ABSTRACT
The expression pattern of manganese peroxidases (MnPs) in nitrogen-limited cultures of the saline-tolerant fungus Phlebia sp. strain MG-60 is differentially regulated under hypersaline conditions at the mRNA level. When MG-60 was cultured in nitrogen-limited medium (LNM) containing 3% (wt/vol) sea salts (LN-SSM), higher activity of MnPs was observed than that observed in normal medium (LNM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that two MnP isoenzymes were de novo synthesized in the culture of LN-SSM. Three MnP-encoding genes (MGmnp1, MGmnp2, and MGmnp3) were isolated by reverse transcription (RT)-PCR and rapid amplification of cDNA ends PCR techniques. The corresponding isozymes were identified by peptide mass fingerprinting analysis. MnP isozymes encoded by MGmnp2 and MGmnp3 were observed mainly in LN-SSM. Real-time RT-PCR analysis revealed high levels of MGmnp2 and MGmnp3 transcripts in LN-SSM 48 h after the addition of 2% NaCl. The induction of MnP production and the accumulation of gene transcripts by saline were well correlated in the presence of Mn2+. However, in the absence of Mn2+, there was no clear correlation between mnp transcripts levels and MnP activity, suggesting posttranscriptional regulation by Mn2+.
Mangroves are trees that grow in saline habitats in the tropics and subtropics. Plants in mangrove forests have developed a set of physiological adaptations in response to frequent tidal inundation. Mangroves and sea grasses provide a natural habitat for marine fungi. Marine fungi are often found on decayed lignocellulosic substrates such as prop roots, pneumatophores, branches, leaves, and driftwood in the intertidal region of mangrove stands. They are thought to play an important role in lignocellulosic degradation in such marine ecosystems (9). Commonly isolated marine fungi belong to ascomycetes and deuteromycetes, while basidiomycetes are relatively rarely reported (22, 27, 36). The lignocellulolytic enzymes of marine fungi have potential industrial and environmental applications (22, 27, 29).
White rot fungi have a unique ability to decompose wood lignin via the secretion of extracellular lignin-degrading enzymes such as manganese peroxidase (MnP), lignin peroxidase, versatile peroxidase, and laccase. MnP is considered to be one of the key enzymes involved in lignin degradation caused by white rot fungi. MnP oxidizes Mn2+ to Mn3+ in an H2O2-dependent reaction, and Mn3+ organic acid chelates oxidize monomeric phenol, phenolic lignin dimers, and synthetic lignin via the formation of a phenoxy radical (7, 19). Additionally, MnP participates in lignin biodegradation via thiol and lipid-derived free radicals that are able to oxidize a variety of nonphenolic aromatic compounds (1, 35). Although many genes encoding MnP have been cloned from several white rot fungi, there has been no report focusing on marine fungi.
The marine fungus Phlebia sp. strain MG-60 was selected from 28 mushrooms and driftwoods collected from mangrove stands in Okinawa, Japan, based on PolyR-478 decolorization and lignin biodegradation under hypersaline conditions (15). Phlebia sp. strain MG-60 produces MnP mainly under hypersaline conditions. It was able to brighten the unbleached hardwood kraft pulp extensively even under conditions of 5% (wt/vol) sea salts. In contrast, pulp was only slightly brightened by the widely studied white rot fungus Phanerochaete chrysosporium at 3% (wt/vol) and 5% (wt/vol) sea salt concentrations (15, 16). Thus, MG-60 has significant bleaching ability, especially in a hypersaline environment.
To clarify the effect of hypersaline conditions on MnP production from Phlebia sp. strain MG-60, herein, we compare the productions of MnP activity and different MnP isozymes under normal and hypersaline conditions. We also provide the full sequences of three new MnP-encoding genes, MGmnp1, MGmnp2, and MGmnp3, which are differentially regulated in response to saline stress.
MATERIALS AND METHODS
Fungal cultures. Phlebia sp. strain MG-60 TUFC40001 (Fungus/Mushroom Resource and Research Center, Tottori, Japan), Trametes versicolor NBRC6482, and Phanerochaete chrysosporium ATCC 34541 were maintained on potato dextrose agar (PDA) plates. Mycelium mats on an agar plate were transferred into a sterilized blender cup containing 50 ml of sterilized water and were homogenized with a Waring blender for 30 s. To monitor the MnP activity and isozyme expression pattern, 5 ml of homogenate was inoculated into a 500-ml Erlenmeyer flask containing 300 ml of low-nitrogen basal III medium (LNM) that contained 1.0% (wt/vol) glucose as a carbon source, 1.2 mM ammonium tartrate as a nitrogen source, and 20 mM sodium acetate at pH 4.5 (33) or LNM containing 3% (wt/vol) sea salts (LN-SSM) (Sigma). One gram of sea salts contained 482.25 mg of chloride, 269.5 mg of sodium, 66.5 mg of sulfate, 33 mg of magnesium, 10.5 mg of potassium, 10 mg of calcium, 5 mg of carbonate/bicarbonate, 0.22 mg of strontium, 0.14 mg of boron, 1.4 mg of bromide, and <0.5 mg of other total trace element (manufacturer's analysis). Flasks were incubated on a rotary shaker at 150 rpm in the dark at 30°C. After the prescribed incubation period, the whole culture was separated into biomass and extracellular fluid by centrifugation (12,000 rpm for 10 min). The mycelial dry weight and extracellular MnP activity were then measured. In order to investigate the effect of sea salt and its main component, NaCl, on the transcription of MGmnp genes, LNM containing 2% NaCl (LN-NaClM) was also used.
Enzyme activity and SDS-PAGE.MnP activity was determined spectrophotometrically at 270 nm by monitoring the formation of the Mn3+-malonate complex at pH 4.5 in 50 mM sodium malonate buffer (34). For analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), culture fluids were concentrated in an Amicon ultrafiltration unit with a 10-kDa-cutoff Omega membrane filter (Filtron), and the concentrates were desalted using a PD-10 column (GE Healthcare). An equal amount of protein of the resulting fraction was loaded onto each lane and separated by 10% (wt/vol) SDS-PAGE. The proteins were visualized by staining with 0.2% (wt/vol) Coomassie brilliant blue R-250.
Isolation of nucleic acid and cDNA preparation.The mycelia from 10-ml liquid cultures were filtered through a Miracloth (Calbiochem), semidried with a sterile paper towel, frozen rapidly in liquid nitrogen, and stored at −80°C. The frozen mycelium was ground into a powder in a mortar containing liquid nitrogen. DNA was isolated from the mycelium powder with an extraction buffer (2% CTAB [N-cetyl-N,N,N-trimethylammonium bromide], 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA, and 0.2% β-mercaptoethanol) and purified with chloroform-isoamyl alcohol (24:1). Total RNA was prepared by a combination of plant RNA isolation reagent (Invitrogen Corp., Carlsbad, CA) and TRIzol reagent (Invitrogen Corp., Carlsbad, CA). The obtained RNA was washed with 75% ethanol and dissolved in diethylpyrocarbonate-treated water. The amount and quality of the RNA were calculated by measuring the absorbances at 260 and 280 nm.
cDNA was synthesized in a 20-μl reaction mixture that included 1 μg of total RNA, 1 μM oligo(dT) adapter primer containing an M13 primer M4 sequence, 10 U of RNase inhibitor, and 10 U of avian myeloblastosis virus reverse transcriptase (TaKaRa, Japan) according to the manufacturer's instructions. The reaction was carried out for 60 min at 45°C, and the samples were the heated for 5 min at 95°C to terminate the reaction. Finally, the reaction mixture was diluted 1:100 with diethylpyrocarbonate-treated water, and a 1-μl sample was used for real-time reverse transcription-PCR (RT-PCR) analysis as described below.
Gene identification and characterization.To isolate the partial sequence of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) in Phlebia sp. strain MG-60, a 629-bp fragment of gpd cDNA was amplified with a pair of degenerate primers (forward primer 5′-GGTCGYATYGGCCGYATYGT-3′ and reverse primer 5′-ATRACCTTKCCGACRGCCTT-3′) (5). The PCR amplification was performed with a denaturation step at 94°C for 3 min, followed by 35 cycles that consisted of 94°C for 30 s, 54°C for 30 s, and 72°C for 2 min and, finally, an extension step at 72°C for 5 min. To identify the MnP-encoding genes, 1,075-bp and 1,081-bp fragments of MGmnp2 and MGmnp3 cDNA containing a poly(A) sequence were amplified with a degenerate sense primer (5′-TSCGYCTYAYKTTCCACGA-3′) and M13 primer M4 (5′-GTTTTCCCAGTCACACGAC-3′). The degenerate sense primer was designed according to the amino acid sequence conserved in the Mn-binding region (RLTFHD). A fragment of MGmnp1 cDNA (909 bp) was amplified with a degenerate primer (5′-AACTGYCCYGGYGCDCCMCR-3′) and M13 primer M4. The degenerate sense primer was designed according to the conserved sequence (NCPGAP) among mnp genes. Each consensus amino acid sequence was searched from alignment data reported previously (19). The PCR amplification was performed with a denaturation step at 94°C for 3 min, followed by 35 cycles that consisted of 94°C for 30 s, 57°C for 30 s, and 72°C for 2 min and, finally, an extension step at 72°C for 5 min.
A 5′ rapid amplification of cDNA ends system (Invitrogen) was used to amplify the missing 5′ ends of the transcripts according to the manufacturer's instructions. The full-length genomic DNA and open reading frame of cDNA of the mnp genes were amplified with primers designed according to the nucleotide sequence data obtained from the rapid amplification of cDNA ends PCR fragments.
Ex-Taq polymerase (TaKaRa, Japan) or Pfu polymerase (Stratagene, La Jolla, CA) was used for PCR amplifications. The PCR products were subcloned into vector pXcmkn12, and the resulting ligation products were transformed into Escherichia coli strain DH5α according to the manufacturer's protocol (Invitrogen, Carlsbad, CA). The clones were sequenced by a dideoxy method (Thermo Sequence cycle sequence kit; Amersham Bioscience) with a sequencer (LIC-4000; Aloka, Japan).
Peptide mass fingerprinting analysis.To identify the mnp gene encoding the isozyme observed by SDS-PAGE, gel tryptic digestion was performed as previously described (30). The target band was excised and cut into 2-mm cubes, and the gel pieces were then transferred into a 200-μl microcentrifuge tube and washed with 40% (vol/vol) 1-propanol in water at room temperature for 15 min. After removal of the 1-propanol solution, 200 mM ammonium bicarbonate in 50% (vol/vol) acetonitrile in water was added, and the sample was incubated at room temperature for 15 min. The gel pieces were then dried and covered with 20 ng/μl modified trypsin (Promega) in a minimal volume of 100 mM ammonium bicarbonate to rehydrate the pieces. After a 12-h incubation, the supernatant was collected, and the gel pieces were extracted with 100 mM ammonium bicarbonate, followed by two extractions with 80% (vol/vol) acetonitrile containing 0.05% trifluoroacetic acid. The supernatant and extracts were combined, and the acetonitrile was allowed to evaporate in a desiccator at room temperature. The resulting peptide mixtures were desalted using C18 ZipTips (Millipore) and eluted onto a 96-well matrix-assisted laser desorption ionization target plate. A 1-μl sample on the plate was mixed with 1 μl of 10 mg/ml α-cyano-4-hydroxycinnamic acid solution in H2O-acetonitrile (1:1) containing 0.1% trifluoroacetic acid. Samples were then dried at room temperature. Mass spectral data were obtained using a Voyager DE mass spectrometer equipped with a 337-nm N2 laser in the positive-ion reflectron mode (Applied Biosystems). Spectral data were obtained by averaging 64 spectra, each of which was the composite of 64 laser firings. The internal mass calibration was performed using bradykinin (904.45 Da) and adrenocorticotropic hormone (2,465.75 Da).
Incubation for transcriptional analysis.Expression of the MnP-encoding genes was assessed under conditions using several media. Mycelium was preincubated in 500-ml Erlenmeyer flasks containing 300 ml of LNM without MnSO4 for 5 days as described above. Preincubated mycelium was transferred into a sterilized blender cup and homogenized with a Waring blender for 30 s. One milliliter of homogenate was inoculated into 100-ml Erlenmeyer flasks containing 10 ml of LNM, LN-SSM, LN-NaClM, or MnSO4-free versions of each medium. Flasks were incubated statically at 30°C in the ambient atmosphere. After incubation, the whole culture was separated into biomass and extracellular fluid by centrifugation. The total RNA was then extracted from mycelium, and the extracellular MnP activity was measured as described above.
Real-time RT-PCR.Real-time, fluorescence-based RT-PCR was performed using a final volume of 10 μl with a Line Gene apparatus (Bio Flux Corporation, Japan). The total RNA was prepared from the mycelium harvested from the above-mentioned 10-ml cultures of LNM, LN-SSM, or LN-NaClM. cDNA was synthesized in a final volume of 20 μl that included 1 μg of total RNA, 1 μM oligo(dT) 18-mer primer, 10 U of RNase inhibitor, and 10 U of avian myeloblastosis virus reverse transcriptase (TaKaRa, Japan) according to the manufacturer's instructions. After RT for 60 min at 45°C, the samples were heated for 5 min at 95°C to terminate the reaction. The Sybr Premix Ex-Taq kit (TaKaRa, Japan) was used for real-time PCR according to the manufacturer's instructions, with a final concentration of 0.2 μM for each gene-specific primer. The PCR amplification was performed as follows: (i) an initial denaturation step at 95°C for 1 min and (ii) 45 cycles, with 1 cycle consisting of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and elongation at 72°C for 30 s. Amplicon specificity was verified by melting-curve analysis conducted at 65°C to 95°C with stepwise fluorescence acquisition and by 2% (wt/vol) agarose gel electrophoresis staining with ethidium bromide. No fluorescence was detected from real-time RT-PCR amplification without a template. Primer sequences and amplicon lengths for the MnPs and reference gene are listed in Table 1. In the cases of MGmnp2 and MGmnp3, the forward primer sequence was designed from a coding region as close as possible to the stop codon. The reverse primer was constructed in the 3′ untranslated region to achieve specificity. Although the possibility of the existence of alternative splicing cannot be completely excluded, gel electrophoresis of PCR products using each forward primer and a poly(A) primer confirmed that a single product was amplified with each primer pair. Besides that of the melting curve, gel electrophoresis analysis confirmed single-product amplification for gpd, MGmnp1, MGmnp2, and MGmnp3 under all conditions tested here. The ratio of the gene-specific expression was defined as expression relative to gpd gene expression. Data are presented as means of triplicate PCRs, each containing an aliquot of the same template.
Gene-specific PCR primers used for real-time RT-PCR
Transcriptional induction of MGmnp genes in Phlebia sp. strain MG-60.To monitor the expression of the MnP-encoding genes after the addition of NaCl to MnSO4-free LNM, mycelium was preincubated statically at 30°C in 100-ml Erlenmeyer flasks containing 10 ml MnSO4-free LNM for 6 days, at which time the maximum activity of MnP in LNM was produced. After this preincubation period, 0.2 g of solid NaCl (equivalent to 2%) was added to the culture, mixed gently, and then incubated. Every day, each culture was harvested, and the whole culture was separated into biomass and extracellular fluid by centrifugation. Total RNA was extracted from mycelium, and the extracellular MnP activity was measured.
RESULTS
Effect of 3% (wt/vol) sea salts on MnP production. Phlebia sp. strain MG-60, P. chrysosporium, and T. versicolor were cultured in LNM (with MnSO4) with or without sea salts. The time course of MnP production is shown in Fig. 1. In the case of P. chrysosporium, MnP production in LN-SSM (with MnSO4) was decreased to about one-half of the level of MnP production in LNM (with MnSO4) (Fig. 1A). There was no growth of T. versicolor in LN-SSM (data not shown). On the other hand, when MG-60 was cultured in LN-SSM (with MnSO4), the MnP activity was four times higher than that in LNM (with MnSO4) (Fig. 1B). When this fungus was incubated in LNM (without MnSO4) or LN-SSM (without MnSO4), there was no MnP activity in the extracellular fluid under stationary conditions (data not shown). SDS-PAGE analysis of the extracellular proteins that were harvested from the 10-day shaking culture gave one main band (45 kDa) in the LNM (with MnSO4) culture and two main bands (47 kDa and 50 kDa) in LN-SSM (with MnSO4) (Fig. 2B).
Extracellular MnP activities of P. chrysosporium (A) and Phlebia sp. strain MG-60 (B) in LNM (black triangles) and LN-SSM (open circles) under shaking conditions. Values are means ± standard deviations of two duplicates.
Induction of extracellular MnP activity after the addition of sea salts (A) and SDS-PAGE of concentrated extracellular culture fluid (B). The lanes of time zero and LN-SSM correspond to the cultures incubated for 10 days with LNM and LN-SSM, respectively. Values are means ± standard deviations of two duplicates.
Phlebia sp. strain MG-60 was incubated in LNM (with MnSO4) with shaking for 10 days, and sea salts at 3% (wt/vol) of the final concentration were then added to the culture. The MnP activity was increased rapidly 48 h after the addition of sea salts (Fig. 2A). SDS-PAGE analysis of the extracellular concentrates revealed that two de novo-synthesized proteins (47 kDa and 50 kDa) appeared within 48 h of the addition of sea salts (Fig. 2B). Primary protein (45 kDa) disappeared, coinciding with the induction of the other two proteins (47 kDa and 50 kDa). These proteins are identical to the main proteins observed in the 10-day LN-SSM (with MnSO4) culture.
Cloning of mnp genes from Phlebia sp. strain MG-60.Three full-length cDNA clones of mnp genes, MGmnp1 (1,095 bp), MGmnp2 (1,173 bp), and MGmnp3 (1,170 bp), were obtained based on the PCR strategy. The nucleotide sequence of MGmnp1 predicts a 365-amino-acid (aa) sequence containing a putative signal peptide (26 aa) at the N terminus. The predicted amino acid sequence of MGmnp1 was 92% and 80% identical to Phlebia radiata MnP3 and Trametes versicolor MP2, respectively. The cDNA sequences of MGmnp2 and MGmnp3 were 77% identical, and their predicted amino acid sequences were 80% identical. MGmnp2 and MGmnp3 encode 390- and 389-aa sequences, respectively, and each one features a 23-aa putative secretion signal at the N terminus. The amino acid sequence of MGmnp2 was 74%, 72%, and 65% identical to P. radiata MnP2, Dichomitus squalens MnP1, and Ceriporiopsis subvermispora MnP-2, respectively. The amino acid sequence of MGmnp3 was 82%, 77%, and 70% identical to P. radiata MnP2, D. squalens MnP2, and C. subvermispora MnP-2, respectively. Multiple alignments revealed conserved catalytic and Mn binding residues (19; data not shown). The genomic nucleotide sequences of MGmnp1, MGmnp2, and MGmnp3 were also obtained by PCR using specific primer sets constructed on the 5′ and 3′ untranslated region sequences. Comparisons of cDNA and genomic sequences showed 100% identity within coding regions and allowed the unambiguous assignment of exon-intron boundaries. MGmnp1 contains 10 short introns, whereas MGmnp2 and MGmnp3 contain seven short introns. The intron-exon organizations of genes were compared with other selected genes encoding MnP (data not shown). The intron positions of MGmnp2 and MGmnp3 corresponded with that of Prmnp2 completely. The intron position of MGmnp1 closely resembled that of Prmnp3, although the eighth intron of Prmnp3 was absent in MGmnp1. From the phylogenetic grouping (Fig. 3), MG-MnP1 appears to be more closely related to the versatile peroxidase group, whereas MG-MnP2 and MG-MnP3 are clustered in the classical manganese peroxidase group.
Neighbor-joining tree of the evolutionary relations of deduced amino acid sequences of selected class II fungal secretory heme peroxidases. Ab, Agaricus bisporus; Ba, Bjerkandera adusta; B, Bjerkandera sp.; Cc, Coprinus cinereus; Cs, Ceriporiopsis subvermispora; Ds, Dichomitus squalens; Ga, Ganoderma applanatum; Pc, Phanerochaete chrysosporium; Pe, Pleurotus eryngii; Po, Pleurotus ostreatus; Pr, P. radiata; Ps, Phanerochaete sordida; Tv, Trametes versicolor. Sequence accession numbers were retrieved from the DDBJ Nucleotide Sequence Databank and are as follows: AJ699058 (Ab-MNP), AY217015 (B-RBPa), E03952/E51135 (Ba-LiP), Q12575 (Cc-CiP), AF013257 (Cs-MnP1), AF036254 (Cs-MnP2), AF161585 (Cs-MnP3), AF157474 (Ds-MnP1), AF157475 (Ds-MnP2), AB035734 (Ga-Ea.mnp1), M74229 (Pc-LIG2), X51590 (Pc-LIG3), M18794 (Pc-LIG5), M37701 (Pc-LIGH8), AF140062 (Pc-LiPJ), M60672 (Pc-MnP1), L29039 (Pc-MnP2), U70998 (Pc-MnP3), AF007223 (Pe-VPL1), AF007222 (Pe-VPL2), AF175710 (Pe-PS1), AJ243977 (Po-MnP2), AB011546 (Po-MNP3), X14446 (Pr-LIG3), AJ315701 (Pr-MnP2), AJ310930 (Pr-MnP3), AB078604 (Ps-MNP1), AB078605 (Ps-MNP2), AB078606 (Ps-MNP3), Z31011 (Tv-LP12), Z30667 (Tv-LP7), Z54279 (Tv-MNP2), Z30668 (Tv-MP2), AF008585 (Tv-MrP), AB360587 (MG-MnP1), AB360588 (MG-MnP2), and AB360589 (MG-MnP3).
Identification of MnP-encoding genes.Protein digestion was performed using trypsin, and a peptide mass map was obtained using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The peptide mass map was compared with the predicted amino acid sequences from MGmnp genes. In the case of the 45-kDa protein that is expressed mainly in LNM, 48% sequence coverage (10/15 masses matched) was achieved with MG-MnP1. Additionally, 47-kDa and 50-kDa proteins showed 38% (8/16 masses matched) and 30% (4/11 masses matched) sequence coverages with MG-MnP3 and MG-MnP2, respectively. These results indicated that the 45-, 47-, and 50-kDa proteins are encoded by the MGmnp1, MGmnp3, and MGmnp2 genes, respectively. Furthermore, the results showed that MG-MnP3 and MG-MnP2 were found mainly under hypersaline conditions.
Effect of sea salts and NaCl on transcription of MGmnp genes.Higher levels of MnP production in LN-SSM were observed under static culture conditions (Fig. 4A) as well as in the shaking culture (Fig. 1B). The main component of sea salts is NaCl, and consistent with its major role, MnP production was also increased in the culture containing 2% (wt/vol) NaCl in LN-NaClM (Fig. 4A). SDS-PAGE analysis showed that MG-MnP2 and MG-MnP3 were also produced mainly in the LN-SSM static culture (Fig. 4B) as well as in the shaking culture (Fig. 2B). Under the LNM condition, the transcription of each gene was observed on days 4, 5, 6, and 7 (Fig. 5). However, in the medium containing sea salts (LN-SSM) or NaCl (LN-NaClM), MGmnp2 and MGmnp3 transcripts increased substantially on days 6 and 7, while the MGmnp1 transcript levels remained low.
Extracellular MnP activities of Phlebia sp. strain MG-60 in LNM (black triangles), LN-SSM (open circles), and LN-NaClM (open squares) under static conditions (A) and SDS-PAGE of concentrated extracellular culture fluid at day 8 (B). Lane LNM refers to the 6-day-incubation culture in LNM, and lane LN-SSM refers to the 8-day-incubation culture in LN-SSM (B). Values are means ± standard deviations of triplicate cultures. Lane M, molecular mass marker.
Relative transcription levels of MGmnp1, MGmnp2, and MGmnp3 in LNM (white bars), LN-SSM (light gray bars), and LN-NaClM (dark gray bars) under static culture conditions. Gene expression was determined by real-time RT-PCR and normalized to the gpd expression. Error bars show the standard deviations for triplicate samples.
Effect of Mn2+ on transcription of MGmnp genes.Extracellular MnP activities under MnSO4-sufficient and -free culture conditions were compared. Although the highest MnP activity was observed on day 6 using MnSO4-sufficient medium, a lack of detectable extracellular MnP activity was observed in the absence of MnSO4 (data not shown). In the case of MnSO4-free LN-SSM, no MnP activity was observed in these static cultures. The highest level of mnp mRNA for each gene was obtained on day 5 in MnSO4-sufficient LNM, whereas the transcript was undetectable in MnSO4-free LNM (Fig. 6). On the other hand, mRNA of MGmnp2 was observed on day 7 in MnSO4-free LN-SSM irrespective of the lack of detectable extracellular MnP activity. Transcripts of MGmnp1 or MGmnp3 were not observed in MnSO4-free LN-SSM (Fig. 6). In another experiment in which the culture was preincubated for 5 days in MnSO4-free LNM and NaCl as 2% (wt/vol) of the final concentration was then added to the culture, the transcript of mRNA of MGmnp2 was also induced 48 h after the addition of NaCl and MnSO4-free LN-SSM (Fig. 7). These results indicate that the transcription of MGmnp2 was responsive to NaCl but independent of MnSO4.
Relative transcription levels of MGmnp1, MGmnp2, and MGmnp3 in LNM (white bars), MnSO4-free LNM (light gray bars), and MnSO4-free LN-SSM (dark gray bars) under static culture conditions. Gene expression was determined by real-rime RT-PCR and normalized to gpd expression. Error bars show the standard deviations for triplicate samples.
Induction of MGmnp2 transcription in MnSO4-free LNM by the addition of NaCl. The fungus was cultured for 5 days in MnSO4-deficient LNM (10 ml), and NaCl at a final concentration of 2% (wt/vol) was then added to the culture. Gene expression was determined by real-time PCR and normalized to the gpd expression. Error bars show the standard deviations for triplicate samples.
DISCUSSION
The mineralization of 14C-labeled lignin to 14CO2 by marine fungi was reported previously (25, 31). Isolated from decaying sea grass from a coral lagoon off the west coast of India, the white rot fungus Flavodon flavus produced lignin peroxidase, MnP, and laccase in nitrogen-limited medium prepared with distilled water or with synthetic seawater using Instant Ocean salts (Instant Ocean, Mentor, OH). However, enzyme production in LN medium containing Instant Ocean salts was reduced by about 50% relative to that in LN medium (26). In the present study, the growth of T. versicolor was severely inhibited and MnP production by P. chrysosporium was reduced in LN-SSM. These results indicate that the seawater condition is unsuitable for growth and enzyme production by these lignin-degrading fungi. When MG-60 was incubated in PDA medium containing several concentrations of sea salts, the growth of this fungus was observed even at a 5% (wt/vol) sea salt concentration (15). Furthermore, the growth of MG-60 in PDA medium containing 1.5% (wt/vol) sea salts was faster than that in normal PDA medium, while the growths of P. chrysosporium and T. versicolor were inhibited using the same concentration of sea salts (unpublished data). MG-60 had better tolerance to a hypersaline environment, and MnP production by this fungus was strongly enhanced (Fig. 1). Therefore, it seems that Phlebia sp. strain MG-60 has a special mechanism for the expression of MnP under hypersaline conditions.
In the present study, the results show that saline stress up-regulated the specific MnP isozymes MGmnp2 and MGmnp3 of Phlebia sp. strain MG-60 at the transcriptional level. To our knowledge, this is a unique system among the white rot fungi characterized so far. Figure 2 clearly shows that MnP production was enhanced in response to the addition of sea salts. Moreover, MG-MnP2 and MG-MnP3 were de novo-synthesized MnP isoforms under saline conditions. These characteristics were similarly observed under either shaking or static culture conditions, suggesting that the production of MG-MnP1, MG-MnP2, and MG-MnP3 isozymes was differentially regulated by hypersaline conditions.
Genetic analysis revealed three MnP-encoding genes of Phlebia sp. strain MG-60, MGmnp1, MGmnp2, and MGmnp3. All mnp genes were closely related to the mnp genes described previously for P. radiata strain 79 (ATCC 64658) (8). Recently, phylogenetic analysis based on the internal transcribed spacer (ITS) region (containing 5.8S ribosomal DNA, ITS1, and ITS2) was carried out (11). The ITS sequence of MG-60 (DDBJ accession number AB210077) showed only 90% identity to P. radiata strains (ATCC 64658 [accession number DQ056859], HHB-5324-sp [accession number AB084619], JLL-15608-sp [accession number AY219366], and 345B [accession number AY089740]), while the sequences of P. radiata strains shared identities of approximately 99 to 100% to each other. These results indicate that Phlebia sp. strain MG-60 is not Phlebia radiata.
MGmnp2 and MGmnp3 were classified as being typical MnP group I representatives (Fig. 3) (19). The calculated molecular masses of MG-MnP1, MG-MnP2, and MG-MnP3 were 32 kDa, 36 kDa, and 36 kDa, respectively, although actual masses determined by SDS-PAGE were 44 kDa, 50 kDa, and 47 kDa. This difference may be caused by glycosylation, a common feature of MnP isozymes. Such differences were also reported for MnP from P. chrysosporium (21, 24). According to the general eukaryotic rule for N glycosylation (Asn-X-Ser/Thr) (13), two and three asparagines potentially involved in glycan linkage were found in MG-MnP2 (N132 and N218) and MG-MnP3 (N132, N218, and N348), respectively, whereas only one (N103) was found in MG-MnP1. Glycans generally modify protein conformation, leading to stabilization toward thermal denaturation or protease attack (20).
The production of three MnP isozymes by Phlebia sp. strain MG-60 was regulated by Mn2+ at the mRNA level (Fig. 6). Typically, lignin degradation and peroxidase production occur as a secondary metabolic event triggered by nutrient nitrogen and/or carbon limitation. In several fungi, a sufficient amount of Mn2+ in the medium is required for MnP expression at the mRNA level (19). Differential regulation of mnp genes by manganese in Pleurotus ostreatus (4), C. subvermispora (18, 32), and T. versicolor (6, 10, 12) was previously described. Additionally, there is a report of the existence of Mn-responsive cis-acting sequences found in the upstream region of P. chrysosporium mnp1 (17). In the present study, real-time RT-PCR analysis shows that the accumulation of MGmnp3 transcripts in sea salts/NaCl medium was dependent on Mn2+ regulation. However, it should be noted that the MGmnp2 transcripts were observed under MnSO4-free conditions (Fig. 6 and 7), indicating that Mn2+ is not essential for the expression of MGmnp2.
Higher levels of MGmnp2 and MGmnp3 mRNAs were obtained with simultaneous induction by Mn2+ and sea salts than by sea salts alone: the maximum transcription ratios (mnp/gpd) of MGmnp2 and MGmnp3 in LNM were approximately 2 (Fig. 5), whereas those in MnSO4-free LN-SSM were approximately 5 and 0 (Fig. 6) and those in LN-SSM were approximately 15 and 21 (Fig. 5), respectively. These results imply that sea salts and Mn2+ activate the transcription of MGmnp2 and MGmnp3 synergistically, while sea salt-driven activation of MGmnp3 transcription follows in an Mn2+-dependent transcriptional manner. The accumulation of MGmnp2 transcripts occurred within 48 h of the addition of NaCl. This induction time seems longer than those of other known stress-driven mnp inductions or osmotic responses in yeast. In previous studies, several stresses on mnp gene expression by P. chrysosporium were examined. The accumulation of mnp transcripts by heat shock (3), hydrogen peroxide, oxygen, and chemical agents (14) was reported. In these studies, the accumulation of mnp transcripts occurs rapidly at 0.25 h after treatment with stress agent. Osmotic stress responses in Saccharomyces cerevisiae have been well studied. A high-osmolarity glycerol pathway mediates the response to hyperosmotic stress (2). The activation of the high-osmolarity glycerol pathway leads to a massive and rapid transcriptional response in which the expression of several hundred genes was induced within 5 to 10 min (23, 28). Little is known about the influence of saline stress on gene expression on basidiomycetes, and the observed delay in the induction of MGmnp2 remains to be investigated.
There was a clear correlation between mnp mRNA levels, MnP proteins, and MnP activity under Mn2+-sufficient conditions. On the other hand, there was no clear correlation between mnp mRNA levels and MnP activity under Mn-free conditions. MnP activity was undetectable even after the addition of NaCl in the absence of Mn2+, although higher transcripts of the MGmnp2 gene were observed. Based on this result, besides the transcriptional regulational function of Mn2+, Mn2+ also seems to be involved in posttranscriptional regulation. A similar effect was reported previously for oxidative stress induction of MnP in the case of P. chrysosporium (14) and for metal induction of MnP in the case of Ceriporiopsis subvermispora (18). Those authors observed that hydrogen peroxide and some metals, Cu2+, Zn2+, Ag2+, and Cd2+, induce mnp mRNA accumulation but not the production of extracellular MnP activity in medium where MnSO4 is absent.
This is the first report of the isolation and characterization of mnp genes from marine fungus and the first observation that hypersaline conditions regulate mnp gene expression. A marine habitat requires adaptation to cation toxicity and to osmotic stress. Phlebia sp. strain MG-60 might be evolutionarily adapted to saline-rich conditions for the sufficient expression of its lignin degradation ability. Further investigations of this marine fungus may provide insight into physiological adaptations to saline stress and into lignin degradation in severe environments.
ACKNOWLEDGMENTS
We are grateful to Hiroyuki Wariishi, Motoyuki Shimizu, and Shinya Sasaki, Kyushu University, for technical support in the peptide mass fingerprinting analysis of MG-MnPs.
This work was partially supported by a research fellowship of the Japan Society for the Promotion of Science for Young Scientists (I.K.).
FOOTNOTES
- Received 3 October 2007.
- Accepted 15 February 2008.
- Copyright © 2008 American Society for Microbiology
