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Stress response of Nidula niveo-tomentosa to UV-A light

     Zentrum Angewandte Chemie, Institut für, Lebensmittelchemie der Leibniz Universität Hannover, Wunstorfer Straße 14, D-30453 Hannover, Germany

Manfred Nimtz

     Helmholtz-Zentrum für Infektionsforschung, Abteilung, Biophysikalische Analytik, Inhoffenstr. 7, D-38124, Braunschweig, Germany

Ralf G. Berger

     Zentrum Angewandte Chemie, Institut für, Lebensmittelchemie der Leibniz Universität Hannover, Wunstorfer Straße 14, D-30453 Hannover, Germany

Holger Zorn ¹

     AG Technische Biochemie, Universität Dortmund, Emil-Figge-Straße 68, D-44221 Dortmund, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Exposition to UV-A light stimulated the growth and synthesis of raspberry ketone in submerged cultures of the basidiomycete Nidula niveotomentosa. To investigate the fungus’ response to UV-A light differentially expressed proteins were identified by means of 2D-electrophoresis. Light induced proteins were de novo sequenced by ESI-MS/MS spectrometry, and the encoding nucleotide sequences were cloned from cDNA or genomic DNA. The spectrum of UV-A light-induced proteins comprised several stress-related proteins including a catalase, heat-shock proteins, glutathione S-transferases and proteasomes. In addition, growth-related enzymes of the citric cycle were found to be up-regulated as a response to irradiation with UV-A.

Key words: Basidiomycete, cDNA, de novo sequencing, 2-D electrophoresis

	INTRODUCTION

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Effects of light on fungi have been investigated for a number of model species, including basidiomycetes (e.g. Coprinopsis), fungi incertae sedis (e.g. Phycomyces), and ascomycetes (e.g. Neurospora) (Kües 2000, Cerdá-Olmedo 2001). In these species, light was shown to influence the rate and direction of growth, the sexual and asexual reproduction and the formation of pigments (Idnurm and Heitman 2005a). On a molecular level the mechanisms of photoreception and photo response are best understood for Neurospora crassa (Liu et al 2004). All established light responses in Neurospora are specific to blue light, while the phytopathogenic fungus Exserophilum turcicum responded to wavelengths of the entire UV-VIS spectrum (Flaherty and Dunkle 2005). In N. crassa blue light regulates inter alia the biosynthesis of carotenoids, the formation of sexual fruiting bodies (protoperithecia) and the circadian rhythm (Loros and Dunlap 2006, Linden et al 1997). The genome of Neurospora exhibits a number of possible photoreceptors including several bacteriophytochromes and a cryptochrome (Borkovich et al 2004, Dunlap and Loros 2004).

The basidiomycete Nidula niveo-tomentosa is the only known microbial producer of 4-(4-hydroxyphenyl)-butan-2-one (p-HPB, raspberry ketone), the character impact compound of raspberry flavor (Ayer and Singer 1980). As the concentration of p-HPB in raspberries is a few milligrams per kg wet weight, at best, efforts have been made to use the basidiomycete as a biotechnological source of natural raspberry ketone. Submerged cultures of N. niveo-tomentosa respond to the supplementation of the peptone nutrient medium with L-phenylalanine by producing elevated levels of p-HPB (Böker et al 2001). Stable isotope labelling studies showed that the fungal biosynthetic pathway starts from L-phenylalanine and glucose (Zorn et al 2003). To our surprise ambient light was found to increase both the biomass formation and the production of p-HPB (Böker et al 2001).

To investigate the proteome response of N. niveo-tomentosa to UV-A light, intracellular enzymes of exposed and dark grown cultures were compared by means of two-dimensional (2-D) electrophoresis. Identification of differentially expressed enzymes was performed by mass spectrometric de novo sequencing, homology searches against public protein databases and cloning of the encoding cDNA or genomic DNA sequences. Our results provide a first insight into the cellular defense and repair mechanisms of a basidiomycete against UV-A stress.

	MATERIALS AND METHODS

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General.— – The Nidula niveo-tomentosa strain (CBS strain 380.80) was obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands.

Chemicals.— – The constituents of the nutrient media were purchased from Merck (Darmstadt, Germany), Fluka (Neu-Ulm, Germany), Riedel-de Haen (Seelze, Germany) and Sigma-Aldrich (Taufkirchen, Germany). Chemicals and materials for 2-D electrophoresis were from Roth (Karlsruhe, Germany), Serva (Heidelberg, Germany) and Bio-Rad (München, Germany). Solvents were provided by BASF (Ludwigshafen, Germany) and Baker (Deventer, The Netherlands). All solvents were distilled before use. Highly purified water was prepared with an E-pure water purification system (Barnstead, Dubuque, Iowa).

Cultivation of N. niveo-tomentosa.— – Strains on agar plates were inoculated (homogenized mycelium) into 100 mL of culture medium and grown aerobically at 24 C in 300 mL Erlenmeyer flasks on a rotary shaker (Multitron; Infors, Bottmingen/Basel, Switzerland). The standard culture period was 12–14 d, after which the formation of volatile metabolites leveled off. Each day aliquots of the culture broth were centrifuged, glucose consumption and pH were measured according to standard protocols, and the supernatants were extracted immediately for metabolite analysis. The culture medium contained 30 g glucose H₂O L^–1, 6 g soy peptone L^–1, 1.5 g yeast extract L^–1, 2.5 g KH₂PO₄ L^–1, 0.5 g MgSO₄ L^–1, 73.5 mg CaCl₂ 2H₂O L^–1, and 1.0 mL of trace element solution (Fe, Zn, Cu and Mn ions) L^–1 (soy peptone medium, pH 6.0). The biosynthesis of raspberry compounds was increased by addition of 10 mM L-phenylalanine as a precursor on the third culture day. The cultures were irradiated with UV light (10 h UV light and 14 h dark regimen) or cultivated in the dark. Illumination was performed with UV-A (TL 44 D 25/09 N) lamps (irradiation maximum 350 nm; energy at maximum 560 mW 5 nm² 1000 lm^–1) from Philips, Eindhoven, the Netherlands.

General DNA procedures.— – Total RNA was isolated with the RNeasy Plant Mini Kit of QIAGEN, Hilden, Germany. For isolation of genomic DNA, the genomic DNA from plant kit (Macherey & Nagel, Düren, Germany) was used. Mycelia were disrupted by grinding under liquid nitrogen. cDNA and cDNA-library synthesis were accomplished by the SMART^TM cDNA Library Construction Kit (CLONTECH, Mountain View, California). To amplify differentially expressed genes, several sets of PCR primers (TABLE I) were designed with a codon usage table of Nidula niveo-tomentosa (supplementary material available). PCR primers were synthesized by Roth (Karlsruhe, Germany) and by Operon Biotechnologies (Köln, Germany). For PCR reactions, 50 ng of genomic DNA, cDNA or subtracted cDNA, respectively, were used as template in 50 µL reaction mixtures containing 1x PCR Buffer (QIAGEN), 0.2 mM dNTPs, 3.0 mM MgCl₂, 0.5 µM of each primer, and 2.5 U HotStarTaq DNA polymerase (QIAGEN). Amplification experiments were performed in a master cycler gradient (Eppendorf, Hamburg, Germany) with these conditions: denaturation for 5 min at 95 C, 40 cycles of 1 min at 95 C, 1 min at 64 C (pros_for and CDS3rev3), 63 C (hsp1_for and hsp1_rev), 65 C (hsp2for and nested primer 2R), or 69 C (cat_for and cat_rev), and 2 min at 72 C; final extension for 10 min at 72 C and storage at 4 C.

Protein concentration.— – It was estimated by the method of Bradford (1976) with bovine serum albumin as a standard.

2-D-electrophoresis.— – Sample preparation: 1 g mycelium was ground in liquid nitrogen and dissolved in 1 mL of sterile bidistilled H₂O. For protein precipitation, 400 µL methanol, 100 µL chloroform, and 300 µL H₂O were added to 100 µL protein solution. After mixing the samples were incubated 5 min at 4 C and centrifuged (9000 g, 4 C, 2 min). The aqueous phase was removed without disturbing the interphase, and another 300 µL methanol were added. The sample was incubated at 4 C for 5 min, and the proteins were turned into pellets by centrifugation (13 000 g, 4 C, 5 min).

Electrophoresis: The precipitated proteins (250 µg/10 µL) were redissolved in lysis buffer (8 M urea, 4% [m/v] CHAPS, 40 mM TRIS, 5 mg mL^–1 DTT). To remove insoluble particles the sample was centrifuged (15 000 g, 4 C, 20 min). Rehydration solution (8 M urea, 4% (m/v) CHAPS, traces of bromophenol blue, 10 µL mL^–1 Bio-Lyte ampholyte solution [Bio-RAD, München, Germany], 2.8 mg mL^–1 DTT) was used to dilute the sample to a final volume of 330 µL. ReadyStrip IPG strips (Bio-RAD) were employed for the first electrophoretic dimension in a Protean IEF-Cell (Bio-RAD). Before SDS-PAGE, IPG strips were treated with SDS equilibration buffer (1.5 M Tris-HCl, pH 8.8, 6 M urea, 30% [v/v] glycerol, 2% [w/v] SDS, traces of bromophenol blue) twice at least 20 min. In the first step the equilibration buffer also contained 10 mg mL^–1 DTT, in the second step 25 mg mL^–1 iodoacetamide. After fixing with 30% ethanol (v/v) and 10% acetic acid (v/v), the proteins first were stained with ruthenium II tris (bath-ophenanthroline disulfonate) according to Rabilloud et al (2001). A fluorescence scanner (Fuji FLA 3000, Fujifilm, Düsseldorf, Germany) was used for gel documentation. Protein staining afterward with colloidal coomassie brilliant blue was accomplished according to Neuhoff et al (1990). For documentation the proteins were stained with silver according to Blum et al (1987).

ESI-MS/MS.— – Analyses were performed as reported by Zorn et al (2005). Spots from 2-D gels were excised and digested with trypsin. The resulting peptides were extracted and purified according to standard protocols. A QTof II mass spectrometer (Micromass, Manchester, England) equipped with a nanospray ion source and gold coated capillaries (Protana, Odense, Denmark) was used for electrospray MS of peptides. For collision-induced dissociation experiments multiple charged parent ions were transmitted selectively from the quadrupole mass analyzer into the collision cell (collision energy 25–30 eV for optimal fragmentation). The resulting daughter ions were separated by an orthogonal time-of-flight mass analyzer. The acquired MS-MS spectra were enhanced (Max. Ent. 3, Micromass) and used for de novo sequencing of tryptic peptides. The fasts3 algorithm, which compares linked peptides to a protein databank, was used for homology queries.

Metabolite micro-extraction.— – From the centrifuged culture broth 200 µL aliquots were saturated with sodium chloride and extracted twice with 1 mL of ethyl acetate. The combined organic phases were used directly for GC analysis. 2-(3,4-dimethoxyphenyl)-ethanol was added as an internal standard. The oven temperature was programmed as follows: 160 C isothermal for 3 min, linear gradient to 240 C at 3 C min^–1, isothermal at 240 C for 10 min. GC analysis was performed on a CP-WAX 52 CB column (30 m, 0.32 mm ID and 0.25 µm FD) (Varian, Darmstadt, Germany). GC-MS analysis was carried out with the same chromatographic conditions as for GC-FID analysis and helium as the carrier gas (38 m s^–1). Identification of raspberry ketone was achieved by comparison of EI mass spectra with reference data (Wiley and NIST spectral library) using a Fisons GC 8000 gas chromatograph and a Fisons MD 800 mass selective detector (interface: 230 C; ion source: 200 C; quadrupole: 100 C: EI ionization (70 eV); scan range m/z 33–400 amu).

	RESULTS

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For the characterization of differentially expressed proteins N. niveo-tomentosa was grown in submerged cultures. Compared to cultures grown in the dark the biomass of the cultures irradiated with UV-A nearly doubled within 13 d. The consumption of glucose and the biosynthesis of raspberry compounds also were increased in the cultures exposed to UV-A (FIG. 1). UV-A grown fungal pellets turned from brownish to a pale yellow after 3–4 d. These data indicate a stress-like stimulating effect of UV-A light.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Production of biomass and raspberry compounds in irradiated and dark grown cultures of Nidula niveo-tomentosa; biomass (dark), biomass (UV-A irradiated), raspberry compounds (dark), raspberry compounds (UV-A irradiated).

View larger version (76K): [in this window] [in a new window]   FIG. 2. 2-DE gels of cytosolic proteins expressed by N. niveo-tomentosa growing with (a) and without (b) UV-A irradiation (13th culture day).

	DISCUSSION

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UV-A effects also were observed with the basidiomycete Inonotus hispidus (formerly Polyporus hispidus). Compared to N. niveo-tomentosa a somewhat less pronounced growth-promoting effect of light was found. The mycelial dry weight increased by a factor of 1.3. Compared to mycelia grown in the dark the nonoxidative deamination of phenylalanine to cinnamic acid was considerably favored in mycelia exposed to light, and a yellow pigmentation was formed as a response to illumination (Nambudiri et al 1973).

For the aquatic fungus Blastocladiella emersonii (Blastocladiomycetes), illumination with blue light improved the uptake of glucose (Cantino and Turian 1961). In the filamentous fungus Phycomyces blakesleeanus, sporangiophore growth is regulated by light and shows phototropism in a manner similar to plant phototropic responses (Idnurm et al 2006). Red light, by contrast, triggered asexual development and repressed sexual development in the ascomycete Aspergillus nidulans. This response required a phytochrome red/far-red light photoreceptor with a tetrapyrrole chromophore (Idnurm and Heitman 2005b).

Numerous studies have been performed to investigate the TCA of plants. Because light is an essential factor for plant growth it also affects the citric acid cycle. For example in leaf chloroplast of spinach (Spinacia oleracea L.) a light-dependent activation of the NADP-malate dehydrogenase has become apparent (Miginiac-Maslow et al 1988, Jackson et al 1992).

Heat-shock proteins.— – Because excessive UV light may impair growth and productivity of secondary metabolites via deterioration of DNA, RNA and proteins (Teramura 1983, Stapleton 1992) plants and fungi up-regulate enzymes involved in the defense against UV light upon irradiation. So called heat-shock (hsp) or "stress-induced" proteins are synthesized during temperature or other cellular stresses to protect proteins from denaturation. In the basidiomycete Phanerochaete chrysosporium the heat shock proteins hsp70 and hsp80 were up-regulated after addition of vanillin (Shimizu et al 2005), and an analog fungal response to chemical stress has been described for the white-rot fungus Coriolus versicolor (Ichinose et al 2002, Iimura and Tatsumi 1997). In exposed cultures of N. niveo-tomentosa two heat shock proteins (spots 5 and 9) strongly related to hsp70 were expressed. Hsp70 proteins are able to bind damaged or abnormal protein complexes and aggregates to resolve and refold them by iterative cycles of ATP-binding and release (Burnie et al 2006).

Glutathione S-transferases.— – Glutathione S-transferases (GST), identified in spots 12 and 13, represent a further class of stress-related cytosolic proteins. UV light triggers the formation of active oxygen species (AOS), which are known to damage membranes and enzymes (Foyer et al 1997). Because glutathione is a key component of the antioxidant defense system in most aerobic organisms (Marrs 1996, May 1998) GST essentially participate in the detoxification of critical oxidation products and thus protect the cell (Dixon et al 1998). In parsley cell suspension cultures, a glutathione S-transferase (PcGST1), which may activate the phenylpropanoid metabolism, was expressed within 2 h of an UV-B pulse (Loyall et al 2000). For the class of basidiomycetes this is the first report on the induction of heat shock proteins and GST by UV-A.

Proteasome related events.— – In addition to heat-shock proteins and GST the exposition of N. niveo-tomentosa to UV-A induced enzymes involved in the degradation of proteins Proteasome subunits were identified in spots 8, 10 and 11 and an ubiquitin activating enzyme in spot 1. Ubiquitin mediated proteolysis plays an important role in the cell cycle, cell differentiation and development, in the modulation of cell surface receptors and ion channels, in the cellular response to extracellular effectors and stress, in DNA repair and in the biogenesis of diverse organelles. The modification with ubiquitin targets the substrate for degradation by proteasomes (Ciechanover et al 2000), peptidase complexes responsible for removing most transient intracellular proteins (Smalle et al 2003). The ubiquitin/proteasome pathway has been studied intensely for a number of species, also with regard to its induction by UV light. Human cells respond to UV light by a rapid proteasome-dependent degradation of human securin (hSecurin), a protein involved in the organization of the cell cycle (Romero et al 2004). hSecurin degradation results in increased cell growth, and the UV light induced proteasome thus is an activator of the cell cycle (Romero et al 2004). In Saccharomyces cerevisiae the lack of an intact 20S proteasome caused increased sensitivity to UV light. Mutant cells of S. cerevisiae with a deletion in the UMP1 gene, which encodes a small transient protein necessary for 20S proteasome formation ("proteasome maturation factor"), were highly sensitive to UV irradiation (Mieczkowski et al 2000). The proteasome presumably degrades a phosphatase that antagonizes a mitogen-activated protein (MAP) kinase, which is involved in the UV resistance mechanism of S. cerevisiae (Stitzel et al 2001). UMP1 mutants therefore exhibit remarkably decreased levels of ubiquitin-mediated proteolysis (Mieczkowski et al 2000). No other UV light induction of proteasomes has become known for a basidiomycete up to now.

With the exception of spots 5 and 10 (cf. TABLE II) all stress-related proteins discussed above already were traceable on 2-D gels of illuminated cultures of the 5th culture day (supplemental material available). The respective spots were absent from the gels of the reference cultures grown in the dark. Thus illumination and not senescence is the key factor that stimulates the production of the stress-induced proteins.

Growth stimulation.— – Light significantly stimulated the growth of N. niveo-tomentosa in submerged cultures. Nevertheless the comparison of illuminated and dark grow cultures revealed similar growth profiles. On culture day 13, when the concentration of raspberry compounds peaked, the biomass was still increasing under both growth conditions (supplemental material available). This suggests similar culture phases for both culture series on the 13th culture day. In addition to the stress-induced proteins discussed above, typical growth-related enzymes were up-regulated in the irradiated cultures. Spots 6 and 7, citrate synthase and malate dehydrogenase respectively, both represent enzymes of the citric acid cycle. The so-called tricarboxylic acid cycle (TCA) is a series of chemical reactions of central importance for all living cells that use oxygen in cellular respiration. Only fragmentary knowledge is available on the effects of light on the fungal TCA. Light expedited the reaction of isocitrate to ketoglutarate via isocitric dehydrogenase and the reaction of isocitrate to succinate and glyoxylate catalyzed by isocitrate lyase in the aquatic phycomycete Blastoeladiella emersonii (Cantino and Turian 1961).

In summary the basidiomycete N. niveo-tomentosa disposes of a highly complex proteome response to UV light. Kinetic and quantitative proteome analyses will be necessary to better understand the complex interactions of the enzymes involved in the protection of the fungus against UV exposition. Because the secondary metabolite raspberry ketone represents an interesting target compound for the flavor industry, identifying and cloning of the enzymes catalyzing its light-stimulated formation might be a worthwhile task.

	ACKNOWLEDGMENTS

 
Financial support by the Deutsche Forschungsgemeinschaft (DFG, project ZO 122/2-1) is gratefully acknowledged. The authors thank M. Dünßer for support in construction of the codon usage table of N. niveo-tomentosa.

	FOOTNOTES

 
Accepted for publication March 30, 2006.

¹ Corresponding author. E-mail: H.Zorn{at}bci.uni-dortmund.de. Phone: ++49(0)231 755 7487; fax: ++49(0)231 755 7489

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