2.7.1 Sterigmatocystin analysis
For analysis of sterigmatocystin, plates were inoculated with 107 spores and grown 3 days at 37°C. An agarpiece (Ø = 1.5 cm) was homogenized in 3 ml water and subsequently extracted with an equivalent volume of chloroform. The organic phase was evaporated and the residual metabolites were dissolved in 50 µl methanol. 15 µl of the extract was separated on TLC in acetone/chloroform 4:1 (v/v) and derivatized with an alcoholic aluminum chloride solution (20% (w/v)). Metabolites were visualized under λ = 366 nm. As standard, sterigmatocystin
(Sigma-AldrichChemie GmbH, Steinheim, Germany) was used. The data were documented using CAMAG TLC Visualizer (CAMAG, Muttenz, Switzerland).
2.7.2 Analysis of DHMBA and DHPDI 2.7.2.1 General experimental procedures
1H-NMR spectra were recorded on a Varian Mercury-Vx 300 (300 MHz) or a Varian VNMRS-300 (300 MHz) spectrometer, respectively. 13C-NMR spectra were recorded on a Varian Inova-500 spectrometer (125.7 MHz). ESI-MS data were acquired using a Finnigan LC-Q mass spectrometer. HPLC was performed using a system by Instrumentelle Analytik Goebel GmbH (analytical HPLC: HPLC pump 420, autosampler SA 360, HPLC detector Celeno DAD UV, evaporative light scattering detector ELSD-Sedex 85, ERC, column Nucleodur 100-5 C18 ec, 250 mm x 3 mm, solvent system: A = H2O + 0.1% TFA, B = acetonitrile + 0.1% TFA. Preparative HPLC: HPLC pump RAININ Dynamax SD-1, HPLC detector RAININ Dynamyx UV-1, column Nucleodur 100-5 C18 ec, 250 mm x 20 mm, solvent system: A = H2O, B = acetonitrile).
2.7.2.2 Cultivation
For analysis of secondary metabolites, 1 l liquid minimal medium with nitrate or ammonium as nitrogen source was inoculated with 109 spores and grown at 37°C for 36 hours.
2.7.2.3 Extraction
Mycelia of cultures were removed by filtering with Miracloth and pH of the culture filtrate was adjusted to 5 by adding NaOH or HCl. The culture filtrate was extracted twice with an equivalent amount of ethyl acetate. The combined extracts were dried to yield the crude extract.
2.7.2.4 Analysis in HPLC/UV-DAD
The crude extracts were dissolved in 2 ml methanol and analyzed in HPLC using an analytical column (Nucleodur 100-5 C18 ec, 250 mm x 3 mm) under gradient conditions (20% B to 100% B in 20 minutes).
2.7.2.5 Isolation and chemical characterization of DHMBA
The crude extract of 2 l cultures of strain AGB527 (grown in inducing medium) was extracted with CH2Cl2. The resulting extract was concentrated and chromatographed by preparative HPLC under gradient conditions (20% B to 100% B in 20 minutes, flowrate: 14 ml/min).
Detection was carried out at 230 nm. DHMBA (3.9 mg) was eluted at 14.4 min.
2,4-Dihydroxy-3-methyl-6-(2-oxopropyl)benzaldehyde (DHMBA): light yellow solid, ESI-MS: m/z (%) 231 [M + Na]+, 207 [M - H]-. 1H-NMR (300 MHz, CD2Cl2): δ = 2.06 (s, 3H, 2‘-H), 2.22 (s, 3H, 5’-2‘-H), 3.89 (s, 2H, 3’-2‘-H), 6.21 (s, 1H, 5-2‘-H), 9.85 (s, 1H, 1‘-2‘-H), 12.64 (s, 1H, 2-OH). 13C-NMR (125.7 MHz, CD2Cl2): δ = 6.98 (C-2’), 29.53 (C-5’), 46,78 (C-3’), 110.71 (C-3), 110.76 (C-5), 112.82 (C-1), 136.88 (C-6), 161.36 (C-4), 164.35 (C-2), 192.53 (C-1’), 205.15 (C-4‘). UV λmax (acetonitrile/H2O/formic acid)/nm: 221, 296. NMR data are in good agreement with reported data (Suzuki et al., 2001).
2.7.2.6 Isolation and chemical characterization of DHPDI
The crude extract of 7 l cultures of strain TNO (grown in inducing medium) was extracted with CH2Cl2. The resulting extract was concentrated and fractionated by preparative HPLC under gradient conditions (20% B to 100% B in 20 minutes, flowrate: 14 ml/min). Detection was carried out at 230 nm. The fraction eluted at 12.0 min was further chromatographed on a Sephadex column using methanol as solvent and yielded in 1.4 mg of compound DHPDI.
3,3-(2,3-Dihydroxypropyl)diindole (DHPDI): white solid, ESI-MS: m/z (%) 329 [M + Na]+, 305 [M - H]-. 1H-NMR (300 MHz, CD2Cl2): δ = 3.55 (m, 2H, 2‘-H), 4.47 (m, 1H, 5’-H), 4.70 (d, 1H, 3’-H), 6.90 (m, 2H, 5-H), 7.0 (m, 2H, 5-H), 7.15 (s, 1H, 1‘-H), 7.30 (dd, 2H, 2-OH), 7.32 (s, 1H, 1‘-H), 7.55 (dd, 2H, 2-OH). UV λmax (acetonitrile/H2O/formic acid)/nm: 229, 279. MS, NMR and UV data were consistent with the reported data (Porter et al., 1977, Schröder, 2001).
2.7.3 Bioactivity tests
A potential antibiotic or antifungal activity of the isolated metabolites DHMBA and DHPDI was tested by agardiffusion tests. 25 µl of a methanolic solution of the substances (c = 1 mg/ml) was added on sterile filter discs (Ø = 9 mm) and put on agar plates inoculated with Escherichia coli, Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Salmonella enterica serovar typhimurium, Aspergillus fumigatus, Aspergillus nidulans,
Verticillium longisporum, Neurospora crassa and Sordaria macrospora. Inhibition zones were measured after 1 to 5 days at 37°C or 25°C, respectively.
2.7.4 Metabolic fingerprinting by UPLC TOF-MS
For metabolic fingerprinting 109 spores of the wild type and the mutant strains (ΔdbaI, ΔcsnE, ΔcsnE/ΔdbaI) were inoculated in 1 l liquid minimal medium (with ammonium as N-source) in P-flasks and grown 10 days in dark. The culture filtrates (pH 5) were extracted twice with ethyl acetate. Two biological replicates of each sample were analyzed three times by Ultra Performance Liquid Chromatography (UPLC, ACQUITY UPLCTM System, Waters Corporation, Milford, USA) coupled with an photo diode array detector (UPLC eLambda 800 nm, Waters Corporation, Milford, USA) and with an orthogonal time-of-flight mass spectrometer (TOF-MS, LCT PremierTM, Waters Corporation, Milford). For LC an ACQUITY UPLC™ BEH SHIELD RP18 column (1 x 100 mm, 1.7 μm particle size, Waters Corporation, Milford, USA) was used at a temperature of 40°C, a flow rate of 0.2 ml/min and with the following gradient for the analysis of the polar phase: 0-0.5 min 0% B, 0.5-3 min from 0% B to 20% B, 3-6 min from 20% B to 99% B, 6-9.5 min 99% B and 9.5-13 min 40%
B (solvent system A: water/methanol/acetonitrile/formic acid (90:5:5:0.1 (v/v/v/v)); B:
acetonitrile/formic acid (100:0.1 (v/v)). The TOF-MS was operated in negative as well as positive electrospray ionization (ESI) mode in W optics and with a mass resolution larger than 10,000. Data were acquired by MassLynxTM software (Waters Corporation, Milford, USA) in centroided format over a mass range of m/z 50-1200 with scan duration of 0.5 sec. and an interscan delay of 0.1 sec. The capillary and the cone voltage were maintained at 2,700 V and 30 V and the desolvation and source temperature at 250°C and 80°C, respectively. Nitrogen was used as cone (30 l/h) and desolvation gas (600 l/h). The Dynamic Range Enhancement (DRE) mode was used for data recording. All analyses were monitored by using Leucine-enkephaline ([M+H]+ 556.2771 or [M+H]- 554.2615 as well as its 13C-isotopomer [M+H]+ 557.2803 or [M+H]- 555.2615, Sigma-Aldrich, Deisenheim, Germany) as lock spray reference compound at a concentration of 0.5 μg/ml in acetonitrile/water (50:50 (v/v)) and a flow rate of 30 μl/min. The raw mass spectrometry data of all samples were processed by the MarkerLynxTM Application Manager for MassLynxTM software (Waters Corporation, Milford, USA). The toolbox MarVis (http://marvis.gobics.de) was used for ranking, filtering, adduct correcting and combining the data as well as for clustering and visualization, respectively (Kaever et al., 2009, Meinicke et al., 2008). An ANOVA test was applied to extract a subset of high-quality marker candidates with a p-value < 1x10-6. The filtered data sets were adduct
corrected according to the following rules ([M+H]+, [M+Na]+, [M+NH4]+ for the positive and [M-H]-, [M+CH2O2-H]-, [M+CH2O2+Na-2H]- for the negative ionization mode). The combined data led to an overall data set with 895 marker candidates, which were used for clustering and visualization by means of a one-dimensional self-organizing-map (1D-SOM) model and for data base search within a mass window of 5 mDa (In-house-data base;
KNApSAcK, http://kanaya.naist.jp/KNApSAcK/; Metacyc, http://metacyc.org; LIPID MAPS, http://www.lipidmaps.org; KEGG http://www.genome.jp/kegg/ligand.html). The identity of DHMBA, and orsellinic acid were confirmed by comparison of exact mass, retention time and UV/VIS spectra with authentic standards.
To identify metabolites specifically secreted by dbaA OE strain, the medium polar fractions (pre-separated by preparative HPLC) of ethyl acetate extracts of wild type and dbaA OE culture filtrates were also analyzed and compared by the metabolite fingerprinting approach.
The UPLC ESI TOF-MS analysis was done as described above with the exception of the use of ACQUITY UPLC™ HSS T3 column (1 x 100 mm, 1.7 μm particle size, Waters Corporation, Milford, USA) for LC separation of more polar compounds with the following solvent gradient: 0-0.5 min 1% B, 0.5-3 min from 1% B to 20% B, 3-8 min from 20% B to 95% B, 8-10 min 95% B and 10-14 min 1% B (solvent system A: water/formic acid (100:0.1 (v/v)); B: acetonitrile/formic acid (100:0.1 (v/v)).
The raw mass spectrometry data of all samples were processed within a retention time range from 2.5 to 6 min using the MarkerLynxTM Application Manager. After filtering by Kruskal-Wallis test and adduct correction the data sets from the positive and the negative ionization mode were combined. Clustering and visualization of the resulting data set by the MarVis tool enabled us to detect 21 metabolite marker specifically secreted by ΔdbaA OE strain. Exact mass, retention time and UV/VIS maxima of these compounds are shown in Tab. 10.