INTRODUCTION

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The ligninolytic basidiomycete Pleurotus eryngii degrades wheat lignin preferentially (26) under conditions used to treat straw in bio-semichemical pulping laboratory experiments (6). This fungus secretes laccase, aryl-alcohol oxidase (AAO), and peroxidase enzymes in liquid culture (16, 28, 30) and during lignocellulose solid-state fermentation (8), although different peroxidase isoenzymes have been identified under the two different growth conditions (9, 27, 28). Biochemical and molecular characterization revealed that they are versatile enzymes possessing catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (or manganese-dependent peroxidase [MnP]) from Phanerochaete chrysosporium and other white-rot fungi. These properties include the ability to oxidize Mn²⁺, substituted hydroquinones and phenols, veratryl alcohol, dimethoxybenzene, -keto--thiomethylbutyric acid, and phenolic or nonphenolic lignin model dimers (10, 19). Moreover, it has been found that these Pleurotus peroxidases have higher sequence and structural affinity with LiP than with MnP from P. chrysosporium but that their molecular structure includes an Mn²⁺ interaction site accounting for the ability to oxidize very low Mn²⁺ concentrations (9, 34).

All attempts to detect peroxidase activity in Pleurotus cultures grown under conditions similar to those used to produce P. chrysosporium LiP and MnP failed. However, the above-mentioned versatile peroxidases were purified from liquid cultures of different Pleurotus species when peptone was used as the N source (without Mn²⁺ addition) (7, 28, 35). These results suggest that not only are the Pleurotus peroxidases different from P. chrysosporium LiP and MnP in terms of catalytic properties and molecular structure but also that their expression is regulated in a different way. In the present study, regulation by N source, Mn²⁺, and oxidative stress of the transcript levels of the unique ligninolytic peroxidase produced in peptone-containing liquid cultures of P. eryngii (34) was investigated by Northern blotting.

	MATERIALS AND METHODS

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Culture conditions. P. eryngii CBS 613.91 (IJFM A169) was grown in two N-sufficient media containing (wt/vol) 2% glucose, 0.2% yeast extract (Difco), and 0.5% peptone (Bacto Peptone [Difco]) (glucose-peptone medium), or ammonium tartrate (glucose-ammonium medium) (28). N-limited glucose-ammonium medium (containing 0.05% ammonium tartrate) was used in preliminary experiments. The effect of adding different Mn²⁺ concentrations to the above media was also determined. Finally, peptone was fractionated by molecular exclusion chromatography in Sephadex G15, and the resulting fractions were dried, weighed, and added to glucose-ammonium medium at concentrations corresponding to 5 g of peptone/liter. The results were compared with those obtained after the addition of 5 g of peptone or Casamino Acids/liter (Difco). In all cases the pH was adjusted to 5.5 after the addition of salts (0.1% KH₂PO₄ and 0.05% MgSO₄ · 7 H₂O), and cultures were incubated at 28°C and 180 rpm.

Gene regulation experiments. Studies on peroxidase transcript levels were carried out by including different compounds in the culture media or by adding them to 6-day-old mycelium from glucose-peptone or glucose-ammonium cultures which was separated by filtration, suspended in 20 mM sodium tartrate (pH 5), and incubated at 28°C and 180 rpm for up to 120 min after the addition of the potential transcription regulators. These included 5 g of peptone/liter, 100 µM Mn²⁺ (as MnSO₄), and 500 µM H₂O₂ (final concentration). Moreover, hydroxyl radical (OH · ) was generated in situ through redox cycling of 500 µM 2-methyl-1,4-naphthoquinone (menadione) in the presence of P. eryngii mycelium from 6-day-old cultures in glucose-ammonium medium, and 100 µM Fe³⁺ (17, 18). Peroxidase activity and mnpl mRNA were quantified as described below.

Enzymatic activities. Peroxidase activity was estimated by the formation of Mn³⁺-tartrate complex (₂₃₈, 6,500 M¹ cm¹) during the oxidation of 100 µM MnSO₄ in 0.1 M sodium tartrate (pH 5) containing 100 µM H₂O₂. One unit of enzymatic activity was defined as the amount of enzyme transforming 1 µmol of substrate per min.

Analysis of H₂O₂. H₂O₂ concentration was determined by using peroxidase and phenol red (31). The reaction mixture contained 0.01% phenol red, 2.5 U of horseradish peroxidase (Sigma, type II)/ml, and 0.1 M sodium phosphate buffer (pH 6). After 10 min, NaOH (0.2 M final concentration) was added, and the absorbance was read at 610 nm. Samples preincubated with 30 U of catalase (Sigma)/ml were used as blanks. A standard curve of H₂O₂ was prepared with dilutions of Perhydrol 30% (Merck) processed in the same way. The H₂O₂ concentration in the commercial solution was calculated from its absorbance at 230 nm (₂₃₀, 81 M¹ cm¹).

RNA isolation and Northern analysis. After gene regulation experiments, mycelium was recovered by filtration, washed with distilled water, frozen, and stored at 80°C. It was disrupted in liquid N₂, and total RNA was isolated by using the Ultraspec RNA isolation system (Biotecx). RNA samples were solubilized in water and denatured in the presence of 40% formamide, 4% formaldehyde, 40 mM morpholinepropanesulfonic acid (MOPS) (pH 7), 10 mM sodium acetate, and 1 mM EDTA for 10 min at 65°C. Ten micrograms of each sample was electrophoresed overnight in 1.2% agarose-6% formaldehyde gels by using 40 mM MOPS (pH 7), 10 mM sodium acetate, and 1 mM EDTA. Gels were washed with water and transferred to nitrocellulose in 20× SSC (1× SSC is 0.15 M NaCl and 15 mM sodium citrate [pH 7]). RNA was cross-linked by using Stratalinker-UV. Then filters were hybridized in 5× SSC, 2.5× Denhardt's, 10% dextran sulfate, 20 mM sodium phosphate (pH 7.5), 50 µg of carrier single-strand DNA ml¹ and 50% formamide, at 42°C with probes labeled by using the rediprime DNA random labeling system (Amersham). Two probes were used in Northern blot analysis, as follows: the first corresponding to the 648-bp cDNA fragment from mRNA encoded by P. eryngii allele mnpl2 (GenBank accession no. AF007224), which corresponds to the portion encoding Thr⁹-Pro²²¹ (34), and the second corresponding to a 12-kb EcoRI fragment of the 28S rRNA gene from Drosophila melanogaster included in pDm238 (33). The filters were sequentially hybridized with the mnpl probe and, after exhaustive washing removing labeling, with the rRNA probe. After each hybridization, the filters were washed (the final step consisted of 0.2× SSC, 0.1% SDS at 58°C), and both the europium screen of a PhosphorImager (Molecular Dynamics) and Kodak X-OMAT-AR-ray film were exposed (the latter for different periods of time). The films were scanned, and digital images were imported by the PhosphorImager software (program IQ) for processing and quantitation, together with the images obtained with this equipment. The mnpl mRNA values obtained were referred to the intensity of the signal of 28S rRNA in the same sample, which was used as an internal standard (to normalize differences due to sample loading, etc.). Moreover, an RNA sample corresponding to the highest production of mnpl transcripts (i.e., day 5 in peptone medium) was included in all the electrophoresis and (after normalization of the mnpl mRNA signal to rRNA) used as an external reference for the transcript levels, which were presented as percentages of the maximal transcript level obtained.

	RESULTS

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Effect of peptone on peroxidase production and transcript levels. No peroxidase activity was detected in P. eryngii cultures grown in either N-limited or N-sufficient glucose-ammonium media. However, high activity was obtained in N-sufficient glucose-peptone medium. The two proteins with peroxidase activity, MnPL1 and MnPL2 (28), in peptone-containing cultures were found to be 99% identical variants encoded by two alleles of gene mnpl (GenBank accession no. AF007223 and AF007224) cloned from dikaryotic mycelium of P. eryngii (34). They represent a new type of peroxidase oxidizing both Mn²⁺ and aromatic substrates including typical LiP substrates. Recently, a second gene encoding peroxidase PS1 with similar catalytic properties (and 74% identity) was cloned from P. eryngii (9). It was found that both are differentially expressed, the two peroxidase variants encoded by gene mnpl being the only ones produced in liquid cultures, whereas the peroxidase PS1 was found during fungal growth on lignocellulosic substrates (9, 34). Southern blot experiments with the mnpl probe (data not shown) showed a unique hybridization band after digestion of P. eryngii DNA with EcoRI and EcoRV, suggesting that the probe was specific for a unique gene. This gene is different from that encoding P. eryngii peroxidase PS1 or P. chrysosporium LiP, as confirmed by the different hybridization pattern obtained with the ps1 probe and the lack of hybridization signals with the lpo probe corresponding to the gene encoding LiP-H8 (as expected by the absence of LiP-type enzymes in Pleurotus species).

View larger version (39K): [in this window] [in a new window]   FIG. 1.   Influence of N source (peptone versus ammonium tartrate) on the level of mnpl transcripts (dashed line) and peroxidase activity (continuous line) in N-sufficient cultures of P. eryngii. (A) Northern blot analysis of total RNA from mycelium samples with mnpl2 cDNA and ribosomal DNA from Drosophila melanogaster as probes. (B) Time course of normalized mnpl mRNA levels (as percentages of the maximal level obtained, after normalization to the same rRNA in each sample) and Mn2+-oxidizing peroxidase activity (MnP) estimated by formation of Mn3+-tartrate complex in glucose-peptone () and glucose-ammonium () media.

View larger version (21K): [in this window] [in a new window]   FIG. 2.   Effect of peptone fractions on peroxidase activity in cultures of P. eryngii. (A) Peptone fractionation in Sephadex G15 (profiles at 205, dashed line, and 280 nm, continuous line, monitoring total and aromatic amino acids, respectively). (B) Peroxidase activity (estimated by formation of Mn3+ tartrate, MnP) after the addition of fractions I to VI, obtained during peptone fractionation (A) and free amino acids (5 g of Casamino Acids/liter from Difco) to glucose-ammonium medium used as a control (for each fraction, the amount obtained from 5 g of peptone was added to cultures, expressed in grams per liter).

View larger version (52K): [in this window] [in a new window]   FIG. 3.   mnpl mRNA levels maintained in peroxidase-expressing mycelium of P. eryngii after the addition of peptone (B) and Mn2+ (C) compared with the corresponding control, showing rapid decline of mnpl mRNA (A). Northern blot analysis of total RNA from samples of washed mycelium from glucose-peptone medium incubated for 30 min in the presence of 5 g of peptone/liter or 100 µM Mn2+ (in 20 mM sodium tartrate [pH 5]) and the corresponding control, with mnpl2 cDNA and ribosomal DNA from D. melanogaster used as probes.

Effect of Mn²⁺ addition. The effect of Mn²⁺ on the levels of mnpl transcripts was first studied in liquid cultures with peptone or ammonium as N sources. Neither mnpl mRNA (Northern blotting) nor extracellular peroxidase activity was detected in glucose-peptone medium when Mn²⁺ concentrations 25 µM or higher were added (data not shown). In this medium, the highest levels of mnpl mRNA and peroxidase activity were obtained without added Mn²⁺ (the total manganese content in the peptone used, estimated by atomic absorption, was less than 0.5 ppm). Neither peroxidase activity nor mnpl mRNA levels were significant in glucose-ammonium medium with or without Mn²⁺.

Effect of oxidative stress. The influence of reduced oxygen species on the expression of gene mnpl was studied. As shown in Fig. 4, induction was demonstrated by using mycelium isolated from N-sufficient ammonium medium, in which the gene is not expressed. H₂O₂ was added to washed mycelium to a final concentration of 500 µM, and samples were harvested after 15, 30, 60, and 120 min. Northern blotting analysis showed that the maximum accumulation of mnpl mRNA was after 1 h of incubation, when H₂O₂ was already exhausted.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   Induction of mnpl transcription in the presence of H2O2. (A) Northern blot analysis of total RNA from samples of washed mycelium from glucose-ammonium medium incubated for 120 min after the addition of 500 µM H2O2 (in 20 mM sodium tartrate, pH 5) and the corresponding control (without inducer), with mnpl2 cDNA and ribosomal DNA from D. melanogaster (control) used as probes. (B) Time course of normalized mnpl mRNA levels (as percentages of maximal transcript levels in peptone medium after normalization to same rRNA in each sample) in the presence () and absence () of H2O2 (evolution of H2O2 levels is also shown as dashed line).

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Induction of mnpl transcription in the presence of OH · . (A to D) Northern blot analysis of total RNA from samples of washed mycelium from glucose-ammonium medium incubated for 120 min in the presence of 500 µM menadione and 100 µM Fe3+ generating OH · (B), 500 µM menadione generating H2O2 (C), 100 µM Fe3+ (D), and the corresponding control without the addition of the above-mentioned compounds (A), with mnpl2 cDNA and ribosomal DNA from D. melanogaster (control) (all samples were incubated in 20 mM sodium tartrate, pH 5). (E) Time course of normalized mnpl mRNA levels (as percentages of maximal transcript levels in peptone medium after normalization to same rRNA in each sample) corresponding to B (), C (), and A (). The H2O2 level is also shown (dashed line).

	DISCUSSION

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The optimal conditions for peroxidase production in liquid cultures of P. eryngii were described previously (28). No significant activity was detected in cultures grown in glucose-ammonium medium, with the maximal activity being produced in low-manganese N-sufficient glucose-peptone medium. The stimulation of peroxidase levels by peptone has also been reported in other white-rot basidiomycetes (21). The above-mentioned conditions are different from those established for LiP and MnP production in P. chrysosporium cultures (14, 23, 36), in which the maximal activity of ligninolytic peroxidases is obtained in N-limited media containing glucose and ammonium tartrate, the highest MnP and LiP levels being obtained in high- and low-Mn²⁺ media, respectively. Subsequent studies demonstrated that LiP and MnP production in P. chrysosporium is regulated at the level of gene transcription by nutrient N (25, 32). Moreover, MnP of this fungus is regulated at the same level by Mn²⁺, H₂O₂, chemical agents, O₂, and heat shock (only in N-limited cultures) (2-4, 15, 24, 29). Recently, differential expression of the three mnp genes in response to Mn²⁺ has been shown in P. chrysosporium (13).

The results obtained here demonstrate that the levels of transcripts of P. eryngii versatile peroxidase are controlled by N source, Mn²⁺, and oxidative stress. The mnpl mRNA was present at very low levels in N-sufficient cultures in glucose-ammonium medium. This could be due to gene repression by this N source, but even under conditions involving a limited concentration of ammonium, no peroxidase activity was detected in P. eryngii. However, when ammonium was replaced by peptone, the induction of gene mnpl transcription was strong, and extracellular activity was detected. A similar effect on peroxidase activity was observed when the highest-molecular-weight peptone fraction was added at the same ratio as peptone. By contrast, the addition of free amino acids did not result in detectable peroxidase activity. These results suggest that the effect of peptone (in the culture medium) on peroxidase activity was due to peptides and not to free amino acids. A second effect of peptone added to peroxidase-expressing mycelium was the slower decline in the level of mnpl mRNA.

An investigation of the effect of Mn²⁺ on transcript levels indicated no peroxidase activity in peptone-containing cultures at Mn²⁺ concentrations over 25 µM. The addition of Mn²⁺ to mycelium grown in glucose-ammonium medium had no effect on the expression of gene mnpl. A peroxidase has recently been described in Trametes versicolor whose transcript levels are repressed by low concentrations of Mn²⁺ in the culture medium (11). On the other hand, the results obtained after the addition of Mn²⁺ to peroxidase-expressing mycelium of P. eryngii suggested that Mn²⁺ could also be implicated in the stabilization of mnpl mRNA. The stabilization of mRNA by metals has been reported for ferredoxin I from the cyanobacterium Synechococcus sp. (1).

The interest of studying the effect of reduced oxygen species on the transcript levels of ligninolytic peroxidases is related to the oxidative nature of lignin biodegradation (22). This process requires H₂O₂ (12) as a cosubstrate of peroxidases or a precursor of OH · , which can be directly involved in lignin attack (20). As demonstrated in the present study, both H₂O₂ and OH · can also be involved in the induction of ligninolytic peroxidases. The action of H₂O₂ (500 µM) was demonstrated by using P. eryngii mycelium from glucose-ammonium medium. After an mnpl mRNA maximum, the induction effect disappeared, because most H₂O₂ was destroyed by the mycelium. A positive effect of H₂O₂ on the transcript levels of P. chrysosporium mnp has been reported (24).

The effect of OH · on peroxidase transcript levels had not been previously shown, although it was suggested that some cell responses to the oxidative stress produced by exogenous H₂O₂ could be mediated by OH · (5). In the present study, this strong oxidant was generated by menadione added to fungal mycelium in the presence of Fe³⁺. Quinone redox cycling involving mycelium-associated reductases provided a continuous supply of O₂ ·  (17, 18). This radical dismutase generates H₂O₂, which is reduced by Fe²⁺ (from Fe³⁺ reduction by semiquinone or O₂ · ), yielding OH · (Fenton-type reaction). OH · formation has been confirmed under these experimental conditions (18), and the reaction mechanism was supported by the formation of H₂O₂ when only menadione was added to the fungal mycelium. Using the above-described system, we showed for the first time that OH · elicits the transcriptional expression of a ligninolytic peroxidase, probably via a stress response mechanism. It is interesting that stimulation of P. eryngii peroxidase activity (in glucose-peptone medium) has been observed in the presence of sublethal doses (0.05 to 0.1 mg/ml) of several toxic compounds which can also induce stress response, such as -amanitin and hycanthone, as well as with actinomycin D (data not shown). Even though the response observed is indirect, it is notable that the presence of OH · triggers the expression of gene mnpl faster than the addition of H₂O₂. The undetectable levels of H₂O₂, which is reduced in a Fenton-type reaction, support the notion that mainly OH · , and not H₂O₂, was involved in gene induction response under the experimental conditions used. The possibility of an effect of the semiquinone, either in the presence of Fe³⁺, resulting in the formation of OH · , or in its absence, resulting in the formation of H₂O₂, cannot be completely ruled out. However, this aromatic radical tends to auto-oxidize, as revealed by the reduction of Fe³⁺ in the first case (data not shown) and by the formation of H₂O₂ in the second. In the latter case, the response was very similar to that previously obtained with exogenous H₂O₂, suggesting that peroxide is involved. The rapid peroxidase induction in the former case also suggests induction by a stronger chemical oxidant, as formed in the Fenton-type reaction.

Finally, it should be mentioned that the promoter region of gene mnpl includes some putative response elements (34) which could be involved in the above regulation of transcript levels of the new ligninolytic peroxidase produced by P. eryngii. Additional studies are necessary to elucidate this and other aspects of ligninolytic peroxidase regulation in these white-rot fungi.