Mycol. Res. 109 (1): 115–124 (January 2005). f The British Mycological Society
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DOI: 10.1017/S0953756204001376 Printed in the United Kingdom.
Screening of basidiomycetes and xylariaceous fungi for lignin peroxidase and laccase gene-specific sequences
Stephen B. POINTING1*, Anna L. PELLING2, Gavin J. D. SMITH1, Kevin D. HYDE1 and C. Adinaryana REDDY3 1
Department of Ecology and Biodiversity, University of Hong Kong, Pokfulam Road, Hong Kong, People’s Republic of China. Department of Biochemistry, University of Hong Kong, Sassoon Road, Hong Kong, People’s Republic of China. 3 Department of Microbiology and Molecular Genetics, and NSF Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824-4320, USA. E-mail :
[email protected] 2
Received 21 March 2003; accepted 16 August 2004.
Lignin peroxidase and laccase gene-specific PCR primers were used to screen 38 diverse basidiomycetes and xylariaceous fungi. Lignin peroxidase gene-specific sequences were obtained for basidiomycetes only and were highly divergent. Possession of laccase genes was relatively widespread among basidiomycetes, and is shown for the first time in Xylariaceae. All sequences were highly conserved with no variation resulting in changes to predicted amino acid sequence. Those basidiomycetes shown to possess lignin peroxidase and laccase genes also produced the enzyme in vitro. Conversely none of the xylariaceous fungi shown to possess laccase genes were able to do so, whilst others decolorized Poly R yet yielded no PCR amplicons.
INTRODUCTION Lignocellulose is the most abundant biopolymer in nature, comprising the polysaccharides cellulose and hemicellulose, plus a polyphenolic heteropolymer, lignin (Eaton & Hale 1993). The ability to catabolise cellulose and hemicellulose is fairly common as a primary metabolic process among fungi and other organisms, and occurs under a range of environmental conditions. Conversely, lignin is extremely recalcitrant and is mineralized in an obligately aerobic oxidative process, carried out appreciably only by the white-rot fungi during secondary metabolism (Boominathan & Reddy 1992). This recalcitrance, possession of ligninolytic ability among relatively few species, and annual lignin production estimated at 20.3r1012 kg annually (Bassham 1975) contribute to lignin degradation being regarded as the rate limiting step to carbon turnover in lignocellulose dominated environments. White-rot fungi variously secrete one or more of three extracellular enzymes that are essential for lignin degradation, and combine with other processes to effect lignin mineralization. They are often referred to as lignin-modifying enzymes or LMEs. The three enzymes * Corresponding author.
comprise two glycosylated heme-containing peroxidases, lignin peroxidase (lip, E.C. 1.11.1.14) and Mn dependant peroxidase (Mnp, E.C. 1.11.1.13) (Orth & Tien 1995), and a copper-containing phenoloxidase, laccase (lcc, E.C. 1.10.3.2) (Thurston 1994). LME production occurs during secondary metabolism and is subject to complex regulation. Limited nutrient levels induce LMEs although responses to nutrient and other factors vary between taxa (Wariishi et al. 1992, Hatakka 1994, Reddy & D’Souza 1994, Thurston 1994, Orth & Tien 1995). Nutrient nitrogen levels, mediator compounds and required-metal (i.e. Mn2+ for Mnp, Cu2+ for lcc) concentrations variously affect transcription levels of lip genes (e.g. Li et al. 1994), Mnp genes (e.g. Ruiz-Duenas et al. 1999) and lcc genes (e.g. Collins & Dobson 1997, Palmieri et al. 2000) in whiterot taxa including Phanerochaete chrysosporium, Pleurotus sp., and Trametes versicolor. Such complex and varied physiology suggests in vitro studies designed to assess enzyme production or lignin mineralization may not truly reflect which taxa are capable of lignin mineralization in the environment (Pointing 1999). Nevertheless, such assays remain the most frequently used technique for screening fungi for LME-producing ability due to their low cost and simple methodology. The in vitro production of LMEs has
Ligninolytic enzyme genes in diverse fungi been recorded for several species of white-rot basidiomycetes using both defined liquid growth medium (Hatakka 1994) and undefined agar growth medium (Leung & Pointing 2002) although different responses were observed between taxa. Certain xylariaceous and diatrypacous wood decay ascomycetes are also capable of white-rot decay (Pointing, Parungao & Hyde 2003) and in vitro LME production has been recorded for some taxa using undefined agar growth medium (Abe et al. 1989, Pointing et al. 2003). Genes encoding Lip, Mnp (e.g. Gold & Alic 1993, Reddy & D’Souza 1994) and lcc (e.g. Mansur et al. 1997) have been characterized for P. chrysosporium and a few other white-rot basidiomycetes. Attempts at molecular screening for the presence of LME genes have involved hybridization studies using oligonucleotide probes for lignin peroxidase (Kimura, Asada & Kuwahara 1990, Varela, Martinez & Martinez 2000) and PCR screening for lignin peroxidase, Mn-dependant peroxidase (Chen et al. 2001) and laccase (D’Souza, Boominathan & Reddy 1996). The variability in results compared to in vitro enzyme production assays highlights the need for further study among a large number of taxa, and refinement of molecular screening techniques. The aim of this study was to carry out PCR-based screening for possession of genes encoding LMEs together with enzyme production tests, for a number of white-rot basidiomycetes and xylariaceous fungi from tropical forests, including many previously untested species.
MATERIALS AND METHODS Organisms and culture conditions The 18 basidiomycetes and 20 xylariaceous taxa were used in this study are detailed in Table 1. Cultures were maintained on potato-dextrose agar (PDA ; Difco, Kansas City) at 25 xC in darkness. For basidiomycetes, heterokaryons were used for all experiments. Genomic DNA extraction Mycelium was harvested from agar plates after 7 days growth. Genomic DNA was isolated by standard phenol-chloroform extraction followed by ethanol precipitation, and resuspended in 100 ml TE buffer. PCR amplification Genomic DNA from each of the fungal isolates was used as the template in PCR amplification reactions consisting of : 10r PCR buffer (Promega, Madison, WI), 10 ml; template DNA (1–2 ng mlx1), 20 ml ; MgCl2 (50 mM), 3 ml ; dNTPs (10 mM) (Promega), 2 ml each; forward and reverse primers (10 mM) (Invitrogen, Carlsbad), 5 ml each ; Taq polymerase (5 U mlx1) (Promega), 0.5 ml; sterile deionized distilled water to
116 100 ml. Sterile mineral oil (100 ml) was added as an overlay to each PCR reaction mixture. PCR amplification was carried out in a thermal cycler (Geneamp 2700, Applied Biosystems, Foster City, CA) with an initial cycle of: denaturation (3 min at 94 x), annealing (1 min at 50 x) and extension (1 min at 72 x). A further 45 cycles of denaturation (1 min at 94 x), annealing (1 min at 50 x) and extension (1 min at 72 x) were carried out. The following primers encoding catalytic and conserved domains for each gene were used : Laccase, LccF (forward primer) 5k – CA(T/C) TGG CA(T/C) GGN TT(T/C) TT(T/C) CA ; LccR (reverse primer) 5k – (A/ G)TG (A/G)CT (A/G)TG (A/G)TA CCA (A/G)AA (G/A/T/C)GT (D’Souza et al. 1996). Lignin peroxidase, LipF (forward primer) 5k – (G/C)C(G/T/C) AAC AT(T/C) GG(T/C) CT(T/C) GAC GA ; LipR (reverse primer) 5k – TC(G/C) A(G/T/C)G AAG AAC TG(G/ C) G(A/T)G TC (derived from Reddy & D’Souza 1998). Manganese dependant peroxidase, MnpF (forward primer) 5k – G(A/C)(G/A) ATG GCC TTC (A/ G)(A/G)T TC(T/C)T ; MnpR (Reverse primer) – TTA (G/T)GC AGG (G/A)CC (G/A)T(T/C) GAA CT (Bogan et al. 1996). After amplification, PCR products were electrophoresed on a 1 % w/v agarose gel with a 100 bp ladder (Gibco BRL, New York) run in a separate lane. DNA was visualized under UV light after EtBr staining. Cloning and sequencing of PCR-amplified products Bands corresponding to PCR amplicons were excised from gels and purified using the GFX gel band purification kit (Amersham Biosciences, Chalfont, Bucks). DNA was cloned into a pDrive vector using the Cloning Plus kit (Qiagen, Cologne). Plasmids were transformed into competent cells and transformants selected on the basis of LccZ a-peptide expression, according to manufacturers instructions (Qiagen). The cloned lcc and lip gene fragments were sequenced using M13 forward (x20) and M13 reverse primers, with an ABI PRISM 377 automated DNA Sequencer (Applied Biosystems) according to manufacturers instructions. Sequence assembly, alignment and analysis Sequences obtained for each primer were used to create a consensus sequence using BioEdit version 5.0.9 (Hall 1999). During assembly, individual bases from each sequence were checked against the original fluorescence signal. Sequence identity was then confirmed using the GenBank BLASTn search (Altschul et al. 1997). Consensus sequences, and those obtained from GenBank, were then aligned using ClustalW (Thompson et al. 1997), as implemented in BioEdit, before being aligned manually. Neighbour-joining (NJ) analysis of the data, with additional sequences as listed in Table 2, was conducted with PAUP* 4.0b8 (Swofford 2001) using the HKY85 model (Hasegawa,
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Table 1. Lignin modifying enzyme production by basidiomycetes and Xylariaceae.
Fungus and source
HKUCCa (unless stated)
Poly Rb
Azure bb
Syringaldazineb
Basidiomycota Cyathus sp. Ganoderma lucidum Hymenochaete sp. Marasmius sp. Microporus sp. Mycena sp. Panus sp. Peniophora sp. Pereniporia medulla-panis Phanerochaete chrysosporium P. cinnabarinus P. coccineus P. sanguineus P. cinnabarinus P. sanguineus Stereum sp. Trametes versicolor (brown) T. versicolor (purple)
Hong Kong Austria Hong Kong Hong Kong Hong Kong Hong Kong Hong Kong Australia Australia USA Australia Solomon Isl. Sri Lanka Australia Thailand Hong Kong Hong Kong Hong Kong
4069 CBS 251.61 4124 4068 4070 4067 4062 4108 4115 IMI 284010 CBS 311.33 CBS 355.63 CBS 614.73 4066 4122 4127 4063 4081
+ + 0 0 + 0 + 0 + + + + + + + + + +
0 + 0 0 + 0 + 0 + + 0 0 0 0 + + + +
+ + 0 + + + + + + + + + + + + + + +
Xylariaceae Biscognauxia doidgaeae Hypoxylon bovei var. macrospora Hypoxylon fragiforme Hypoxylon monticulosum Hypoxylon nitens Hypoxylon sp. nov. Hypoxylon sp. Xylaria cfr pallida Xylaria hypoxylon Xylaria schweinitzii Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp. Xylaria sp.
Hong Kong Australia Denmark Hong Kong Australia Hong Kong Hong Kong Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia Australia
3178 2814 1022 3217 2817 3179 3196 2842 3716 2894 2778 2787 2797 2807 2822 2830 2880 2885 2782 2896
0 + + + + 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 + 0 + 0 0 0 0 0 0 + 0 0
a HKUCC, University of Hong Kong culture collection; CBS, Centraalbureau voor Schimmelcultures, utrecht; IMI, CABI Bioscience, Egham. b +, positive reaction; 0, negative reaction.
Kishino & Yano 1985). Two different analyses were conducted for both datasets. The lcc data was analyzed with the full sequence (intron plus exon) and with the exon only (D’Souza et al. 1996). The lip data was analyzed using the full sequence, and also with the removal of inserts common only to larger fragments. Unrooted NJ trees were constructed using TREEVIEW (Page 1996). Translation of DNA sequence data Nucleic acid sequences for the lcc gene fragments were translated into their predicted amino acid sequences using BioEdit. These were aligned as described above. Determination of intron and exon boundaries for lcc sequences was made according to previously published sequences (D’Souza et al. 1996).
Enzyme production assays The following qualitative tests were used to assess production of LME’s ; Poly R agar, comprised glucose (Sigma, St Louis) 0.2 % w/v, Peptone (Difco) 0.1 % w/v, yeast extract (Difco) 0.01 % w/v, Poly R 478 (Sigma) 0.02 % w/v, agar (Difco) 1.6 % w/v. A positive result for production of LME’s was indicated by clearance of the purple growth medium (Boominathan & Reddy 1992). Azure B agar, comprised glucose 0.2 % w/v, Peptone 0.1 % w/v, yeast extract 0.01 % w/v, Azure B (Sigma) 0.02 % w/v, agar 1.6 % w/v. Azure B is a substrate for lignin peroxidase only (Archibald 1992). A positive result was indicated by clearance of the blue growth medium. Syringaldazine well test, cultures were grown for 7 d on agar growth medium comprising glucose 0.2 % w/v, Peptone 0.1 % w/v, yeast extract
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Table 2. Fungal strains, their GenBank accession numbers, and summary of PCR amplification results.
Fungus Lignin peroxidase Panus sp. Perenniporia medulla-panis Phanerochaete chrysoporium H8 P. chrysoporium H8 P. chrysosporium P. chrysosporium H10 P. chrysosporium H2 P. chrysosporium H8 Pycnoporus coccineus P. sanguineus Trametes versicolor T. versicolor Laccase Coriolus versicolor Cyathus sp. Ganoderma lucidum G. lucidum Hypoxylon sp. Lentinula edodes Lentinus tigrinus Panus sp. Panus sp. Panus sp. Phlebia brevispora P. radiata PM1 Pycnoporus cinnabarinus P. cinnabarinus P. coccineus P. sanguineus P. sanguineus Trametes versicolor T. versicolor Xylaria sp. Xylaria sp. Xylaria sp.
Culturea
GenBank accession no.b
Fragment size ( bp)
HKUCC 4062 HKUCC 4115 FPL BKM-F-1767 FPL BKM-F-1767 IMI 284010 FPL BKM-F-1767 FPL BKM-F-1767 FPL BKM-F-1767 CBS 355.63 CBS 614.73 HKUCC 4063 HKUCC 4081
AY243873* AY243869* M27401 M27884 AY243870* X55343 AF140062 AF140063 AY243868* AY243867* AY243871* AY243872*
500 544 530 530 538 528 531 533 431 534 429 429
DBR HKUCC 4069 CBS 251.61 FPL 58537 HKUCC 3196 FPL RA-3-2-E CAR HKUCC 4062 HKUCC 4062 HKUCC 4062 FPL HHB-7099-Sp ATCC 64658 CECT 2971 CBS 311.33 HKUCC 4066 CBS 355.63 CBS 614.73 HKUCC 4122 HKUCC 4063 ATCC 12679 HKUCC 2782 HKUCC 2797 HKUCC 2880
DBR AY243862* AY243858* DBR AY243866* DBR DBR AY243859* AY243860* AY243861* DBR X52134 Z12156 AY243853* AY243855* AY243854* AY243852* AY243856* AY243857* DBR AY243863* AY243864* AY243865*
199 195 195 198 203 144 144 249 198 148 197 201 206 194 194 203 199 203 194 199 220 194 201
a ATCC, American Type Culture Collection; CAR, private collection of C. A. Reddy; CECT, La Coleccio´n Espan˜ola de Cultivos Tipo; DBR, Sequence information taken from D’Souza et al. (1996); FPL, USDA Forest Products Laboratory; HKUCC, Hong Kong University Culture Collection. b *, sequences obtained in this study.
0.01 % w/v and agar 1.6 % w/v. Wells (5 mm diam) were cut in the agar and filled with a syringaldazine solution (0.1 % w/v). A positive result for laccase production was indicated by formation of a pink colour in and around wells (Pointing 1999). All assays were performed in triplicate, with a positive result for each fungus recorded only in the case of all three tests yielding positive reactions.
RESULTS PCR amplification of lip and lcc-specific sequences The lip primers were expected to yield amplicons of y450 bp. PCR products were obtained for 39 % of basidiomycetes screened (Fig. 1A), but none of the xylariaceous taxa yielded products. Two distinct size amplicons were generated, of y450 bp and 550 bp. The
450 bp PCR product was obtained for Pycnoporus coccineus, Trametes versicolor (HKUCC 4063) and T. versicolor (HKUCC 4075), whilst the 550 bp PCR product was obtained for Pycnoporus sanguineus (CBS 614.73), Perenniporia medulla-panis, and Phanerochaete chrysosporium. Panus sp. yielded two PCR products, one each corresponding to the 450 bp and 550 bp amplicons observed for other taxa. All amplicons had high sequence similarity (by BLASTn search) to published GenBank lip sequences. The lcc primers were expected to generate amplicons of y200 bp. PCR products of this size were obtained for 50% of basidiomycetes and 20% of xylariaceous fungi (Fig. 1B). The PCR generated a single band of y200 bp in all cases except for Panus sp. where three bands of y150, 200 and 250 bp were obtained. All amplicons had high sequence similarity (by BLASTn search) to published GenBank lcc gene sequences.
S. B. Pointing and others (A)
(B)
1
2
3 4
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1
2 3 4
5
6
7
8
5
6 7 8
9
10 11 12 13 14 15
Fig. 1. (A) Amplicons achieved using lignin peroxidase genespecific PCR primers in screening diverse basidiomycetes and xylariaceous fungi : Lanes : 1, 100 bp DNA ladder ; 2, Pycnoporus sanguineus (CBS 614.73) ; 3, P. coccineus ; 4, Perenniporia medulla-panis ; 5, Phanerochaete chrysosporium ; 6, Trametes versicolor (HKUCC 4063) ; 7, T. versicolor (HKUCC 4081) ; 8, Panus sp. (B) Amplicons achieved using laccase gene-specific PCR primers : Lanes : 1, 100 bp DNA ladder; 2, Pycnoporus sanguineus (CBS 614.73); 3, P. cinnabarinus (CBS 311.33) ; 4, P. coccineus ; 5, P. cinnabarinus (HKUCC 4066) ; 6, P. sanguineus (HKUCC 4122); 7, T. versicolor (HKUCC 4063) ; 8, T. versicolor (HKUCC 4081); 9, Panus sp. ; 10, Ganoderma lucidum ; 11, Cyathus sp.; 12, Xylaria sp. (HKUCC 2782) ; 13, Xylaria sp. (HKUCC 2797) ; 14, Xylaria sp. (HKUCC 2880) ; and 15, Hypoxylon sp. (HKUCC 3196).
Five of the basidiomycetes produced PCR products for both lip and lcc-specific PCR, these were Pycnoporus coccineus, P. sanguineus (CBS 614.73), Trametes versicolor (HKUCC 4063), T. versicolor (HKUCC 4075) and Panus sp. Satisfactory amplification using primers designed from mnp sequences of Phanerochaete chrysosporium (BKM-F 1767) could not be obtained for any of the taxa used in this study and suggests they are too specific to detect Mnp genes among diverse taxa, as a result of their design based upon a single isozyme of Mnp in Phanerochaete chrysosporium. Sequence analysis of PCR amplicons All amplicons were successfully sequenced, and sequences lodged in the GenBank database with accession numbers as shown in Table 2. Lignin peroxidase : The final lip dataset alignment was 563 bp. One hundred and seventeen character positions were excluded for the second analysis. Two main groups are present in the NJ analysis of the full lip dataset (Fig. 2A). Group A consists of all the Phanerochaete chrysosporium GenBank sequences of approx. 530 bp. Group B contains all 3 y430 bp sequences from
Pycnoporus coccineus and Trametes versicolor, plus the 501 bp product of Panus sp. The remaining three taxa, all with approx. 540 bp fragments, did not group together. Phanerochaete chrysosporium (HKUCC 284010) is basal to Groups A and B while Pycnoporus sanguineus (614.73) and Perenniporia medulla-panis are at the base of the tree. Fig. 2B shows the NJ tree of the reduced lip dataset. Relationships differ slightly compared to the previous analysis. Group B remains intact with Phanerochaete chrysosporium (284010) basal to this group. Group A, however, is now organized differently, with 3 of its members now sister groups to Group B, with P. chrysosporium (284010), and the remaining two members basal to the rest of Group A. The position of Pycnoporus sanguineus (614.73) and Perenniporia medulla-panis did not vary. The final alignment of the laccase lcc dataset was 251 bp long, with the exon in positions 1–123 and 228–258 (intron position 124–227 bp). NJ analysis of the full lcc sequence data revealed 3 main groups that have been further divided into subgroups (Fig. 3A). Group A contains 13 taxa, including species of Cyathus, Coriolus/Trametes, Ganoderma, Hypoxylon, Lentinus, Panus (148 and 219 bp), Phlebia and Xylaria, plus the unidentified fungus PM1. Within this group there is no obvious grouping of lcc sequences based on taxonomic relationships among the fungi. For example, the two Ganoderma sequences do not group together, and Xylaria sp. (2782) is seperated from the other Xylariaceae sequences in Group A. Group B contains six basidiomycete species including two species of Pycnoporus, Trametes versicolor (4063) and the third Panus sp. amplicon (198 bp). Group C consists of the remaining two species of Pycnoporus plus Xylaria sp. (2797). Pycnoporus sanguineus (614.73) is at the base of the tree. Fig. 3B shows the NJ analysis of the lcc exon sequence. Tree topology varies somewhat from that in Fig. 3B. Cyathus sp. and Xylaria sp. (2782) from Group A1 are now clustered with Group C at the base of the tree. The remaining members of Group A1 still group together but are now associated with Group B1. These two groups contain the three Panus sp. lcc fragments. Groups A1 and A2 no longer cluster together as Group B2 has moved into a basal position to Groups A1 and B1. No patterns of lcc fragment association based on taxonomy are apparent, but the exon only analysis has moved the Lentinus, Panu, and Phlebia lcc fragments into closer association. In vitro enzyme production The Poly R assay for production of LME’s produced unambiguous positive results for 78 % of basdiomycetes and 25 % of Xylariaceae tested. The azure B assay specific for production of lip yielded positive results for 50 % of basidiomycetes but no Xylariaceous taxa. The syringaldazine assay for lcc production produced positive results for 94 % of basidiomycetes
Phanerochaete chrysosporium AF140062
Trametes versicolor AY243871
Trametes versicolor AY243872
B Phanerochaete chrysosporium X55343
Phanerochaete chrysosporium AF140063
Pycnoporus coccineus AY243868
A
Panus sp. AY243873
Phanerochaete chrysosporium M27884 Phanerochaete chrysosporium AY243870
Ligninolytic enzyme genes in diverse fungi
(B )
(A)
Phanerochaete chrysosporium M27401 Phanerochaete chrysosporium AF140062 Trametes versicolor AY243871 Phanerochaete chrysosporium X55343 Trametes versicolor AY243872 B
Phanerochaete chrysosporium AF140063
Pycnoporus coccineus AY243868
A
Phanerochaete chrysosporium M27884 Panus sp. AY243873
Phanerochaete chrysosporium AY243870
Phanerochaete chrysosporium M27401
Pycnoporus sanguineus AY243867
Pycnoporus sanguineus AY243867
Perenniporia medulla panis AY243869
Perenniporia medulla panis AY243869
Fig. 2. Neighbour Joining trees of lip partial gene sequence data produced using the HKY85 measure of distance. No outgroup was specified. (A) Full sequence. (B) With 117 characters excluded.
120
Panus sp. AY243859
(B)
Lentinula edodes DBR
Xylaria sp. AY243863 Lentinus tigrinus DBR
Panus sp. AY243860 B1
Panus sp. AY243859 A1
Cyathus sp. AY243862
Lentinus tigrinus DBR
Phlebia brevispora DBR
Phlebia brevispora DBR
Panus sp. AY243861
Panus sp. AY243861
Xylaria sp. AY243865
Phlebia radiata DBR
Ganoderma lucidum DBR
Pycnoporus coccineus AY243854
Hypoxylon sp. AY243866
Pycnoporus sanguineus AY243856
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(A)
A1
B2
Trametes versicolor DBR
A2
Trametes versicolor AY243857
Coriolus versicolor DBR
Ganoderma lucidum AY243858
Ganoderma lucidum AY243858
PM1 DBR
PM1 DBR
Trametes versicolor DBR
Pycnoporus coccineus AY243854
Coriolus versicolor DBR Xylaria sp. AY243865
Pycnoporus sanguineus AY243856 Trametes versicolor AY243857
A2
B2
Ganoderma lucidum DBR Hypoxylon sp. AY243866
Phlebia radiata DBR
Pycnoporus cinnabarinus AY243853
Panus sp. AY243860 B1
Lentinula edodes DBR
Xylaria sp. AY243864
Pycnoporus cinnabarinus AY243853
Pycnoporus cinnabarinus AY243855
Xylaria sp. AY243864
C
C
Cyathus sp. AY243862 A1
Pycnoporus cinnabarinus AY243855
Xylaria sp. AY243863
Pycnoporus sanguineus AY243852
Pycnoporus sanguineus AY243852
Fig. 3. Neighbour Joining trees of lcc partial gene sequence data produced using the HKY85 measure of distance. No outgroup was specified. (A) Intron and Exon. (B) Exon only.
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Ligninolytic enzyme genes in diverse fungi Cyathus sp. AY243862 Ganoderma lucidum AY243858 Ganoderma lucidum DBR Hypoxylon sp. AY243866 Lentinula edodes DBR Lentinus tigrinus DBR Panus sp. AY243859 Panus sp. AY243860 Panus sp. AY243861 Phlebia brevispora DBR P. radiata DBR PM1 DBR Pycnoporus cinnabarinus AY243853 P. cinnabarinus AY243855 P. coccineus AY243854 P. sanguineus AY243852 P. sanguineus AY243856 Trametes versicolor AY243857 T. versicolor DBR Xylaria sp. AY243864 Xylaria sp. AY243865
122
HWHGFFQHGTNWADGPAFVNQCPIASGNSFLYDFTVPDQAGTFWYHSH HWHGFFQKGTNWADGPAFVNQCPIATGHSFLYDFQVPDQAGTFWYHSH HWHGFFQKGTNWADGPAFVNQCPIASGNSFLYDFQVPDQSGTFWYHSH HWHGFFQEGTNWADGPAFVTQCPIASGDSFLYDFRVPDQAGTFWYHSH HWHGFFQKTTNYADGVSFVSQCPIVANHSFMYDFQVPDQAGTFWYHSH HWHGFFQHGTAWADGTAFVTQCPIQPFNAFLYNFTAVGQAGTFWYHSH HWHGFFQKGTNWADGPASVNQCPVATNHSFLYQFSSQGQPGTFWYHSH HWHGFFQRGTNWADGPAFVTQCPIVANDSFLYNFTVPNQAGTFWYHSQ HWHGFFQKTTAWADGPAFVTQCPIISGDSFLYDFNVPDQAGTFWYHSH HWHGFFQHTTNWADGPAFVTQCPIAPGNSFLYDFTVPDQAGTFWYHSH HWHGFFQHGTNWADGPAFINQCPIASGDSFLYNFQVPDQAGTFWYHSH HWHGFFQHGTNWADGPAFVNQCPISTGHAFLYDFQVPDQAGTFWYHSH HWHGFFQHGTNWADGAAFVNQCPIATGNSFLYDFSVPDQAGTFWYHSH HWHGFFQHGTNWADGAAFVNQCPIATGNSFLYDFSVPDQAGTFWYHSH HWHGFFQHGTNWADGVSFVNQCPIASGHSFLYDFQVPDQAGTFWYHSH HWHGFFQHGTNWADGAAFVNQCPIATGNSXLYDXSVPDQAGTFWYHSH HWHGFFQHGTNWADGVSFVNQCPIASGHSYLYDFQVPDQAGTFWYHSH HWHGFFQHGTNWADGVPFINQCPIASGHSFLYDFQVPDQAGTFWYHSH HWHGFFQKGTNWADGPAFINQCPISSGHSFLYDFQVPDQAGTFWYHSH HWHGFFQHGTNWADGAAFVNQCPIATGNSFLYDFSVPDQAGTFWYHSQ HWHGFFQKGTNWADGPAFVNQCPISTGHSFLYDFQVPDQAGTFWYHSH
Fig. 4. Alignment of predicted amino acid sequences from PCR-amplified lcc gene fragments. Invariant amino acids are shown in bold, those with >50 % match are highlighted. Xylaria sp. (AY243863) is omitted due to sequence divergence from all others.
and 15 % of Xylariaceae (Table 1). All Xylariaceae produced weaker reactions in all tests than basidiomycetes. In comparing PCR screening to enzyme production results, all lignin peroxidase-producing basidiomycetes also possessed lip-like sequences, although Pycnoporus coccineus and P. sanguineus were unable to produce lignin peroxidase despite possessing lip-like sequences. None of the xylariaceous fungi tested were capable of producing lignin peroxidase or possessed lip-like sequences. All basidiomycetes that produced laccase also possessed lcc-like sequences. None of the laccase producing xylariaceous fungi yielded positive results in the lcc gene-specific PCR screening. Those xylariaceous fungi shown to possess lcc gene-specific sequences did not produce the enzyme during in vitro assays.
DISCUSSION This study is the first report of lip gene-specific sequences in Pycnoporus sanguineus, Pycnoporus coccineus, Perenniporia medulla-panis and Panus sp. Interestingly members of the genus Pycnoporus have not previously been shown to produce any detectable lignin peroxidase during in vitro cultivation in defined growth medium (Eggert, Temp & Eriksson 1996, Pointing, Jones & Vrijmoed 2000). This is reflected in studies with other fungi, where the possession of liplike genes and RT-PCR evidence of their transcription was not associated with any detectable lignin peroxidase production for Phanerochaete sordida and Ceriporiopsis subvermiposa (Rajakumar et al. 1996). This is also the first report of laccase gene-specific sequences for all species of the genus Pycnoporus, and for the genus Cyathus. Importantly, laccase gene-specific
sequences in the xylariaceous ascomycetes Xylaria sp. and Hypoxylon sp. are reported for the first time. This supports the view that xylariaceous fungi are also capable of white-rot decay, and complements recent evidence of wood delignification by some xylariaceous taxa (Pointing et al. 2003). Few other studies have screened fungi for the presence of LME genes. Southern blot screening was used to identify restriction fragments in Phanerochaete chrysosporium, Bjerkandera adusta and Coriolus consors with sequence similarity to a probe designed from known lip sequences (Kimura et al. 1990). A second southern blot screening revealed nine known white-rot basidiomycetes gave a hybridization signal with an oligonucleotide probe based upon the H8 isozyme of lignin peroxidase for Phanerochaete chrysosporium (Varela et al. 2000). Screening using PCR-based assays has been attempted in two studies. The presence of lcc genes was demonstrated among nine white-rot fungal strains using the same primers as our study. Two species were common to this and the present study, and in both cases these fungi (Ganoderma lucidum and Trametes versicolor) generated Lcc gene-specific sequences of y200 bp. They also revealed a second PCR product for Ganoderma lucidum of 144 bp, which was also observed for Gloeophyllum trabeum, Grifola frondosa, Lentinula edodes and Lentinus tigrinus (D’Souza et al. 1996). A band of this size was produced in our study only by Panus sp. (cfr Lentinus) although in our study this fungus also generated lcc bands of 200 and 250 bp. The Panus sp. in our study was tentatively identified from rDNA-sequence data rather than morphology since it was isolated as vegetative mycelium from rotting wood (GenBank accession no. AY187277). Another study screened a range of ectomycorrhizal
S. B. Pointing and others basidiomycetes for the presence of lip and mnp genes (Chen et al. 2001). This revealed lip-like gene fragments of varying size were widespread whilst relatively few taxa producing amplicons during PCR with mnpspecific primers. The lip sequences grouped according to size class, even after inserts have been accounted for. The lip fragments obtained for the strain of Phanerochaete chrysosporium used in this study had little affinity to H2, H8 and H10 lip genes sequenced from other Phanerochaete chrysosporium isolates. This may reflect the range of different primers used by various authors (Reddy & D’Souza 1998). High sequence diversity among lip genes has been shown among Phanerochaete chrysosporium lip genes (Reddy & D’Souza 1994) and our study suggests this is also the case for lip genes among diverse white-rot basidiomycetes. It is not known whether ligninolytic xylariaceous fungi possess lip genes with divergent sequences that could not be detected with the PCR primers employed. Future studies where PCR-primers for lip and Mnp genes encompass critical residues in the protein products (such as the approach used for lcc genes) will shed more light on diversity of lip and Mnp genes among varied taxa. There was no grouping among lcc sequences on the basis of fungal taxonomy, even between such divergent groups as ascomycetes and basidiomycetes. This implies very high conservation of this region among lcc genes, which is unsurprising considering the region encodes amino acids critical to catalytic function of the enzyme (D’Souza et al. 1996) and it is a relatively short gene fragment. The translation table (Fig. 4) shows that predicted lcc amino acid sequence is, despite nucleic acid variation, highly conserved among the taxa we have studied, including the ascomycetes. The one exception was Xylaria sp. (AY243863) whose partial lcc gene sequence was highly divergent from all others. Since this affected predicted translation of the catalytic region for the enzyme we assume this may be a nonfunctional gene. The additional fragments obtained for Panus sp. varied only by number and size of intron sequences, exon sequence was identical. These may be non-functional pseudogenes (Rajakumar et al. 1996), or if they represent separate gene copies and lcc overexpression occurs as a result, this may explain the extremely rapid dye-decolorization rates observed for this fungus during in vitro enzyme production assays. Recent studies suggest that multiple lcc, lip and Mnp genes may be differentially expressed by white-rot fungi (Janse et al. 1998, Zhao & Kwan 1999, Soden & Dobson 2001) and so this fungus may be ligninolytic over a wider physiological range than other white-rot fungi. This is also supported by enzyme production assays where apparent nitrogen-deregulation of LME production by this fungus has been recorded (Leung & Pointing 2002). The results for enzyme production assays in this study were largely unsurprising for basidiomycetes, and results for individual fungi compared well to their
123 reported LME physiology. One point of interest was the decolorization of Azure B, a substrate specific to lip, by Pycnoporus sanguineus (HKUCC 4122) since members of this genus have been previously assumed not to produce lip (Eggert et al. 1996, Pointing et al. 2000). In all cases, in vitro enzyme production by Xylariaceae appeared weak in comparison to basidiomycetes. This may reflect comparatively low levels of LME production by xylariaceous fungi and evidence from lignin solubilization rates in wood substrates supports this (Abe 1989, Worral, Anagnost & Zabel 1997, Pointing et al. 2003), where lignin solubilization typically occurs at much lower levels than for basidiomycetes. Since no data for LME physiology in xylariaceae is available we employed test methodology that has been optimized for basidiomycete LMEs and it is also possible that conditions were not optimal for enzyme production by these fungi, although our molecular data suggests high similarity between basidiomycete and xylariaceous lcc gene sequences. Future studies focusing on factors influencing expression of LME genes will improve our knowledge in this area.
ACKNOWLEDGEMENTS This research was supported in part by the Hong Kong Research Grants Council, grant number HKU 7235/99M awarded to SBP and CAR. We wish to thank Kelly L. Y. Lau and Marianne S. Castro for technical assistance.
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