Manganese-containing superoxide dismutase from the white-rot fungus Phanerochaete chrysosporium: its...

Enzyme and Microbial Technology 31 (2002) 754–764

Manganese-containing superoxide dismutase from the white-rot fungusPhanerochaete chrysosporium: its function, expression and gene structure

Paula A. Belinkya, Doron Goldberga, Bella Krinfelda, Martin Burgera,Nathan Rothschilda, Uri Coganb, Carlos G. Dosoretzc,∗

a MIGAL-Galilee Technology Center, South Industrial Zone, Kiryat Shmona 10200, Israelb Faculty of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel

c Division of Environmental and Water Resources Engineering, Faculty of Civil Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel

Received 20 March 2002; accepted 2 May 2002

Abstract

The gene structure and expression of the antioxidant enzyme manganese-containing superoxide dismutase (MnSOD1) of the white-rotfungusPhanerochaete chrysosporium was characterized. The MnSOD1 protein was purified from liquid cultures ofP. chrysosporium andthe amino acid sequences of four of its tryptic fragments were determined and used to clone a 3.2-kb genomic fragment encompassingthe entire gene of MnSOD1. Sequence analysis of the new gene,MnSOD1, revealed more than 80% identity with the MnSOD genesfrom the white-rot fungiGanoderma resinaceum, Ganoderma microsporum andAmauroderma rude, forming a clearly discernible cluster.The expression ofMnSOD1 was measured as a function of culture age, and a good correlation between transcript level and enzymeactivity was observed, with a peak at 72 and 96 h of incubation, respectively. Interestingly, as forP. chrysosporium, the only SOD activitydetectable in linear cultures of other white-rot fungi tested (Pleurotus florida, Lentinus edodes, Trametes versicolor, Ganoderma lucidum)was cyanide-insensitive SOD activity defined as MnSOD.© 2002 Elsevier Science Inc. All rights reserved.

Keywords: White-rot fungi;Phanerochaete chrysosporium; MnSOD; Fungal physiology

1. Introduction

The white-rot fungus Phanerochaete chrysosporiumcan degrade and metabolize lignin, as well as a broadrange of recalcitrant organopollutants, more extensivelythan any other organism[1,2]. The main mechanism of ini-tial lignin degradation by this fungus involves one-electronoxidation reactions catalyzed by extracellular oxidases andperoxidases. Lignin peroxidase (LIP) is one of the mostdiscernible enzymes associated with the extracellular degra-dation of lignin by P. chrysosporium. It oxidizes phenolsto phenoxy radicals and non-phenolic aromatics to radicalcations [3,4]. Liquid cultures of P. chrysosporium needto be concomitantly starved and exposed to a pure oxy-gen atmosphere in order to trigger LIP expression[5–7].Increasing oxygen availability leads to an increase in thetiter of the ligninolytic enzyme[8,9]. Such a high oxygenconcentration presumably leads to increased production ofreactive oxygen species (ROS) relative to that during normal

∗ Corresponding author. Tel.:+972-4-829-4962; fax:+972-4-822-8898.E-mail address: [email protected] (C.G. Dosoretz).

metabolism, subjecting the fungus to a ROS-rich environ-ment. Oxygen toxicity has been reported to be the principalcause of a major loss in organization of ultrastructure ofcells from cultures ofP. chrysosporium that had been ex-posed to pure oxygen[10]. Numerous chlamydospores aredeveloped when LIP synthesis is triggered[11], and cellswith impaired mitochondrial function express LIP, proba-bly in response to accumulating ROS[12]. Similar levelsof LIP activity were obtained in oxygenated cultures ofP. chrysosporium as in manganese-deficient cultures underatmospheric air[9]. In the latter, no manganese-containingsuperoxide dismutase (MnSOD) activity was detected, sug-gesting that both cultures are saturated by the ROS neededto trigger LIP expression. In addition to the ROS-rich en-vironment to which the fungus is subjected during lignindegradation, the fungus itself develops mechanisms for theproduction of a main ROS, hydrogen peroxide (H2O2),and is further exposed to high concentrations of the toxicaromatic radical intermediates generated.

Considering the high oxygen requirement of ligninolyticcultures ofP. chrysosporium and the attempts to clarify themechanisms of oxygen’s influence on LIP production, we

0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0141-0229(02)00180-1

P.A. Belinky et al. / Enzyme and Microbial Technology 31 (2002) 754–764 755

found it of great interest to investigate the fungus antioxi-dant enzymes. The survival of the fungus and the extensiveproduction of LIP in an environment extremely rich in ROSand aromatic radical intermediates suggest the existence ofefficient activation of an intracellular antioxidant defencesystem in the fungus.

Low levels of ROS are produced continuously and at ahigh rate as by-products of aerobic metabolism in living or-ganisms and are essential in a plethora of cellular processes.However, the accumulation of ROS, which is connected withdisturbances in the oxidant–antioxidant balance, may dam-age nucleic acids, proteins and membranes, leading to cyto-toxicity, mutagenicity or lethality[13]. Aerobic organismshave developed a protective system to resist these damagingoxygen species, which involves glutathione, iron chelators,Vitamins E and C, ceruloplasmin, and the combined actionof highly specialized antioxidant enzymes, such as SOD,glutathione peroxidase and catalase[14]. SOD plays an es-sential role in allowing organisms to survive in the presenceof molecular oxygen (O2). It catalyzes the dismutation ofthe highly reactive superoxide radical anion O2

− to O2 andH2O2 in all oxygen-metabolizing organisms and in someanaerobes[15], the latter being broken down in turn, to wa-ter by catalase or peroxidase.

SODs are metalloproteins containing iron, manganese,copper plus zinc or nickel as the prosthetic groups[16].MnSOD has been found in the cytosolic fractions ofprokaryotes and in the mitochondrial matrix of eukaryotes.In eukaryotic cells, MnSOD is synthesized in the cytosoland imported post-translationally into the mitochondrialmatrix [16,17]. In eukaryotes, the mitochondrial matrix isthe intracellular site of the final combustion of nutrients andthe reduction of oxygen to water. It is also an importantsite for the single-electron reduction of oxygen to O2

−.Therefore, MnSOD is thought to be a major scavenger ofdamaging reactive oxygen metabolites in the mitochondrialmatrix, like CuZnSOD in the cytoplasm.

Mutations generating a defective SOD cause hypersen-sitivity to oxygen-induced damage in bacteria, yeast andDrosophila melanogaster [18]. Inactivation of MnSODand FeSOD genes inEscherichia coli increased mutationfrequency when grown under aerobic conditions[19]. Elim-ination of the MnSOD gene inSaccharomyces cerevisiaeincreased its sensitivity to oxygen[18,20]. Expression of Fe-SOD fromE. coli or MnSOD from maize in SOD-deficientstrains ofS. cerevisiae protects the cells against oxidativestress[21,22] and its over-expression increases the meanlife span of Coenorhabditis elegans and D. melanogaster[21,23]. Expression of human MnSOD genes in transgenicmice protected the mice against oxygen-induced pulmonaryinjury [24]. Thus, the expression of MnSOD is essentialfor the survival of aerobic life and for the development ofcellular resistance to oxygen radical-mediated toxicity.

The present study characterizes an MnSOD fromP.chrysosporium and the structure and expression of its en-coding gene.

2. Materials and methods

2.1. Organisms, culture conditions and biomass handling

The following organisms were used for measurements ofSOD activity: the white-rot fungiPleurotus florida (MarkPlatt F6),Lentinus edodes (Somycel 4072),P. chrysospo-rium Burds BKM-F-1767 (ATCC 24725),Trametes versi-color and Ganoderma lucidum; the non-ligninolytic fila-mentous fungiNeurospora crassa and Rhizoctonia solani(the last four organisms were obtained from the Division ofPlant Pathology and Microbiology at the Faculty of Agri-cultural, Food and Environmental Quality Sciences of theHebrew University, Rehovot, Israel); and the yeastS. cere-visiae (MATa ura3-52 leu2-3 leu2-112 his3-200, YGO535).

Fungi were maintained on 2% malt-extract agar slants andwere grown in potato-dextrose agar (PDA) in petri dishes(linear growth) at 28◦C (except forP. chrysosporium whichwas grown at 37◦C) and harvested after the entire surface ofthe medium was covered. The yeastS. cerevisiae was grownin liquid medium containing yeast extract-potato dextrose(YEPD) at 30◦C overnight. The biomass harvested from thedifferent cultures was homogenized in the cold for 1 min in50 mM phosphate buffer, pH 7, with an Ultra-Turrax T25homogenizer (IKA, Germany). The homogenate was cen-trifuged at 2000× g for 30 min. The resultant filtrate wascentrifuged at 12,500×g for 10 min and the suspension wasstored at−80◦C until use. Phenylmethanesulfonyl fluoride(1 mM; PMSF, Sigma) was added to each extract.

For purification and isolation of SOD,P. chrysosporiumwas grown in 250 ml flasks, containing 90 ml medium, inshaken cultures (175 rpm) at 37◦C under conditions of freeair exchange. The growth medium was prepared as previ-ously described[6,9] except that glycerol, at an initial con-centration of 65 mM, was used as the carbon source insteadof glucose. The initial concentration of nitrogen (as diammo-nium tartrate) was 2.4 mM. Manganese (as manganese sul-fate) was added at a concentration of 1.2 mM to induce theformation of MnSOD. Unless otherwise specified, the cul-tures were incubated for 5 days.

E. coli strain DH10 was used for most plasmid construc-tion and maintenance.E. coli cells were grown at 37◦C inLuria–Bertani (LB) medium with or without ampicillin.

2.2. Isolation and purification of MnSOD from P.chrysosporium

MnSOD fromP. chrysosporium was purified by a mod-ification of the method described by Oeztuerk et al.[25].Cell extracts were prepared from 72-h-grown mycelium,collected by filtration, and washed with double-distilledwater. About 50–70 g of wet biomass were resuspendedin 50 mM potassium–phosphate buffer, pH 7.4, containing1 mM PMSF. The cell suspension was then homogenizedin the cold for 2 min as before. The suspension was furtherdisrupted according to Rothschild et al.[9] by extrusion

756 P.A. Belinky et al. / Enzyme and Microbial Technology 31 (2002) 754–764

via several passages through two syringes connected by asmall-orifice valve. The extract was then clarified by cen-trifugation at 17,000×g for 15 min at 4◦C, and the proteinswere precipitated with ammonium sulfate (65% saturationfor 30 min, with stirring). The precipitate was collected bycentrifugation, as above, redissolved in 20 ml of 20 mMTris-buffer, pH 8.8, and centrifuged again.

The clarified supernatant was then concentrated in an Am-icon ultrafiltration system with a 10-kDa-cutoff type PM-10membrane and redissolved in 2 ml of 20 mM Tris-buffer,pH 8.8. The concentrate was subjected to anion-exchangehigh-performance liquid chromatography (HPLC) on a 1-mlMonoQ column (HR 5/5, Pharmacia, NJ) at a flow rate of1 ml/min (20 mM Tris-buffer pH 8.8 with a linear gradientof 0–1 M NaCl over 40 min). Absorbance was monitoredat 280 nm and 2-ml aliquots were collected. Samples dis-playing the highest SOD activity were further purified byisoelectric focusing (IEF) electrophoresis using broad-range(pI 3–10) polyacrylamide gels (Bio-Rad). A broad-rangeBio-Rad pI calibration kit (pI 4.45–9.6) was used as thestandard. After electrophoresis, half of the gel was stainedwith Coomassie blue for protein identification and the otherhalf was stained for SOD activity. The lanes displaying thehighest SOD activity were further processed for amino acidsequencing.

2.3. Determination of MnSOD activity

SOD activity was tested by two assays. For samples ana-lyzed by non-denaturing polyacrylamide gel electrophoresis(PAGE) or IEF electrophoresis, the assay for SOD activitywas based on its ability to inhibit the reduction of nitrob-lue tetrazolium (NBT) by superoxide anions, producedphotochemically by riboflavin. For liquid samples, the as-say for SOD activity was based on the auto-oxidation ofhematoxylin[26].

Activity staining for SOD on a non-denaturing poly-acrylamide or IEF gel was performed according to themethod proposed by Beauchamp and Fridovich[27]. Thegels were incubated in the dark for 30 min in 80 ml reac-tion mixture containing 0.1 M potassium–phosphate buffer(pH 7.8), 1 mM EDTA, 33�M riboflavin (Sigma), 245�MNBT (Sigma), 17 mM TEMED. The gel was then incubatedin 0.1 M potassium–phosphate buffer (pH 7.8) with 1 mMEDTA and exposed to light for 30 min[9]. SOD activitywas detected by the appearance of transparent bands, rep-resenting the inhibition of NBT reduction by superoxideanions, on a blue background (reduced NBT).

The assay of SOD activity in liquid samples was basedon the SOD-mediated increase in the rate of auto-oxidationof hematoxylin in aqueous alkaline solution, which yields achromophore with maximum absorbance at 560 nm. Fortymicroliters of cell-free extract were mixed with 930-�laliquot of 50 mM Tris (pH 8.8) and 30�l of 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate (Bioxytech,Oxis International, OR) were added to eliminate mercap-

tans. The mixture was then vortexed and incubated for1 min at 37◦C. Afterwards, 30�l of 0.66 mM hematoxylin(Sigma) in 32 mM HCl was added to start the reactionwith immediate vortexing. The change in absorbance of themixture at 560 nm was monitored for 1 min.

CuZnSOD activity is sensitive to both H2O2 and cyanide.In contrast, MnSOD activity is not inhibited by either. Todistinguish it from that of CuZnSOD, MnSOD activity wasassayed in all cases in the presence of 5 mM KCN. CuZn-SOD from bovine erythrocytes (Sigma) and MnSOD fromE. coli (Sigma) were used as controls.

2.4. Amino acid sequencing and the design of PCR primers

The protein was recovered from the piece of the IEF gelcorresponding to the fraction with the highest SOD activ-ity, and subjected to proteolytic digestion with trypsin. Theamino acid sequences of three internal peptides were deter-mined by electrospray ion-trap mass spectrometry using aliquid chromatograph quadrupole ion-trap mass spectrome-ter (LCQ, Finnigan, San Jose, CA at the National Laboratoryof Biotechnology, Technion, Israel). The mass spectrometrydata was compared to simulated proteolysis and collisioninduce dissociation (CID) of the proteins in the NR-NCBIdatabase using the Sequest software (University of Wash-ington and Finnigan, San Jose).

The amino acid sequence of a fourth peptide was de-termined by Edman degradation. The sequence informationwas then used to design and synthesize five degenerate PCRprimers (Biotechnology General, Nes-Ziona, Israel), whichare listed inTable 1together with their corresponding aminoacid sequences.

2.5. Genomic DNA extraction

Genomic DNA fromP. chrysosporium was extracted bya method for the rapid isolation of genomic DNA from fila-mentous fungi[28] and used for probe preparation by PCRand for Southern blot analyses.

2.6. PCR

Optimized PCR amplification reactions (100�l) con-tained about 20 ng of genomic DNA fromP. chrysosporium,

Table 1Amino acid and oligonucleotide sequences of primers used for sequencingthe MnSOD ofP. chrysosporium

Primer Sequence Amino acidsequence

Al CGT CAG ATC ATG GAG CT(C,G) RQIMELHHCAY CAC

A2 CAR ATY ATG GAR YTN CAY C QIMELHB AAG CAC CAY CAR ACY TAC KHHQTYB2 AAR CAY CAY CAR CAN TAY G KHHQTYCl—reverse NGG RTC YTG RTT NGC ANQDP

N: A,C,G,T; Y: C,T; R: A,G.


10 mM Tris–HCl buffer, pH 8.3, 1.5 mM MgCl2, 25 mMKCl, 100 pmol of each primer, 2.5 U Klen Taq polymeraseand 0.25 mM of each dNTP. PCR amplification was donefor 35 cycles under the following conditions: denaturationat 94◦C for 1 min (3 min for the first cycle), annealing at46–56◦C for 1 min and extension at 72◦C for 1 min in aRobocycler (Stratagene).

2.7. Southern blot analysis and construction of amini-genomic library

Genomic DNA was completely digested by PstI(Boehringer) and electrophoresed in duplicates in 0.8%(w/v) agarose. After electrophoresis, half of the gel wastaken for Southern blotting and the other half was storedat 4◦C. Southern blotting was performed, according toSambrook et al.[29], onto a Hybond-N+ nylon membrane(Amersham Pharmacia Biotech, UK) and the blots werefixed and hybridized with a radioactively labeled probe. Theprobe was a 413-bp PCR product, encompassing the mid-dle portion of theP. chrysosporium MnSOD gene, and thelabeling was done by the random-primed labeling system,using a kit (Rediprime II, Amersham Pharmacia Biotech,UK). Hybridization was carried out at 65◦C in a solu-tion containing 5× SSC, 5× Denhardt’s reagent and 0.5%sodium dodecyl sulfate (SDS). Final washes were done at37◦C in 2× SSC containing 0.1% SDS, twice.

The autoradiogram was then used to locate, in the remain-ing half of the gel, the positions of genomic DNA fragmentsthat contained the MnSOD gene. The genomic DNA, sur-rounding a 3.2-kb PstI fragment, which hybridized with theprobe, was extracted from the gel and cloned into a plasmid(pGEM 3Zf, Promega) to create a mini-genomic library. Thelibrary was then screened by colony hybridization.

2.8. Colony hybridization

Colony hybridization was performed as described by Ger-gen et al.[30], using the 413-bp PCR fragment that con-tained the middle portion of the MnSOD gene, as a probe.

2.9. DNA sequencing and analysis

Positive clones were sequenced at Molecular BiologyCenter Ltd., Nes-Ziona, Israel. Almost 2 kb of the PstI3.2-kb genomic fragment that contained the MnSOD genewere sequenced. The sequencing was performed on bothstrands of the coding region and its proximal flanks. Se-quence assembly was performed with the aid of the Stadensoftware package Version 2000[31]. Sequence analysiswas performed with the aid of the GCG Wisconsin Package(Wisconsin Package, Version 10.1[32]) and the Clustal Wsoftware [33]. Database searching was done by BLASTusing the NCBI website[34]. Identification of regulatory

sequences was done by MatInspector Version 2.2[35] usingthe TRANSFAC website[36].

2.10. Measurement of MnSOD mRNA levels

Total RNA was isolated fromP. chrysosporium cellcultures by means of Tri Reagent (Sigma), according tomanufacturer’s instructions. The relative levels of MnSODwere measured by RT-PCR, usingP. chrysosporium 18SrRNA as an internal standard. The 18S rRNA was chosenas the internal standard since it is abundant and its expres-sion is stable[37]. First-strand cDNA synthesis and theamplification reaction were carried out in the same tube(Access RT-PCR System, Promega). The 50-�l reactionmixture contained a buffer, 100�g of total RNA, 0.2 mMdNTP mix, 5 units of AMV reverse transcriptase and 5units of the thermostableTfl DNA polymerase. The mixturewas incubated at 48◦C for 45 min for first-strand cDNAsynthesis, and then heated to 94◦C for 2 min to inactivatethe reverse transcriptase. Second-strand cDNA synthesisand 40 cycles of PCR amplification with MnSOD specificprimers (5′ ACC ACA AGA AGC ACC ACC AG 3′ and5′ GAA GTT CTC AAA GGA GCC GAA G 3′) wereperformed in a minicycler (MJ Research, MA) under thefollowing conditions: denaturation at 94◦C for 30 s, anneal-ing at 53◦C for 1 min and extension at 68◦C for 2 min. Theamplification of the internal standard 18S rRNA cDNA wasdone in a similar manner but with 20 cycles, using two 18SrRNA-specific primers (5′ CAA ATT ACC CAA TCC CGACAC 3′ and 5′ CAA CTA CGA GCT TTT TAA CTG C 3′).Forty cycles of amplification of the MnSOD cDNA and 20cycles of amplification of the 18S rRNA cDNA producedenough PCR products for the semi-quantitative analysis,while maintaining linear dependence on the concentrationof both transcripts. The PCR products (5�l) were elec-trophoresed on a 2% agarose gel at 100 V for about 45 min.All reaction mixtures were electrophoresed on the same gel.The DNA was then stained with 0.03 mg ethidium bromide(Sigma) in 100 ml distilled water, and imaged and analyzedwith the Fluor-S MultiImager (Bio-Rad).

2.11. Nucleotide sequence accession number

The nucleotide and deduced amino acid sequences of theP. chrysosporium MnSOD gene were deposited in GenBankunder the accession number AF388395.

3. Results

3.1. SOD activity in filamentous fungi

The only SOD activity detectable in cell extracts ofP.chrysosporium was that of MnSOD. Surprisingly, we werenever been able to detect any CuZnSOD activity in those


Fig. 1. SOD activity in the clarified cell extract of seven filamentous fungi and yeast. The fungi were grown on PDA in petri dishes and were harvestedwhen the surface was completely covered. Samples of clarified cell extract (20�g of protein per lane) were loaded onto a polyacrylamide gel and stainedfor SOD activity with NBT and riboflavin; 5 mM KCN was added to inactivate CuZnSOD.−KCN: whole SOD activity;+KCN: KCN-insensitive SODactivity.

extracts. To determine whether this pattern of expression isunique for P. chrysosporium or shared by other fungi, inparticular white-rot fungi, SOD activity in cell extractsfrom linear cultures of seven filamentous fungi and theyeastS. cerevisiae was measured on polyacrylamide gels(Fig. 1). The six basidiomycetes tested (five white-rot fungi:P. florida, P. chrysosporium, L. edodes, T. versicolor, G.lucidum and one non-lignin-degrading fungus:R. solani),exhibited only MnSOD activity, as no band disappearedwhen the gels were incubated in the presence of 5 mM KCN(Fig. 1, +KCN versus−KCN). The non-lignin-degradingfungusN. crassa, as well as the yeastS. cerevisiae displayedboth MnSOD and CuZnSOD activities, as one band clearlydisappeared in the cell extract incubated with KCN (Fig. 1,+KCN versus−KCN). Similar results were obtained whenthe activity was measured in cell extracts fractionated byIEF electrophoresis (data not shown).

3.2. MnSOD purification, internal sequence determinationand primer design

The protein samples from the purified SOD were analyzedby IEF electrophoresis, followed by staining for proteinsand for SOD activity. A predominant protein band of pI 7.42exhibiting SOD activity (Fig. 2) was excised from the gel,subjected to trypsin digestion and analyzed by mass spec-trometry and Edman degradation. The mass spectral analysisof three tryptic peptides resulted in the following amino acidsequences: KHHQTYVNALNAAEQAYAK, FNGGGH-

INHSLFWK and RQIMELHHK. Analysis of a fourth pep-tide by Edman degradation yielded the sequence YVELAIX-ANQDPLLTXV, where X marks unknown amino acids.

A database search for amino acid sequences similar to thefour peptides resulted in sequences of MnSODs from var-ious fungi, and confirmed the identity of the purified pro-tein. The amino acid sequences of the four peptides werethen used to design and synthesize five degenerate oligonu-cleotides (Table 1). The complete identity of the peptidesto the amino acid sequences of MnSOD proteins from sev-eral differentGanoderma species enabled us to significantlyreduce the degree of degeneracy of two of the oligonu-

Fig. 2. IEF of MnSOD fromP. chrysosporium purified by anion-exchangeHPLC on a MonoQ column. Activity: SOD activity staining with NBTand riboflavin. Protein: protein was stained with Coomassie brilliant blue.


cleotides, by using the corresponding DNA sequences ofthe Ganoderma MnSOD (Table 1, primers A1, B1). As itturned out, the first three peptides were completely identi-cal to the deduced amino acid sequence of MnSOD1, whilethe fourth peptide obtained by Edman degradation had dif-ferent amino acids in two positions, tyrosine (Y) in the firstposition and isoleucin (I) in the sixth position. This discrep-ancy most probably reflects experimental errors as no fun-gal MnSOD contains these residues in the correspondingpositions.

3.3. Cloning of the MnSOD gene in P. chrysosporium

The set of five degenerate primers (Table 1) was used toclone, by PCR, an internal fragment of theP. chrysosporium

Fig. 3. The structure of theP. chrysosporium MnSOD gene (MnSOD1). The amino acids of the deduced protein are shown in their three-letter code.The positions of the PCR primers that were used for the initial cloning of the 413-bp fragment (B1 and C1) are marked by arrows and bold letters. Theconsensus sequences of the exon–intron boundaries are marked by bold small letters. A putative TATA transcription initiation site upstream of the ATGstart codon is marked by underlined bold letters. Three putative stress-response elements (STREs), located upstream of the TATA box, are also markedby underlined bold letters (GenBank accession no. AF388395).

MnSOD gene. Four possible combinations of primers weretested and the pair of B1, C1 (Table 1) was chosen sinceit yielded a single product, approximately 400 bp in length(data not shown). The positions of primers B1 and C1, withrespect to the entire gene, are shown inFig. 3. The PCRproduct was isolated, cloned into a plasmid and sequenced.The sequencing revealed a 413-bp DNA fragment which,when compared to the GenBank database, showed highsimilarity to other fungal MnSOD genes. This PCR productwas then used as a probe to identify, by Southern analysis,a 3.2-kb PstI fragment. This PstI fragment was then clonedby constructing a mini-genomic library and screening it bycolony hybridization. The cloned fragment was sequencedand found to encompass the entireP. chrysosporiumMnSOD gene.


3.4. Sequence analysis of the MnSOD gene

The P. chrysosporium MnSOD gene (MnSOD1, Gen-Bank accession no. AF388395), and its flanking regions,are shown inFig. 3. The coding region consists of threeexons separated by two introns. The exons were located by

Fig. 4. Multiple sequence alignment of fungal MnSOD proteins. Sequences fromGanoderma microsporum (G. MICROSPORUM) (GenBank accessionnumber U56403),Ganoderma resinaceum (G. RESINACEUM) (GenBank accession number U56123),Amauroderma rude (A. RUDE) (GenBankaccession number U56109),Phanerochaete chrysosporium (P. CHRYSOSPORIUM),Agaricus bisporus (A. BISPORUS) (GenBank accession numberAJ404469),Neurospora crassa (N. CRASSA) (GenBank accession number AF118809),Penicillium chrysogenum (P. CRHYSOGENUM) (GenBankaccession number AF026523),Aspergillus fumigatus (A. FUMIGATUS) (GenBank accession number U53561),Schizosaccharomyces pombe (S. POMBE)(GenBank accession number AF069292),Aspergillus nidulans (A. NIDULANS) (GenBank accession number AY027564),Saccharomyces cerevisiae (S.CEREVISIAE) (GenBank accession number NC-001142) were compared. The alignment was created by the Clustal W software[33]. Identical aminoacids are marked by white letters on a dark gray background. Similar residues are marked by black letters on a light gray background. Residues conservedin all aligned proteins are marked by white letters on a black background.

searching for similarity between the new gene and its de-duced protein, and MnSOD genes and proteins from otherfungi (Ganoderma resinaceum, Amauroderma rude, Peni-cillium chrysogenum, Aspergillus fumigatus). Exon–intronjunctions were identified by their conserved sequences[38].The 5′ splice site contained the GT(A/G)NG(CT) consensus


sequence and the 3′ splice site contained the (C/T)N(C/T)AGconsensus sequence. Both consensus sequences are shownin Fig. 3. The TAAA sequence in the 5′ untranslated region,which is marked by underlined bold letters inFig. 3, could bethe TATA transcription-initiation signal (TATA box). Its po-sition upstream of the start codon (ATG) is in agreement withthe position of the TATA box in eukaryotes[38]. In contrast,no possible polyadenylation signal (AATAAA) could befound in the 3′ untranslated region. The three short sequencesupstream of the putative TATA box, which are also markedby underlined bold letters, are similar, although not iden-tical, to the yeast stress-response elements (STREs) found

Fig. 5. A dendrogram depicting the relations among fungal MnSODproteins. Sequences fromGanoderma microsporum (G. microsporum),Ganoderma resinaceum (G. resinaceum), Amauroderma rude (A. rude),Phanerochaete chrysosporium (P. chrysosporium), Agaricus bisporus (A.bisporus), Neurospora crassa (N. crassa), Penicillium chrysogenum (P.chrysogenum), Aspergillus fumigatus (A. fumigatus), Schizosaccharomycespombe (S. pombe), Aspergillus nidulans (A. nidulans), Saccharomycescerevisiae (S. cerevisiae) were analyzed. The dendrogram was constructedby the Neighbor-Joining method for cluster analysis, from the multiplesequence alignment shown inFig. 4. Only positions between the firstconserved leucine and the last conserved alanine were considered forthe analysis. The units of the scale bar (bottom right) are the estimatednumber of substitutions per 100 amino acids.

upstream of yeast genes known to be induced by oxidativestress, such asSOD1, SOD2 andCTT1 (catalase)[39].

A comparison of the deduced amino acid sequence ofthe P. chrysosporium MnSOD and similarly deduced par-tial protein sequences from three white-rot fungi (i.e.,G. resinaceum, Ganoderma fornicatum and A. rude) re-vealed approximately 80% identity. A comparison of theP. chrysosporium MnSOD with the complete amino acidsequences of MnSODs fromA. fumigatus and P. chryso-genum revealed only 56 and 57% identical amino acids,respectively. A multiple sequence alignment comparing theP. chrysosporium MnSOD1 protein with 10 additional fun-gal MnSODs is shown inFig. 4. A dendrogram depictingthe relationships among these fungal MnSODs is shown inFig. 5. The proteins of the white-rot fungi (G. resinaceum,Ganoderma microsporum, A. rude and P. chrysosporium)form a clearly discernible cluster, which may indicate acommon origin.

3.5. MnSOD expression

The expression ofMnSOD1 as a function of culture agein submerged cultures ofP. chrysosporium was measured atthe level of both enzymatic activity and mRNA transcripts(Figs. 6 and 7). Crude cell extracts from 2 to 6 days ofgrowth, which were fractionated by IEF electrophoresis andassayed for SOD activity, displayed a major band, the in-tensity of which changed with incubation time, with a max-

Fig. 6. SOD activity in crude extracts ofP. chrysosporium as a functionof culture age. The fungus was grown in shaken liquid cultures underfree air exchange with glycerol as the carbon source. Crude extracts wereassayed for SOD activity by two methods: (A) fractionation by IEF elec-trophoresis and staining with NBT and riboflavin and (B) auto-oxidationof hematoxylin. Both methods were performed in the presence of 5 mMKCN. Numbers above lanes indicate incubation time, in hours.


Fig. 7. MnSOD gene expression during growth ofP. chrysosporium insubmerged culture. The fungus was grown in shaken liquid culturesunder free air exchange with glycerol as the carbon source. MnSODgene expression at the mRNA level was determined by RT-PCR analysis.MnSOD gene expression appears in (A) and 18S rRNA calibration ofRT-PCR analysis appears in (B). The relative level of MnSOD mRNAexpression plotted vs. incubation time is given in (C). The relative levelof MnSOD mRNA expression was calculated by dividing the density(expressed in terms of fluorescence intensity per square millimetre) ofeach MnSOD band by the density of its corresponding 18S rRNA band.The density of bands was obtained by fluoro-densitometry analysis withthe Fluor-S MultiImager. Numbers above lanes indicate incubation time,in hours. Bars indicate standard deviation.

imum at 96 h (Fig. 6). This band probably correspondedto MnSOD since it did not disappear after KCN treatment(Fig. 6A). Under the defined culture conditions applied,the highest SOD activity in the cell extracts, measured byauto-oxidation of hematoxylin, also peaked at 96 h of incu-bation (Fig. 6B). MnSOD expression, at the mRNA level incell extracts ofP. chrysosporium was analyzed by RT-PCR(Fig. 7). The amplified fragments of the MnSOD and 18SrRNA cDNAs were electrophoresed and the resulting bandsare shown inFig. 7A and B, respectively. The level of expres-sion of MnSOD was maximal at 72 h, and decreased there-after (Fig. 7A), whereas the level of the 18S rRNA mRNAremained fairly stable during the experiment (Fig. 7B). Forconfirmation of the quantitative level of mRNA, the bandintensities were subjected to specific densitometric analysisby measurement of their fluorescence intensity (Fig. 7C).The relative level of MnSOD mRNA expression was calcu-lated by dividing the intensity of each MnSOD band by theintensity of its corresponding 18S rRNA band.

4. Discussion

MnSODs are inducible enzymes located in the eukary-otic mitochondrial matrix that scavenge superoxide anions.

Genomic sequences of several mitochondrial MnSODs havebeen identified in yeast, such asS. cerevisiae [40], Can-dida albicans [41], Schizosaccharomyces pombe [42], andin higher eukaryotes[43–45], as well as in filamentous fungisuch asA. fumigatus [46] andP. chrysogenum [47]. The Mn-SOD enzyme has been purified from the white-rot fungiG.microsporum [48] andP. chrysosporium [25].

The present study describes the cloning of theP. chry-sosporium gene encoding the protein, which correspondsto the larger band in the IEF gels, and characterizesits expression. The newly described gene, designatedMnSOD1, codes for a 206-amino acid protein—a sizesimilar to that of other eukaryotic MnSODs. Southernanalysis, carried out as part of the cloning process, in-dicated that only one copy ofMnSOD1 exists in theP.chrysosporium genome. However, search by BLAST ofthe current database of theP. chrysosporium genome[http://www.jgi.doe.gov/index.html], carried out during thepreparation of this manuscript, has revealed that it con-tains at least two additional MnSOD genes. Regardless ofthe presence of closely related genes, the primers used forRT-PCR are specific forMnSOD1. We have found that theexpression of theP. chrysosporium MnSOD1 is induced byoxidative stress (unpublished results). In agreement withthis finding, we have identified several short sequences,similar to the yeast STREs, upstream from the putativetranscription initiation site. However, the definite functionof these putative STREs requires further investigation.

Sequence analysis of theP. chrysosporium MnSOD1 hasrevealed more than 80% identity with the MnSOD genesfrom the white-rot fungiG. resinaceum, G. microsporumandA. rude. This high degree of identity strongly points to acommon evolutionary origin, despite the great physiologicaldifferences betweenP. chrysosporium and the rest of thisgroup. This finding is further supported by the predominantMnSOD activity found in five white-rot fungi (P. florida,P. chrysosporium, L. edodes, T. versicolor, G. lucidum) andone non-lignin-degrading fungus,R. solani. These white-rotfungi exhibited SOD activity which was not inhibited byKCN, strongly indicating that only MnSOD was active. Onthe other hand, the non-lignin-degrading fungusN. crassa,as well as the yeastS. cerevisiae, displayed both MnSODand CuZnSOD activities. The predominant expression ofMnSOD may be a characteristic common to the white-rotfungi.

Although SODs are the first line of defence against toxicintracellular radicals during normal metabolism or underoxidative stress, in certain eukaryotic cells containing bothSODs, MnSOD is the one which responds first to thethreat of oxygen toxicity[43]. Expression of MnSOD inP.chrysosporium cultures was maximal between 72 and 96 hof growth, as confirmed by measurements of both mRNAlevels and enzymatic activity. This probably indicates en-hanced respiration of the fungus during this stage, coincid-ing with the transition from the tropophase to the idiophase.In a liquid fermentation ofP. chrysogenum in a complex

http://www.jgi.doe.gov/index.html


medium, the level of MnSOD expression was maximal at48 h, decreasing thereafter[47]. In S. cerevisiae andC. al-bicans, MnSOD is essential for the stationary phase, andits transcription significantly increases during this phase incomplex media[41,49].

The primary sources of superoxide anion are in the mito-chondria, in which most of the respiration activities occur.Several factors regulate MnSOD gene expression in eukary-otes. Environmental conditions, iron chelators, compoundsthat increase the intracellular flux of superoxide anions, andstrong oxidants able to change the redox potential, are allknown to regulatesodA gene expression inE. coli [50]. In-creased leakage of superoxide anions and other ROS fromthe respiratory chain probably enhances the activity of Mn-SOD in the mitochondrial matrix.

Rothschild et al.[9] suggested that LIP formation inP.chrysosporium might be induced by conditions which en-hance the accumulation of ROS. Because MnSOD activityis induced by Mn2+ ions, and no MnSOD has been obtainedunder Mn2+ deficiency inP. chrysosporium, or in other mi-croorganisms, they inferred that Mn-deficient cultures ofP.chrysosporium would be more susceptible to oxidative stressthan non-deficient ones, especially due to the lack of CuZn-SOD activity. Further research is underway in our lab toinvestigate the expression of SODs inP. chrysosporium, to-gether with other antioxidant and detoxification enzymes, inrelation to ligninolytic conditions and LIP expression.

Acknowledgments

This research was supported by the Guastella Fellow-ship for Colleges of Higher Education from the Sacta-RashiFoundation. We thank Osnat Kalati for her technical assis-tance with the SOD screening in fungi.

References

[1] Gold MH, Alic M. Molecular biology of the lignin-degradingbasidiomycete Phanerochaete chrysosporium. Microbiol Rev1993;57:605–22.

[2] Reddy CA, D’Souza TM. Physiology and molecular biology ofthe lignin peroxidases ofPhanerochaete chrysosporium. FEMSMicrobiol Rev 1994;13:137–52.

[3] Bietti M, Baciocchi E, Steenken S. Lifetime, reduction potential andbase-induced fragmentation of the veratryl alcohol radical cation inaqueous solution. Pulse radiolysis studies on a ligninase mediator. JPhys Chem 1998;102:7337–42.

[4] Kersten PJ, Tien M, Kalyanaraman B, Kirk TK. The ligninaseof Phanerochaete chrysosporium generates cation radicals frommethoxybenzenes. J Biol Chem 1985;260:2609–12.

[5] Bar-Lev SS, Kirk TK. Effects of molecular oxygen on lignindegradation byPhanerochaete chrysosporium. Biochem Biophys ResCommun 1981;99:373–8.

[6] Dosoretz CG, Chen AH, Grethlein HE. Effect of oxygenationconditions on submerged cultures ofPhanerochaete chrysosporium.Appl Microbiol Biotechnol 1990;34:131–7.

[7] Leisola MSA, Ulmer D, Fietcher A. Factors affecting lignindegradation in lignocellulose byPhanerochaete chrysosporium. JBiotechnol 1984;3:97–107.

[8] Rothschild N, Hadar Y, Dosoretz CG. Ligninolytic system formationby Phanerochaete chrysosporium in air. Appl Environ Microbiol1995;61:1833–8.

[9] Rothschild N, Levkowitz A, Hadar Y, Dosoretz CG. Manganesedeficiency can replace high oxygen levels needed for ligninperoxidase formation byPhanerochaete chrysosporium. ApplEnviron Microbiol 1999;65:483–8.

[10] Zacchi L, Burla G, Zuolong D, Harvey PJ. Metabolism of celluloseby Phanerochaete chrysosporium in continuously agitated cultureis associated with enhanced production of lignin peroxidase. JBiotechnol 2000;78:185–92.

[11] Zacchi L, Morris I, Harvey PJ. Disordered ultrastructure in lignin-peroxidase secreting hyphae of the white-rot fungusPhanerochaetechrysosporium. Microbiology 2000;146:759–65.

[12] Zacchi L, Palmer JM, Harvey PJ. Respiratory pathways and oxygentoxicity in Phanerochaete chrysosporium. FEMS Microbiol Lett2000;183:153–7.

[13] Halliwell B. Oxygen and nitrogen are pro-carcinogens. Damage toDNA by reactive oxygen chlorine and nitrogen species: measurement,mechanism and the effect of nutrition. Mutant Res 1999;443:37–52.

[14] Rice-Evans C. Free radicals and antioxidants in normal andpathological processes. In: Rice-Evans C, Bruckaorfer KR, editors.Oxidative stress, lipoproteins and cardiovascular dysfunction.London: Portland Press, 1995. p. 1–32.

[15] Fridovich I. Superoxide radical and superoxide dismutases. Ann RevBiochem 1995;64:97–112.

[16] Bannister JV, Bannister WH, Rotilio G. Aspects of the structurefunction and applications of superoxide dismutase. CRC Crit RevBiochem 1987;22:111–80.

[17] Keele Jr BB, McCord JM, Fridovich I. Superoxide dismutasefrom Escherichia coli B. A new manganese-containing enzyme.Superoxide dismutases. J Biol Chem 1970;245:6176–81.

[18] Van Loon AP, Pesold Hurt B, Schatz G. A yeast mutant lackingmitochondrial manganese superoxide dismutase is hypersensitive tooxygen. Proc Natl Acad Sci USA 1986;83:3820–4.

[19] Farr SB, Dari R, Touati D. Oxygen-dependent mutagenesis inE. coli lacking superoxide dismutase. Proc Natl Acad Sci USA1986;83:8268–72.

[20] Guidot DM, McCord JM, Wright RM, Repine JE. Absenceof electron transport (Rho-0 state) restores growth of amanganese superoxide dismutase-deficientSaccharomyces cerevisiaein hyperoxia: evidence for electron transport as a major source ofsuperoxide generation in vivo. J Biol Chem 1993;268:26699–703.

[21] Larsen PL. Aging and resistance to oxidative damage inCaenorhabditis elegans. Proc Natl Acad Sci USA 1993;90:8905–9.

[22] Zhu D, Scandalius JG. The maize mitochondrial MnSODs encodedby multiple genes localized in the mitochondrial matrix oftransformed yeast cells. Free Rad Biol Med 1995;18:179–83.

[23] Reveillaud I, Niedzwiecki A, Bensch KG, Fleming JE. Expression ofbovine superoxide dismutase inDrosophila melanogaster augmentsresistance of oxidative stress. Mol Cell Biol 1991;11:623–40.

[24] Wispe JR, Warner BB, Clark JC, Dey CR, Neuman J, Glasser SW,et al. Human Mn-superoxide dismutase in pulmonary epithelial oftransgenic mice confers protection from oxygen injury. J Biol Chem1992;267:23937–41.

[25] Oeztuerk R, Bozakaya AL, Atav E, Saglam N, Tarhan L. Purificationand characterization of superoxide dismutase fromPhanerochaetechrysosporium. Enzyme Microbial Technol 1999;25:392–9.

[26] Martin JP, Dailey M, Sugarman E. Negative and positive assaysof superoxide dismutase based on hematoxylin autoxidation. ArchBiochem Biophys 1987;255:329–36.

[27] Beauchamp CI, Fridovich I. Superoxide dismutase: improvedassays and an assay applicable to acrylamide gels. Anal Biochem1971;44:276–87.


[28] Raeder U, Broda P. Rapid DNA isolation from filamentous fungi.Lett Appl Microbiol 1985;1:17–20.

[29] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratorymanual. Cold Spring Harbor, New York: Cold Spring HarborLaboratory, 1989.

[30] Gergen JP, Stern RH, Wensink PC. Filter replicas and permanentcollections of recombinant DNA plasmids. Nucleic Acids Res1979;7:2115–36.

[31] Bonfield JK, Smith KF, Staden R. A new DNA sequence assemblyprogram. Nucleic Acids Res 1995;24:4992–9.

[32] Wisconsin Package Version 10.1, Genetics Computer Group (GCG).Madison, WI.

[33] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting positions-specific gap penalties and weightmatrix choice. Nucleic Acids Res 1994;22:4673–80.

[34] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic localalignment search tool. J Mol Biol 1990;215:403–10.

[35] Quandt K, Frech K, Karas H, Wingender E, Werner T. MatIndand MatInspector—new fast and versatile tools for detection ofconsensus matches in nucleotide sequence data. Nucleic Acids Res1995;23:4878–84.

[36] Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE,Kel OV, et al. Databases on transcriptional regulation: TRANSFACTRRD and COMPEL. Nucleic Acids Res 1998;26:364–70.

[37] Amoureux MC, van Gool D, Herrero MT, Dom R, Colpaert FCA,Pauwels PJ. Regulation of metallothionein-III (GIF) mRNA in thebrain of patients with Alzheimer disease is not impaired. Mol ChemNeuropathol 1997;32:101–21.

[38] Ballance DJ. Sequences important for gene expression in filamentousfungi. Yeast 1986;2:229–36.

[39] Ruis H, Schüller C. Stress signaling in yeast. BioEssays 1996;17:959–65.

[40] Marres CA, Van Loon AP, Oudshoorn P, Van Steeg H, Grivell LA,Slater EC. Nucleotide sequence analysis of the nuclear gene codingfor manganese superoxide dismutase of yeast mitochondria, a gene

previously assumed to code for the Rieske iron–sulphur protein. EurJ Biochem 1985;147:153–61.

[41] Rhie GE, Hwang CS, Brady MJ, Kim ST, Kim YR, Huh WK, etal. Manganese-containing superoxide dismutase and its gene fromCandida albicans. Biochim Biophys Acta 1999;1426:409–19.

[42] Jeong JH, Kwon ES, Roe JH. Characterization of the manganese-containing superoxide dismutase and its gene regulation in stressresponse ofSchizosaccharomyces pombe. Biochem Biophys ResCommun 2001;283:908–14.

[43] Henkle-Duhrsen K, Tawe W, Warnecke C, Walter RD. Characteri-zation of the manganese superoxide dismutase cDNA and gene fromthe human parasiteOnchocerca volvulvus. Biochem J 1995;308:441–6.

[44] Jones PL, Kucera G, Gordon H, Boss JM. Cloning and characteri-zation of the murine manganous superoxide dismutase-encodinggene. Gene 1995;153:155–61.

[45] Wan XS, Devalalajara MN, St. Clair DK. Molecular structure andorganization of the human manganese superoxide dismutase gene.DNA Cell Biol 1994;13:1127–36.

[46] Crameri R, Faith A, Hemman S, Jaussi R, Ismail C, Menz G, et al.Humoral and cell-mediated autoimmunity in allergy toAspergillusfumigatus. J Exp Med 1996;184:265–70.

[47] Diez B, Schleissner C, Moreno MA, Rodriguez M, Collados A,Barredo JL. The manganese superoxide dismutase from the penicillinproducerPenicillium chrysogenum. Curr Genet 1998;33:387–94.

[48] Pan SM, Ye JS, Hseu RS. Purification and characterization ofmanganese superoxide dismutase fromGanoderma microsporum.Biochem Mol Biol Int 1997;42:1035–43.

[49] Flattery-O’Brien JA, Grant CM, Dawes IW. Stationary-phaseregulation of theSaccharomyces cerevisiae SOD2 gene is dependenton additive effects of HAP2/3/4/5- and STRE-binding elements. MolMicrobiol 1997;23:303–12.

[50] Hassan HM, Sun HH. Regulatory roles of Fnr, Fur, and Arcin expression of manganese-containing superoxide dismutase inEscherichia coli. Proc Natl Acad Sci USA 1992;89:3217–21.

Manganese-containing superoxide dismutase from the white-rot fungus Phanerochaete chrysosporium: its...

Documents

Transcript of Manganese-containing superoxide dismutase from the white-rot fungus Phanerochaete chrysosporium: its...