Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus

Gene 236 (1999) 169–177 www.elsevier.com/locate/gene

Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus k Ulrike Temp, Uwe Zierold, Claudia Eggert * Institute of General Microbiology and Microbial Genetics, Friedrich-Schiller University Jena, Neugasse 24, D-07743 Jena, Germany Received 27 March 1999; accepted 7 June 1999; Received by W. Martin

Abstract The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporus cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840 bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes were identified. The cDNA of lcc3-2 contains an open reading frame of 1563 bp. The deduced mature laccase protein consisted of 498 amino acids and was preceded by a signal peptide of 23 amino acids. The sequence of lcc3-2 reveals 73% similarity on the protein level to the previously characterized lcc3-1. The new laccase gene shares highest similarity to lcc1 from Trametes villosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (pI ) of 6.1 for the gene product LCC3-2 was in good agreement with the experimentally determined pI of a laccase secreted by P. cinnabarinus grown on cellulose. Transcription analysis using competitive reverse transcription (RT )PCR showed that lcc3-2 was expressed in glucose and cellulose containing cultures. However, in contrast to lcc3-1, lcc3-2 transcription was not increased in response to 2,5-xylidine. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Competitive RT-PCR; Gene regulation; Phenol oxidase; White-rot fungus

1. Introduction Laccases ( p-diphenol:O oxidoreductase; EC 2 1.10.3.2) are multi-copper enzymes that catalyze the oxidation of p-diphenols with the concurrent reduction of dioxygen to water. They are produced by the majority of white-rot fungi described to date, as well as by other fungi, insects, bacteria, and higher plants (Dean and Eriksson, 1994). Under appropriate inductive conditions, many fungi produce multiple laccase isoforms, which are encoded by gene families (Perry et al., 1993, Yaver and Golightly, 1996). Different laccase isoenzymes appear to be specific for particular metabolic, Abbreviations: A, adenosine; C, cytosine; cDNA, DNA complementary to RNA; IEF, isoelectric focusing; E0, redox potential; G, guanosine; gpd, glyceraldehyde-3-phosphate dehydrogenase; LCC, laccase; lcc, gene encoding LCC; nt, nucleotide; N, terminus amino terminus; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; pI, isoelectric point; RACE, rapid amplification of cDNA ends; RT, reverse transcription; T, thymidine. k The sequence described in this report has been deposited in EMBL/GenBank under accession no. AF123571. * Corresponding author. Tel.: +49-3641-949327; fax: +49-3641-949312. E-mail address: [email protected] (C. Eggert)

developmental or environmental situations. In addition to their involvement in lignin degradation, fungal laccases have been implicated in pathogenicity, fruit body formation, and pigmentation ( Thurston, 1994). Laccase genes have been cloned and characterized from ligninolytic and non-ligninolytic fungi, and also from plants. Studies of the structure of laccase encoding genes, as well as the mechanisms regulating laccase gene expression, will contribute to the elucidation of the roles different laccases play in specific developmental processes. Furthermore, potential applications of laccases, e.g. for the removal of lignin in pulp and paper manufacturing and for the bioremediation of xenobiotic compounds, have resulted in an increased interest in the isolation of new laccase genes and the development of efficient heterologous expression systems for large-scale production of laccases. In the white-rot fungus Pycnoporus cinnabarinus, laccase is a major component of the lignin degrading system. The laccase-encoding gene, lcc3-1, and its corresponding cDNA were cloned. The gene product corresponds to the acidic laccase (pI#3.7) previously identified as the predominant isoform secreted by 2,5-xylidine-induced cultures ( Eggert et al., 1996, 1998).

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 23 9 - 5

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This laccase is not only important for lignin degradation; it also catalyzes the synthesis of phenoxazinone pigments that give the fruiting bodies a red color ( Eggert et al., 1995). However, Southern analysis indicated the presence of a small laccase gene family in P. cinnabarinus ( Eggert et al., 1998). In order to characterize further the ligninolytic system of the fungus and understand the regulatory mechanisms controlling the expression of different laccases, a second laccase gene, lcc3-2, and the corresponding cDNA were cloned and sequenced. Transcription of lcc3-2 and lcc3-1 in the presence and absence of 2,5-xylidine was analyzed by competitive RT-PCR.

according to the manufacturers’ instructions. All chemicals and reagents were of at least analytical grade purity. 2,5-Xylidine was purchased from Tokyo Kasei Kogyo ( Tokyo, Japan). Coal-derived humic substances were prepared by alkaline extraction from German lignite. Oligonucleotide primers were obtained from MWG Biotech ( Ebersberg, Germany) and sequencing primers were synthesized at the Molecular Genetics Instrumentation Facility of the University of Georgia (Athens, GA, USA). The sequences of the oligonucleotide primers used in this study are listed in Table 1. 2.2. RNA isolation

2. Materials and methods 2.1. Organisms and reagents P. cinnabarinus PB (ATCC 200478), an isolate recovered from decaying pine wood in the vicinity of Sydney, New South Wales, Australia, was maintained as described previously ( Eggert et al., 1996). The TOPO TA cloning system including the pCR2.1 vector and Escherichia coli (INVaF ∞, One Shot@ competent cells) used for direct cloning of PCR products was purchased from Invitrogen. Unless otherwise indicated, enzymes for manipulating DNA or RNA were obtained from Boehringer or New England Biolabs, and were used

P. cinnabarinus cultures were grown for 3 days in liquid modified Dodson medium ( Eggert et al., 1996) at 30°C on a rotary shaker (125 rpm). The pH of the medium was adjusted to 4.5 with 1 N NaOH. Fungal mycelia were collected by filtration, washed twice with sterile phosphate buffer (20 mM, pH 7.0), and immediately frozen in liquid nitrogen. RNA was isolated after grinding mycelia under liquid nitrogen with the RNeasy Plant Mini kit (Qiagen). 2.3. Genomic DNA isolation Mycelia from P. cinnabarinus grown in 250 ml malt extract medium (15 g/l; pH 5.0) at 30°C for 4 days were

Table 1 Sequences and locations of oligonucleotide primers Primer

Oligonucleotide sequence

Target sequence

Location

AP P 1

5∞-GGTTTTGCCCAGTCACGACTTTTTTTTTTTTTTTTT-3∞ 5∞-ATHCAYTGGCAYGGNTTY-3∞

– 713−(intron II )–763a

P 2

5∞-RTCDATRTGRCARTGGARRAACCA-3∞

P 3 P 4 T3 anchor P 5

5∞-TGCTTCTGGACACTCCTTCCTA-3∞ 5∞-GGTTTTGCCCAGTCACGAC-3∞ 5∞-TCCCTTTAGTGAGGGTTAATTT-NH -3∞ 3 5∞-ATTAACCCTCACTAAAGGGA-3∞

poly(A) for RT degenerate; 1st Cubinding domain degenerate; 4th Cubinding domain lcc3-2 cDNA 3∞-RACE 5∞-RACE 5∞-RACE

P 6 P 7 P 8 P 9 P 10 P 11 P /P C 6 P A∞ P C∞/B∞ P B∞ P GPD1 P GPD2

5∞-GGTCTTCGCATAATCGACGTT-3∞ 5∞-ATGATCAGCATGGGCTTCCGT-3∞ 5∞-GCGATCTGGATGCATTCAAGGAC-3∞ 5∞-CGGTCCCGCATTCGTTCA-3∞ 5∞-CTTCCCAGAGATGAGAGGTCCAG-3∞ 5∞-GCGATGGCGACGAAGAGA-3∞ 5∞-GGTCTTCGCATAATCGACGTTAAGGCCGTTGATGAGGGTAG-3∞ 5∞-AGTCATGTCCAGATTCCAATCTCTCCTC-3∞ 5∞-CCGATACCGCTCAGAGATCGCCGTTGAAGTTGAAGAC-3∞ 5∞-CCGATACCGCTCAGAGATC-3∞ 5∞-CGTCCACGGCCGCTTCAA-3∞ 5∞-GTAGCCCCATTCATTATCGTA-3∞

lcc3-2 cDNA lcc3-2 gDNA lcc3-2 gDNA lcc3-2 gDNA lcc3-2 gDNA lcc3-2 gDNA lcc3-2 mimic fragment lcc3-1 cDNA lcc3-1 mimic fragment lcc3-1 cDNA Pcgpd1 Pcgpd2

2404–2427a 840–861a binds to AP – complementary to T3 anchor 1889–1909a 405–425a 2758–2780a 810–826a 568–590a 434–451a 1247–1266a 304–331b 1812−(intron IX )–1879b 2418–2426b – –

a Positions correspond to the nucleotide sequence of P. cinnabarinus lcc3-2 in Fig. 1 ( EMBL/GenBank database accession no. AF123571). b Positions correspond to the nucleotide sequence of P. cinnabarinus lcc3-1 in Eggert et al. (1998) (EMBL/GenBank database accession no. AF025481).

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harvested, washed, and frozen in liquid nitrogen as described in Section 2.2. High molecular weight genomic DNA was isolated from frozen mycelia according to Xu et al. (1994). 2.4. Isolation and cloning of lcc3-2 cDNA 3∞- and 5∞-RACE protocols were used to obtain the full-length lcc3-2 laccase cDNA sequence. Total RNA (1.0 mg) was primed with an oligo(dT )-anchor primer AP and Superscript II reverse transcriptase (Life Technologies). Degenerate primers P and P , designed 1 2 to match the first and fourth copper-binding domains conserved in laccases, were used to amplify 1200 bp fragments of the P. cinnabarinus laccase gene(s). The PCR temperature program was: 94°C for 4 min, followed by 94°C for 45 s, 53°C for 45 s and 72°C for 90 s for 35 cycles and a final extension at 72°C for 7 min. The resultant products were subcloned into pCR2.1, and six clones were sequenced. Whereas five clones were identical with the previously isolated lcc3-1, one clone corresponded to a new laccase gene. From this resulting sequence, the specific oligonucleotide primer P was 3 derived. For PCR amplification of the AP-primed cDNA with primers P and P , an aliquot (2.0 ml ) of the cDNA 3 4 was used as the template in a 25 ml reaction mixture containing Taq polymerase (Sigma). The PCR temperature program was: 94°C for 4 min, followed by 94°C for 45 s, 53°C for 45 s and 72°C for 120 s for 30 cycles, and a final extension at 72°C for 7 min. To obtain the 5∞ region, the oligonucleotide anchor T3 was ligated to the 5∞ end of the cDNA according to Troutt et al. (1992). The anchor-ligated cDNA was amplified with primer P , which was complementary to the anchor 5 sequence, and a specific primer P , using an aliquot 6 (2.0 ml ) of the ligation mixture as the template in a 25 ml reaction mixture containing Taq polymerase (Sigma). The PCR temperature program was: 94°C for 4 min, followed by 94°C for 45 s, 53°C for 45 s and 72°C for 90 s for 35 cycles, and a final extension at 72°C for 7 min. PCR products were subcloned, and at least two of the resultant clones were sequenced on both strands. 2.5. Isolation and cloning of lcc3-2 gDNA To isolate genomic lcc3-2 sequence, exact match primers P and P were used in PCR mixtures (25 ml ) 7 8 containing genomic DNA (1 mg) and Expand polymerase (Boehringer). The PCR temperature program was: 94°C for 4 min, followed by 94°C for 45 s, 60°C for 45 s and 72°C for 150 s for 35 cycles, and a final extension at 72°C for 7 min. A 2.5 kb product was subcloned, and two of the resultant clones were sequenced on both strands. Isolation of the 5∞-untranslated region of lcc3-2 was obtained by inverse PCR. P. cinnabarinus gDNA was

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digested with selected restriction enzymes, and the digested gDNA was circularized by ligation with T4 DNA ligase. Subsequently, a two-step inverse PCR was performed starting with an aliquot (2 ml ) of the ligation products in a 25 ml reaction containing Expand polymerase (Boehringer) and primers P (which was directed in 9 the 3∞-direction from nucleotides 810), and P and P 10 11 (which were directed in the 5∞-direction from nucleotides 590 and 451 respectively). The PCR temperature program for both reactions was: 94°C for 4 min, followed by 94°C for 45 s, 56°C for 45 s and 72°C for 180 s for 35 cycles, and a final extension at 72°C for 7 min. A 2.4 kb fragment was amplified from BglII-digested gDNA, subcloned, and sequenced on both strands. 2.6. Laccase transcription analysis by competitive RT-PCR 2.6.1. RNA isolation For transcription analysis, P. cinnabarinus was grown for 4 days in modified Dodson medium supplemented with glucose (5 g/l ) and cellulose (10 g/l ), respectively, at 30°C on a rotary shaker (125 rpm). 100 ml Erlenmeyer flasks containing 25 ml modified Dodson medium were inoculated with 300 ml of a P. cinnabarinus spore suspension (6×107 spores) ( Eggert et al., 1995). After 60 h, 2,5-xylidine was added to the fungal cultures to final concentrations of 5 mM or 100 mM. Control flasks without addition of 2,5-xylidine were included in the experiments. All cultures were grown in quadruplicate. In a second set of experiments, coal-derived humic acids (0.3 g/l final concentration) were added as putative laccase inducer. After 96 h, fungal mycelia were collected over gauze, washed with sterile, bidistilled water, and immediately frozen in liquid nitrogen. Total RNA was isolated by a phenol–guanidine-isothiocyanate protocol (peqGOLD RNA pure; PeqLab, Erlangen, Germany) as recommended by the manufacturer and reversely transcribed as described above. RNA was quantified both spectrophotometrically and by ethidium bromide staining after size fractionation on agarose gels under denaturing conditions. cDNA was prepared from RNA isolated from at least two flasks. The reported transcript levels in RNAs extracted from cultures grown under identical conditions varied by less than 25%. The results of competitive PCR experiments performed with RNA of the same culture were essentially identical. 2.6.2. Glyceraldehyde-3-phosphate dehydrogenase ( gpd) transcript levels As a control of RNA quantifications and cDNA syntheses, a 780 bp fragment of the P. cinnabarinus gpd gene ( Temp and Eggert, unpublished data), was amplified from all cDNAs with primers P and P in GPD1 GPD2 semiquantitative PCRs. In preliminary experiments, the optimal number of cycles had been determined to ensure

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that product abundance was evaluated in the exponential phase of the reaction. The PCR temperature program was: 94°C for 4 min, followed by 94°C for 45 s, 53°C for 45 s and 72°C for 30 s for 23 cycles, and a final extension at 72°C for 7 min. 2.6.3. lcc3-2 transcript levels For estimation of lcc3-2 transcript levels in competitive PCRs, a 338 bp mimic-fragment was generated by PCR amplification of the lcc3-2 cDNA integrated in pCR2.1 with antisense hybrid-primer P /P in combinaC 6 tion with sense primer P . The resultant mimic fragment 3 was purified by electroelution from agarose gels. It was homologous to the lcc3-2 cDNA target except for the

introduced binding site for primer P , which resulted in 6 a size reduction of 385 bp compared with the 723 bp product amplified from lcc3-2 cDNA. Fivefold serial dilutions of the mimic-fragment, to which glycogen (10 mg/ml final concentration) was added, were used as internal standards in competitive RT-PCRs. P. cinnabarinus cDNA (2 ml ) and decreasing concentrations of the lcc3-2 mimic fragment (2 ml of each dilution) were coamplified using primer pairs P and P with the PCR 3 6 temperature program: 94°C for 4 min, followed by 94°C for 45 s, 53°C for 45 s and 72°C for 30 s for 25 cycles, and a final extension at 72°C for 7 min. The size reduction of the mimic fragment allowed separation of the cDNAs and the mimic on agarose gels. Also, with

Fig. 1. Nucleotide sequence and deduced amino acid sequence of the P. cinnabarinus lcc3-2 gene. The putative signal peptide is shaded. The 5∞-end of the cDNA is marked by a filled triangle and the polyadenylation site by an open triangle. The numbers in italic type (1, 2, and 3) indicate with which of the three Cu(II ) types the residues coordinate. Residues involved in binding Cu(II ) ions are marked by single shaded boxes. Other residues with importance for the reactivity of the enzyme are indicated by black boxes. Possible glycosylation sites are underlined.

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this approach gDNA contaminations could be detected because the amplified region spans six introns (1069 bp product). Transcript concentrations were estimated by determining the concentration of competitive DNA at which the intensities of the mimic and the cDNA targets (PCR fragments) were equal. 2.6.4. lcc3-1 transcript levels The abundance of lcc3-1 transcripts was estimated using the same competitive RT-PCR strategy. The 1088 bp lcc3-1 mimic fragment was generated by PCR amplification of the lcc3-1 cDNA integrated in pCR2.1 with primers P and P /P , and purified as described A∞ C∞ B∞ above. The resultant size difference between lcc3-1 mimic and lcc3-1 cDNA target (1571 bp) was 483 bp. In competitive PCRs, lcc3-1 mimic and lcc3-1 transcripts were coamplified with primers P and P . Except for the A∞ B∞ annealing temperature of 54°C, the temperature program was as described for lcc3-2 transcript estimation. 2.7. Isoelectric focusing P. cinnabarinus cultures were grown for 6 days in modified Dodson medium supplemented with cellulose (10 g/l ) as the carbon source at 30°C on a rotary shaker (125 rpm). Mycelia and cellulose fibers were separated from the culture broth by filtration over glass fiber filters ( Whatman), and 0.45 mm membrane (Nylon, Millipore) filters. The culture filtrate (2 l ) was concentrated to approximately 4.5 ml by ultrafiltration against a 10 kDa membrane (PM 10, Amicon) and excessively rebuffered against bidistilled H O, using the same ultrafiltration 2 system. The concentrated culture filtrate (2 ml ) was loaded on an isoelectric focusing gel (IEF-PAGE ) with a gradient from pH 3–7 (125 mm×65 mm, 0.4 mm thick; Bio-Lyte, BioRad ). At the completion of the run, lanes were stained for laccase activity with 1 mM panisidine and 1 mM ABTS in 50 mM Na tartrate buffer (pH 4.5). The pI points of the laccases were determined by comparison with a protein standard mixture (Sigma).

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2.9. Nucleotide sequence accession number The nucleotide sequence of P. cinnabarinus lcc3-2 reported in this paper has been deposited in the EMBL/GenBank database under accession no. AF123571.

3. Results and discussion 3.1. Structure of lcc3-2 We isolated a new P. cinnabarinus laccase cDNA from RNA extracted from mycelia that had grown on cellulose for 3 days without addition of 2,5-xylidine by a PCR strategy employing 5∞- and 3∞-RACE protocols. According to the nomenclature suggested in Eggert et al. (1998), it was designated lcc3-2. The longest cDNA clone of the P. cinnabarinus lcc3-2 was 1905 bp exclusive of the poly(A)-tail, and contained a 1563 bp open reading frame (Fig. 1). Sequence comparison of the genomic and cDNA sequences of the P. cinnabarinus lcc3-2 gene revealed that ten introns interrupted the coding region. The introns ranged in size from 52 to 69 bp, and matched the consensus sequence predicted for the 5∞-splice sites of eukaryotic genes, GT(a/g)NG(c/t) (Ballance, 1986), except for intron I (A at position 5 and G at position 6) and intron IV (A at position 5). In general, 3∞-splice sites followed the consensus sequence (c/t)N(c/t)AG, with the exception of position 1 in intron IV (G), intron 7 (A), and intron X (A), and position 3 (A) in intron IIX. The overall exon–intron structure of the lcc3-2 gene is identical to that of the P. cinnabarinus lcc3-1 gene, which also contains ten introns ( Eggert et al., 1998). The number of introns and their position is highly conserved in basidiomycete laccase genes [for a recent comparison see Karahanian et al. (1998)].

2.8. DNA sequencing and sequence analysis Nucleotide sequences were determined by using Taq polymerase cycle sequencing and an automated DNA sequencer (ABI Mdl. 377, Perkin–Elmer, Foster City, CA) in the Molecular Genetics Instrumentation Facility at the University of Georgia. All cloned DNAs were sequenced on both strands, and the encoded amino acid sequence was predicted using Gene Runner (Hastings Software, Hastings-on-the-Hudson, NY ). Sequence similarities were calculated by alignment of laccase gene sequences from EMBL/GenBank databases using the CLUSTAL V algorithm (MegAlign; DNASTAR, Madison, WI, USA).

Fig. 2. Isoelectric focusing (pH 3 to 7) electrophoresis of concentrated culture fluid from P. cinnabarinus cultures grown on cellulose for 6 days. Lanes: M, protein marker (protein stained with Coomassie Brilliant Blue); 1, activity stained laccase with 1 mM p-anisidine and 1 mM ABTS in 50 mM Na tartrate buffer (pH 4.5). Only part of the gel is shown.

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Sequence analysis of an additional 1400 bp 5∞-upstream region of P. cinnabarinus lcc3-2 isolated by inverse PCR from circularized, BglII-digested gDNA revealed typical putative promoter elements. One TATA box was located 253 nucleotides upstream of the start codon ( Fig. 1), and two CAAT boxes were found at nt-600 and nt-1027 (not shown). In a Southern analysis, a 450 bp probe homologous to lcc3-1, which spanned the region from the start of the open reading frame to the second Cu-binding domain, hybridized to four fragments of BamHI-digested P. cinnabarinus gDNA ( Eggert et al., 1998). EcoRI- and HindIII-digested gDNA probed under the same, low stringency conditions, showed signals for three fragments and a single 8.6 kb fragment respectively. The fact that both lcc3-1 and lcc3-2 have no restriction sites for these enzymes within the range of the probe could indicate the presence of, as yet, unidentified laccase genes, which would have to be located on the single 8.6 bp HindIII fragment. Alternatively, as P. cinnabarinus is a dikaryon, allelic variants of lcc3-1 and lcc3-2 could contribute to the observed hybridization pattern.

3.2. Characterization of the deduced protein The lcc3-2 exons code for a protein with 521 amino acids including a 23-residue secretory signal peptide typical for extracellular enzymes, which was predicted by the algorithm of Nielsen et al. (1997) (Fig. 1). The molecular masses derived from the unprocessed and mature LCC3-2 are 58 404 Da and 56 021 Da respectively. P. cinnabarinus LCC3-2 contains eight potential N-glycosylation sites (Asn–Xxx–Ser/Thr) (Gavel and von Heijne, 1990), at positions 77, 190, 231, 240, 285, 314, 357, and 365 of the deduced protein ( Fig. 1), which vary from the seven possible N-glycosylation sites found in LCC3-1 ( Eggert et al., 1998). Typical fungal laccases are glycosylated proteins with a carbohydrate content of about 10 to 20% (Dean and Eriksson, 1994; Thurston, 1994). All of the expected Cu( II ) ligands (ten histidine residues and one cysteine residue) conserved in laccases were present in the lcc3-2 coding sequence of P. cinnabarinus, and are indicated by numbers in Fig. 1 on the basis of whether they coordinate with the type-1, type-2, or

Fig. 3. Phenogram representing the phylogenetic relationships between laccases. The analysis was carried out by using the CLUSTAL program (DNASTAR). Amino acid residues in conserved positions, which have been shown to be critical for the reactivity of the enzyme, are given for each laccase. Coriolus hirsutus ( Kojima et al., 1990), Trametes villosa ( Yaver and Golightly, 1996), basidiomycete 20197 (Mansur et al., 1997), P. cinnabarinus ( Eggert et al., 1998), basidiomycete PM1 (Coll et al., 1993), Trametes versicolor (Jo¨nsson et al., 1995), Cerioporiopsis subvermispora ( Karahanian et al., 1998), Phlebia radiata (Saloheimo et al., 1991), Pleurotus ostreatus (Giardina et al., 1995), Schizophyllum commune (Genbank accession no. AB015758), Rhizoctonia solani ( Wahleithner et al., 1996), Agaricus bisporus (Perry et al., 1993), Podospora anserina (FernandezLarrea and Stahl, 1996), Neurospora crassa (Germann and Lerch, 1986), Cryphonectria parasitica (Choi et al., 1992), Liriodendron tulipifera ( U73103-106), Poplar trichocarpa (Ranocha et al., 1999), Nicotinia tabacum ( Kiefer-Meyer et al., 1996), Acer pseudoplatanus (LaFayette et al., 1995), Cucumis sativus ascorbate oxidase (J04494), Aspergillus nidulans (Aramayo and Timberlake, 1990).

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type-3 Cu(II )-center of the laccase active site (Messerschmidt, 1997). Another residue ten amino acids downstream of the conserved cysteine involved in the coordination of type-1 Cu(II ) can be either leucine, methionine, or phenylalanine. The presence of a phenylalanine in that position has been postulated to be a prerequisite for the high redox potential (E0) of fungal laccases (Messerschmidt, 1997). However, recent advances in our understanding of the laccase active site, which include the analysis of the three-dimensional structure of a laccase (Ducros et al., 1998), as well as spectroscopic and kinetic studies of site-directed mutagenized, recombinant laccases ( Xu et al., 1998), have questioned the connotation that this ligand is essential for the observed E° differences of laccases. A ‘‘leucine– glutamate–alanine’’ tripeptide motif adjacent to the last conserved histidine identified in two ‘‘high E0’’ laccases versus a ‘‘valine–serine–glycine’’ identified in ‘‘low E0’’ laccases was critical for the reactivity of the enzyme ( Xu et al., 1998). The presence of the ‘‘leu–glu–ala’’ motif and the phenylalanine residue in positions +6 to +8 and +10 respectively of the conserved cysteine suggest that both lcc3-1 and lcc3-2 are ‘‘high E0’’ laccases. After separating concentrated cell-free fluid from P. cinnabarinus cultures grown on cellulose without addition of 2,5-xylidine on isoelectric focusing gels, a second laccase isoenzyme with a pI of approximately 5.4 was detected in addition to the acidic laccase previously identified as LCC3-1 ( Eggert et al., 1998) ( Fig. 2). In accordance with our previous results (Eggert et al., 1995), this isoform was not detectable in 2,5-xylidineinduced cultures. A direct relationship between the new laccase and the deduced gene product of lcc3-2 has not been established. However, the pI of the new secreted laccase resembles the pI of 6.1 calculated for the mature LCC3-2.

3.3. Amino acid sequence similarities with other laccases A comparison of the amino acid sequence of the P. cinnabarinus laccase encoded by lcc3-2 with laccase sequences available in databases demonstrates that P. cinnabarinus lcc3-2 is most closely related to lcc1 from Trametes villosa and lcc2 from the unidentified basidiomycete CECT 20197 (both 75% similarity), followed by lcc3-1 from P. cinnabarinus (73%). The derived laccase protein sequences aligned with CLUSTAL were used for a phylogenetic analysis (Fig. 3). The evolutionary distance between the laccases clearly shows the closest relationship to be between basidiomycete, ascomycete, and plant laccases. The notable exception is the laccase of A. nidulans expressed during asexual development, which is less related to fungal and plant laccases than is ascorbate oxidase from cucumber. Even though P. cinna-

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Fig. 4. Transcription analysis of lcc3-1 and lcc3-2 by competitive RT-PCR in total RNA extracted from 4-day-old cultures of P. cinnabarinus containing glucose as the carbon source. After 48 h of cultivation, 0, 5, or 100 mM 2,5-xylidine were added. (A) Separation of equal amounts (0.8 mg) of total RNA on 1.2% formaldehyde–agarose gels. (B) Comparison of gpd-transcript levels after separation of PCR products (5 ml ) on ethidium-bromide-stained agarose (1.0%) gels. Analysis of lcc transcription by competitive RT-PCR. Transcript abundance was estimated after separating the PCR products (5 ml ) on ethidiumbromide-stained agarose (1.2%) gels. (C ) lcc3-2: each cDNA (2 ml ) was coamplified with decreasing concentrations of the lcc3-2 mimic fragment using primers P and P . (1) aliquots (2 ml ) of the mimic 3 6 were added from 10−6, 5×10−7, and 10−7 dilutions. (D) lcc3-1: each cDNA (2 ml ) was coamplified with decreasing concentrations of the lcc3-1 mimic fragment using primers P and P . Aliquots (2 ml ) of A∞ B∞ the mimic were added from (2) 5×10−5, 10−5 and 5×10−6 dilutions, or (3) 10−3, 5×10−4, and 10−4 dilutions. L: 100 bp ladder (Gibco). Landmark intensity bands correspond to 600 bp, 1500 bp, and 2072 bp. Estimated equimolar concentrations of target and mimic fragment are indicated by an asterisk.

barinus and S. commune are both placed within the family Schizophyllacea (http://www.ncbi.nlm.nih.gov/ Taxonomy), their laccases do not share high similarity. The fact that lcc3-1 and lcc3-2 are more closely related to laccases from white-rot basidiomycetes could indicate a functional relationship among ‘‘high E0’’ laccases from ligninolytic basidiomycetes.

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3.4. Transcription analysis of lcc3-1 and lcc3-2 After quantifying the total RNA concentrations (Fig. 4A), synthesis of equal amounts of cDNA was confirmed by PCR amplification of the constitutively expressed P. cinnabarinus gpd (Fig. 4B). Competitive PCR was used to analyze lcc transcript levels in 4-dayold cultures after addition of 0, 5, or 100 mM 2,5-xylidine. Whereas lcc3-2 transcripts remained at a constant level in all cultures (Fig. 4C ), lcc3-1 transcript levels were enhanced approximately 50-fold in cultures supplemented with either 5 mM and 100 mM 2,5-xylidine (Fig. 4D). This correlated with 17- and a 27-fold increases in specific laccase activity measured in the corresponding culture supernatants (data not shown). The enhancing effect of 2,5-xylidine on lcc3-1 transcript abundance and/or mRNA stability was also observed when the fungus was grown on cellulose as the carbon source. Addition of coal-derived humic acids (0.3 g/l ) as a model for more complex, lignin-like compounds had a similar effect on lcc3-1 transcript levels (30-fold increase), whereas lcc3-2 transcripts remained at a constant level (data not shown). According to these results, LCC3-1 is the major laccase produced during growth of the fungus in the presence of lignin. Even when P. cinnabarinus was grown in the absence of phenolic compounds, lcc3-1 constituted the predominant laccase, which significantly exceeded lcc3-2 on the mRNA as well as on the protein level. An increasing of laccase transcription in response to 2,5-xylidine has been reported for several white-rot fungi, including T. versicolor (Collins and Dobson, 1997), and C. subvermispora ( Karahanian et al., 1998). Peculiarly, in A. bisporus, only transcription of the less abundant laccase, lcc2, is enhanced by substrate components, whereas the major laccase, lcc1, is expressed at constant levels (Smith et al., 1998). Like P. cinnabarinus lcc3-2, lcc3 from basidiomycete CECT 20197 is not increased in response to phenolics, but it is repressed by glucose (Mansur et al., 1998). Further work is required to clarify which factors govern differential expression of laccases within gene families and whether there are conditions during the life cycle of the fungus when expression of low-abundant laccases is elevated.

Acknowledgement We thank R. Machmerth for technical assistance.

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