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Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals.

Gressler M, Meyer F, Heine D, Hortschansky P, Hertweck C, Brock M - Elife (2015)

Bottom Line: Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition.Terrein causes fruit surface lesions and inhibits plant seed germination.Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition.

View Article: PubMed Central - PubMed

Affiliation: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany.

ABSTRACT
Secondary metabolites have a great potential as pharmaceuticals, but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer. Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition. Terrein causes fruit surface lesions and inhibits plant seed germination. Additionally, terrein is moderately antifungal and reduces ferric iron, thereby supporting growth of A. terreus under iron starvation. In accordance, the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX. Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition.

No MeSH data available.


Related in: MedlinePlus

1H NMR (500 MHz, MeOD; upper panel) and 13C NMR (150 MHz, MeOD; lower panel) of compound 4, 2-((E)-prop-1-en-1-yl)maleic acid.1H NMR (500 MHz, MeOD): δ 6.22 (1H, d, 3J = 15.8 Hz), 6.15 (1H, dq, 3J = 15.8 Hz, 3J = 6.5 Hz,), 5.73 (1H, s), 1.85 ppm (3H, d, 3J = 6.5 Hz); 13C NMR (600 MHz, MeOH): δ 172.6, 169.5, 151.7, 136.4, 130.2, 118.5, 18.8 ppm; HRMS: (ESI+): m/z calculated for C7H9O4: 157.0495, found 157.0495 [M + H]+.DOI:http://dx.doi.org/10.7554/eLife.07861.023
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fig5s4: 1H NMR (500 MHz, MeOD; upper panel) and 13C NMR (150 MHz, MeOD; lower panel) of compound 4, 2-((E)-prop-1-en-1-yl)maleic acid.1H NMR (500 MHz, MeOD): δ 6.22 (1H, d, 3J = 15.8 Hz), 6.15 (1H, dq, 3J = 15.8 Hz, 3J = 6.5 Hz,), 5.73 (1H, s), 1.85 ppm (3H, d, 3J = 6.5 Hz); 13C NMR (600 MHz, MeOH): δ 172.6, 169.5, 151.7, 136.4, 130.2, 118.5, 18.8 ppm; HRMS: (ESI+): m/z calculated for C7H9O4: 157.0495, found 157.0495 [M + H]+.DOI:http://dx.doi.org/10.7554/eLife.07861.023

Mentions: Metabolites were extracted from culture broth as described previously (Gressler et al., 2011). In brief, an equal volume of ethyl acetate was added and collected after defined shaking of the mixture. The procedure was repeated once. After evaporation of the solvent, residues were taken up in 1 ml methanol each and filtered. Standard extract analyses were performed on an Agilent 1100 series HPLC-DAD system coupled with a MSD trap (Agilent Technologies, Waldbronn, Germany) operating in alternating ionisation mode. Terrein quantification was carried out from 50 ml cultures as described elsewhere (Zaehle et al., 2014). For quantification of the siderophore coprogen, the complete 50 ml culture supernatants were filtered and lyophilised to dryness. The remaining solids were extracted three times with 10 ml MeOH. The solvent from the combined organic extracts was removed under reduced pressure and residues were re-dissolved in 2 ml MeOH. The resulting slurries were filtered and the filtrates analysed by HPLC measurements. HPLC analyses were carried out on an Agilent 1260 device equipped with a quaternary pump and a UV/Vis detector (Agilent Technologies; Column: Zorbax Eclipse XDB-C8, 5 µm, 150 × 4.6 mm; flow rate 1 ml/min; eluent A: H2O/0.1% HCOOH, eluent B: MeOH). The gradient started with 10% B and reached 30% B after 4 min, increased to 55% B within 10 min and reached 100% B after 2 min, where it was retained for an additional 4 min. Quantification of coprogen was performed from a calibration curve of known coprogen concentrations. For correlation of coprogen to the fungal biomass, mycelia from the cultures were dried for 48 hr at 37°C and balanced and coprogen concentrations per gram dried mycelium were calculated. All quantifications were carried out in biological triplicates and technical duplicates. Isolation of coprogen for generation of the calibration curve was performed by semi-preparative HPLC from culture supernatants of the ΔakuB and ΔakuBΔterA strains and fractions were collected by automatic fraction collection. Separation was carried out on a Zorbax Eclipse XDB-C8, 5 µm, 250 × 4.6 mm with a flow rate of 4.0 ml/min using H2O as eluent A and MeOH as eluent B. The gradient started with 10% B, reached 30% B after 6.5 min, increased to 55% B within 16.5 min, reached 100% B after 2 min, and was retained at 100% B for an additional 6 min. For isolation of 2-((E)-prop-1-en-1-yl)maleic acid, the crude product from upscaled terrein reduction assays (see below) was subjected to semi-preparative HPLC using a Zorbax Eclipse XDB-C8, 5 µm, 250 × 4.6 mm with a flow rate of 4.0 ml/min, eluent A: H2O/0.1% HCOOH, eluent B: acetonitrile. The gradient started with 5% B and was held for 14 min, increased to 10% B within 9 min, increased to 100% B within 2 min where it was retained for an additional 7 min. Fractions from the new metabolite formed from ferric iron reduction were collected and evaporated resulting in a white solid which revealed a m/z value of 150.0495 [M + H+] by HRESI-MS that perfectly matched a calculated molecular formula of C7H8O4 containing four double-bond equivalents. 13C-NMR measurements (Figure 5—figure supplement 4) revealed the presence of two carbonyl groups, one terminal methyl group, and four carbons being part of a conjugated system. Two-dimensional NMR data (Figure 5—figure supplement 5) and analysis of all proton coupling constants from the 1H-NMR spectrum (Figure 5—figure supplement 4) finally confirmed the structure of 2-((E)-prop-1-en-1-yl)maleic acid. NMR spectra were recorded on a Bruker Avance III 500 and a Bruker Avance III 600 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a cryoprobe head using DMSO-d6 and methanol-d4 as solvents and internal standards.


Phytotoxin production in Aspergillus terreus is regulated by independent environmental signals.

Gressler M, Meyer F, Heine D, Hortschansky P, Hertweck C, Brock M - Elife (2015)

1H NMR (500 MHz, MeOD; upper panel) and 13C NMR (150 MHz, MeOD; lower panel) of compound 4, 2-((E)-prop-1-en-1-yl)maleic acid.1H NMR (500 MHz, MeOD): δ 6.22 (1H, d, 3J = 15.8 Hz), 6.15 (1H, dq, 3J = 15.8 Hz, 3J = 6.5 Hz,), 5.73 (1H, s), 1.85 ppm (3H, d, 3J = 6.5 Hz); 13C NMR (600 MHz, MeOH): δ 172.6, 169.5, 151.7, 136.4, 130.2, 118.5, 18.8 ppm; HRMS: (ESI+): m/z calculated for C7H9O4: 157.0495, found 157.0495 [M + H]+.DOI:http://dx.doi.org/10.7554/eLife.07861.023
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4528345&req=5

fig5s4: 1H NMR (500 MHz, MeOD; upper panel) and 13C NMR (150 MHz, MeOD; lower panel) of compound 4, 2-((E)-prop-1-en-1-yl)maleic acid.1H NMR (500 MHz, MeOD): δ 6.22 (1H, d, 3J = 15.8 Hz), 6.15 (1H, dq, 3J = 15.8 Hz, 3J = 6.5 Hz,), 5.73 (1H, s), 1.85 ppm (3H, d, 3J = 6.5 Hz); 13C NMR (600 MHz, MeOH): δ 172.6, 169.5, 151.7, 136.4, 130.2, 118.5, 18.8 ppm; HRMS: (ESI+): m/z calculated for C7H9O4: 157.0495, found 157.0495 [M + H]+.DOI:http://dx.doi.org/10.7554/eLife.07861.023
Mentions: Metabolites were extracted from culture broth as described previously (Gressler et al., 2011). In brief, an equal volume of ethyl acetate was added and collected after defined shaking of the mixture. The procedure was repeated once. After evaporation of the solvent, residues were taken up in 1 ml methanol each and filtered. Standard extract analyses were performed on an Agilent 1100 series HPLC-DAD system coupled with a MSD trap (Agilent Technologies, Waldbronn, Germany) operating in alternating ionisation mode. Terrein quantification was carried out from 50 ml cultures as described elsewhere (Zaehle et al., 2014). For quantification of the siderophore coprogen, the complete 50 ml culture supernatants were filtered and lyophilised to dryness. The remaining solids were extracted three times with 10 ml MeOH. The solvent from the combined organic extracts was removed under reduced pressure and residues were re-dissolved in 2 ml MeOH. The resulting slurries were filtered and the filtrates analysed by HPLC measurements. HPLC analyses were carried out on an Agilent 1260 device equipped with a quaternary pump and a UV/Vis detector (Agilent Technologies; Column: Zorbax Eclipse XDB-C8, 5 µm, 150 × 4.6 mm; flow rate 1 ml/min; eluent A: H2O/0.1% HCOOH, eluent B: MeOH). The gradient started with 10% B and reached 30% B after 4 min, increased to 55% B within 10 min and reached 100% B after 2 min, where it was retained for an additional 4 min. Quantification of coprogen was performed from a calibration curve of known coprogen concentrations. For correlation of coprogen to the fungal biomass, mycelia from the cultures were dried for 48 hr at 37°C and balanced and coprogen concentrations per gram dried mycelium were calculated. All quantifications were carried out in biological triplicates and technical duplicates. Isolation of coprogen for generation of the calibration curve was performed by semi-preparative HPLC from culture supernatants of the ΔakuB and ΔakuBΔterA strains and fractions were collected by automatic fraction collection. Separation was carried out on a Zorbax Eclipse XDB-C8, 5 µm, 250 × 4.6 mm with a flow rate of 4.0 ml/min using H2O as eluent A and MeOH as eluent B. The gradient started with 10% B, reached 30% B after 6.5 min, increased to 55% B within 16.5 min, reached 100% B after 2 min, and was retained at 100% B for an additional 6 min. For isolation of 2-((E)-prop-1-en-1-yl)maleic acid, the crude product from upscaled terrein reduction assays (see below) was subjected to semi-preparative HPLC using a Zorbax Eclipse XDB-C8, 5 µm, 250 × 4.6 mm with a flow rate of 4.0 ml/min, eluent A: H2O/0.1% HCOOH, eluent B: acetonitrile. The gradient started with 5% B and was held for 14 min, increased to 10% B within 9 min, increased to 100% B within 2 min where it was retained for an additional 7 min. Fractions from the new metabolite formed from ferric iron reduction were collected and evaporated resulting in a white solid which revealed a m/z value of 150.0495 [M + H+] by HRESI-MS that perfectly matched a calculated molecular formula of C7H8O4 containing four double-bond equivalents. 13C-NMR measurements (Figure 5—figure supplement 4) revealed the presence of two carbonyl groups, one terminal methyl group, and four carbons being part of a conjugated system. Two-dimensional NMR data (Figure 5—figure supplement 5) and analysis of all proton coupling constants from the 1H-NMR spectrum (Figure 5—figure supplement 4) finally confirmed the structure of 2-((E)-prop-1-en-1-yl)maleic acid. NMR spectra were recorded on a Bruker Avance III 500 and a Bruker Avance III 600 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a cryoprobe head using DMSO-d6 and methanol-d4 as solvents and internal standards.

Bottom Line: Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition.Terrein causes fruit surface lesions and inhibits plant seed germination.Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition.

View Article: PubMed Central - PubMed

Affiliation: Microbial Biochemistry and Physiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoell Institute, Jena, Germany.

ABSTRACT
Secondary metabolites have a great potential as pharmaceuticals, but there are only a few examples where regulation of gene cluster expression has been correlated with ecological and physiological relevance for the producer. Here, signals, mediators, and biological effects of terrein production were studied in the fungus Aspergillus terreus to elucidate the contribution of terrein to ecological competition. Terrein causes fruit surface lesions and inhibits plant seed germination. Additionally, terrein is moderately antifungal and reduces ferric iron, thereby supporting growth of A. terreus under iron starvation. In accordance, the lack of nitrogen or iron or elevated methionine levels induced terrein production and was dependent on either the nitrogen response regulators AreA and AtfA or the iron response regulator HapX. Independent signal transduction allows complex sensing of the environment and, combined with its broad spectrum of biological activities, terrein provides a prominent example of adapted secondary metabolite production in response to environmental competition.

No MeSH data available.


Related in: MedlinePlus