathogenic strain ofAlternaria longipes (ATCC 26293). The compounds are N -dimethylated analogs of coprogen, neocoprogen I and isoneocoprogen I. Structures of the compounds have been determined by¹H- and¹³C-NMR, fast-atom-bombardment (FAB) mass spectroscopy and partial hydrolysis. One of the new compounds, N -dimethylcoprogen, is also produced, as the major siderophore, in another fungus,Fusarium dimerum.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Hydroxamate siderophores of the ectomycorrhizal fungi <Emphasis Type="Italic">Suillus granulatus</Emphasis> and <Emphasis Type="Italic">S. luteus</Emphasis>

Despite indications that S. granulatus and S. luteus release iron-chelating compounds, the exact spectrum of ferric hydroxamates synthesized by these two Suillus species remained unclear. Hence the aim of this study was to identify all of the main siderophores produced by these two ectomycorrhizal fungal species under pure culture conditions. By means of HPLC and LC–MS analyses we show that S. granulatus releases cyclic and linear fusigen, ferrichrome, coprogen and triacetylfusarinine C into the nutrient medium, while S. luteus culture filtrates contain cyclic and linear fusigen, ferricrocin and coprogen. All of the different siderophores were identified on basis of reference compounds and their specific MS spectra which were recorded on a high resolution MS in positive electrospray ionisation mode. Initial HPLC separations were performed on a C-18 stationary phase, using an acidic eluent (0.1% formic acid in water and acetonitrile) in gradient mode. The potential of these two ectomycorrhizal fungal species to produce siderophores representing three different groups of hydroxamates is discussed in relation to its ecological significance.     

Isolation and identification of an anticancer drug,taxol from <Emphasis Type="Italic">Phyllosticta tabernaemontanae</Emphasis>, a leaf spot fungus of an angiosperm, <Emphasis Type="Italic">Wrightia tinctoria</Emphasis>

Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (MID) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on MID medium (461 μg/L) followed by PDB medium (150 μg/L). The production rate was increased to 9.2 × 10³ fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.     

Ferrioxamine transport mutants and the identification of the ferrioxamine receptor protein (FoxA) inErwinia herbicola (Enterobacter agglomerans)

Summary Iron deprivation ofErwinia herbicola (Enterobacter agglomerans) induces the biosynthesis of six high-M _r outer-membrane proteins and large amounts of ferrioxamine E. Mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine and selection with ferrimycin A yielded mutants ofE. herbicola K4 (wild type), defective in the expression of a 76-kDa outer-membrane protein, as determined by SDS/polyacrylamide gel electrophoresis. While in bioassays wild-type cells showed growth promotion in the presence of ferrioxamines (B, D₁, D₂, E, G), enterobactin, citrate, ferrichrome and coprogen, these mutants failed to respond to ferrioxamines. Moreover, experiments with⁵⁵Fe-labelled siderophores confirmed that iron transport mediated by ferrioxamine E and B in the mutants was completely inhibited, whereas iron transport by other hydroxamate siderophores, such as ferrichrome and coprogen was unaffected. The results are evidence that the 76-kDa protein in the outer membrane represents the receptor protein (FoxA) for ferrioxamines inE. herbicola.     

Rhizoferrin — a novel siderophore from the fungusRhizopus microsporus var.rhizopodiformis

Summary From a strain ofRhizopus microsporus var.rhizopodiformis a novel siderophore, named rhizoferrin, was isolated by ion-exchange column chromatography, gel filtration and preparative HPLC. Hydrolysis with 6 M HCl and subsequent gas chromatography/mass spectrometry (GUMS) of the esterified/trifluoroacetylated derivatives indicated that citric acid and diaminobutane were the only constituents. From positive fastatom-bombardment (FAB) and ion-spray tandem mass spectrometry, a molecular mass of 436 Da and the assignment of several daughter ion fragments could be obtained, which indicated the presence of two citric acid residues and one diaminobutane residue. NMR studies finally confirmedN ¹,N ⁴-bis(1-oxo-3-hydroxy-3,4-dicarboxybutyl)-diaminobutane as the structure of rhizoferrin. The iron-binding property was demonstrated on chromeazurol S plates and its siderophore activity was confirmed by iron transport measurements in young mycelia ofR. microsporus. While rhizoferrin and also ferrioxamines B and E proved to be effective siderophores, coprogen was a poor siderophore in this fungus.     

Antifungal activity of α-methyl trans cinnamaldehyde,its ligand and metal complexes: promising growth and ergosterol inhibitors

Antifungal effectivity and utility of cinnamaldehyde is limited because of its high MIC and skin sensitivity. In this study, α-methyl trans cinnamaldehyde, a less irritating derivative, have been self coupled and complexed with Co(II) and Ni(II) to generate N, N′–Bis (α-methyl trans cinnamadehyde) ethylenediimine [C₂₂H₂₄N₂], [Co(C₄₄H₄₈N₄)Cl₂] and [Ni(C₄₄H₄₈N₄)Cl₂]. Ligand and complexes were characterized on the basis of FTIR, ESI–MS, IR and ¹HNMR techniques. Synthesized ligand [L] and complexes were investigated for their MICs, inhibition of ergosterol biosynthesis and H⁺ extrusion against three strains of Candida: C. albicans 44829, C. tropicalis 750 and C. krusei 6258. Average of three species MIC of methyl cinnamaldehyde is 317 μg/ml (2168 μM). Compared to methyl cinnamaldehyde ligand [L], Co(II) and Ni(II) complex are found to be 4.48, 17.78 and 21.46 times more effective in liquid medium and 2.73, 8.93 and 10.38 times more effective in solid medium. At their respective MIC₉₀ average inhibition of ergosterol biosynthesis caused by methyl cinnamaldehyde, ligand [L], Co(II) and Ni(II) complex, respectively was 80, 78, 90 and 93%. H⁺ extrusion was also significantly inhibited but did not co-relate well with MIC₉₀. Results indicate ergosterol biosynthesis as site of action of α-methyl cinnamaldehyde, synthesized ligand and complexes. α-methyl cinnamaldehyde and ligand did not show any toxicity against H9c2 rat cardiac myoblast cell, whereas Co(II) and Ni(II) complexes on an average produced 19% cellular toxicity.     

Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624

Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively, after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N ¹-[(6-chloro-3-pyridyl)-methyl]-N ²-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624.     

Physiological relationships between auxin, cytokinin, and a peptide growth factor, phytosulfokine-α, in stimulation of asparagus cell proliferation

The aim of this research was to determine whether the production of the mitogenic peptide, phytosulfokine-α (PSK-α), is affected by auxin and/or cytokinin, and whether the expression of the biological activity of PSK-α requires the presence of these plant hormones. We developed a competition enzyme-linked immunosorbent assay system that measures the amount of PSK-α using a polyclonal antibody. In suspension-cultured mesophyll cells of Asparagus officinalis L., the production of PSK-α was first detected after 48 h of culture, prior to the first cell division which was generally observed after 96 h of culture when both 1-napthaleneacetic acid and N⁶-benzyladenine were present in the medium. No significant amount of PSK-α was, however, produced when one of these plant hormones was eliminated from the medium. We also characterized the progression of the cell cycle triggered by PSK-α using a fluorescent dye and microdensitometry. Asparagus mesophyll cells immediately after isolation were arrested in G₀/G₁, and the cell cycle proceeded only when all three factors, 1-naphthaleneacetic acid, N⁶-benzyladenine, and PSK-α, existed in the medium. These results show that the production and the expression of biological activity of PSK-α is closely correlated with the signal transduction pathway mediated by auxin and cytokinin. Received: 26 June 1998 / Accepted: 11 November 1998     

Siderophores produced by Magnaporthe grisea in the presence and absence of iron

An analysis of siderophores produced by Magnaporthe grisea revealed the presence of one intracellular storage siderophore, ferricrocin, and four coprogen derivatives secreted into the medium under iron depletion. Structural analysis showed that the compounds are coprogen, coprogen B, 2-N-methylcoprogen and 2-N-methylcoprogen B. Siderophore production under low and high iron conditions was quantified.     

Production of taxol from Phyllosticta dioscoreae, a leaf spot fungus isolated from Hibiscus rosa-sinensis

Taxol is a highly functionalized anticancer drug widely used in hospitals and clinics. The leaf spot fungus, Phyllosticta dioscoreae was isolated from diseased leaves of Hibiscus rosa-sinensis and screened for extracellular production of taxol in M1D (Modified liquid medium) and PDB (Potato dextrose broth) medium for the first time. The fungus was identified by its morphological and conidial features in the culture growth. The presence of taxol in the fungal culture filtrate was confirmed by different spectroscopic and chromatographic analyses. The amount of taxol produced was quantified by HPLC. The maximum amount of taxol produced was found to be 298 μg/L in M1D medium. Production rate was 5.96 × 10³ times faster than that found in culture broth of earlier reported fungus, Taxomyces andreanae. The extracted fungal taxol also showed strong cytotoxic activity in vitro in the cultures of human cancer cells tested by apoptotic assay. The results indicate that P. dioscoreae is an excellent source of taxol production, which suggests that the fungus has potential to undergo genetic engineering in order to improve its production level.     

Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

Some nutritional factors affecting α-amylase production by Calvatia gigantea

Summary A number of nutritional factors affecting α-amylase production by the edible fungus Calvatia gigantea, cultivated in a wheat bran liquid medium, were investigated. The concentration and particle size of wheat bran and the relative concentration of three nitrogen and three mineral sources markedly affected enzyme production. A high amylase yield of 60.4 U ml^-1 was obtained when the fungus was cultivated for 5 days at 29°C in a medium containing optimum concentrations of the tested factors. The results provide additional supporting evidence for the versatility of Calvatia gigantea to upgrade a number of low-cost agricultural products to high value biotechnological products.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Biodegradation of soil-applied pesticides by selected strains of plant growth-promoting rhizobacteria (PGPR) and their effects on bacterial growth

A laboratory study was conducted to investigate the influence of four PGPR strains on the degradation of five soil applied pesticides and their effects on bacterial growth. Interactions of Bacillus subtilis GB03, Bacillus subtilis FZB24, Bacillus amyloliquefaciens IN937a and Bacillus pumilus SE34 with two concentrations of acibenzolar-S-methyl, metribuzin, napropamide, propamocarb hydrochloride and thiamethoxam in liquid culture and soil microcosm were studied. The degradation of acibenzolar-S-methyl by all PGPR tested in low and high concentration, was 5.4 and 5.7 times, respectively, faster than that in non-inoculated liquid culture medium. At the end of the 72-h liquid cultured experiments, 8–18, 9–11, 15–36 and 11–22% of metribuzin, napropamide, propamocarb hydrochloride and thiamethoxam, respectively, had disappeared from PGPR inoculated medium. Under the soil microcosm experimental conditions, the half-lives of acibenzolar-S-methyl incubated in the presence of PGPR strains spiked at 1.0 and 10.0 mg kg⁻¹ were 10.3–16.4 and 9.2–15.9 days, respectively, markedly lower compared with >34.2 days in the control. From the rest pesticides studied degradation of propamocarb hydrochloride and thiamethoxam was enhanced in the presence of B. amyloliquefaciens IN937a and B. pumilus SE34. Acibenzolar-S-methyl, propamocarb hydrochloride and thiamethoxam significantly increased the PGPR growth. However, the stimulatory effect was related to the level of pesticide spiked.     

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Summary Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N⁶-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N⁶-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N⁶-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N⁶-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.