Molecular characterization of the basidiomycete isolate Nematoloma frowardii b19 and its manganese peroxidase places the fungus in the corticioid genus Phlebia

INTRODUCTION

Basidiomycetous wood- and litter-degrading white-rot fungi are unique in their ability to decompose the heterogeneous plant polymer lignin by using an array of extracellular lignin-modifying enzymes (LMEs): lignin, manganese and versatile peroxidases (EC 1.11.1.13–16), and laccases (EC 1.10.3.2). Recently, there has been great interest in the potential application of white-rot fungi and their LMEs, in particular manganese peroxidases (MNPs), and laccases in biopulping, biobleaching and bioremediation of toxic compounds in soil and waters (Breen & Singleton, 1999; Hatakka, 2001; Baldrian, 2006).

The South American isolate Nematoloma (Hypholoma) frowardii b19 (Nf b19) is a potent producer of MNP, also on a larger scale for commercial enzyme production, and an efficient degrader of natural aromatic polymers like lignin and humic substances (Hofrichter & Fritsche, 1997; Hofrichter et al., 1999). In addition, the MNP of Nf b19 is capable of mineralizing different aromatics directly, such as trinitrotoluene and polycyclic hydrocarbons (PAHs) (Sack et al., 1997; Hofrichter et al., 1998). Similarities between the lignin polymer degradation patterns and LME profiles of Nf b19 and other white-rot basidiomycetes were previously observed (Hofrichter & Fritsche, 1997) but extensive genetic studies of the fungus or the molecular characterization of its MNPs, or any other LMEs, have not been carried out.

It has been estimated that about 20 % of the fungal gene sequences deposited in public databases (DDBJ, EMBL, GenBank) are misidentified and even more are poorly annotated (Bridge et al., 2003; Nilsson et al., 2006), usually due to incorrect taxonomic identification of the fungus. Taxonomic uncertainty is mostly caused by lack of distinguishable fruiting body, or failures in morphological identification and during the fungal isolation procedure. To avoid more confusion in fungal phylogeny, we believe it is vital to systematically perform comparative functional gene cloning and characterization studies for those fungal strains, such as the Nf b19 basidiomycete, that are stored in microbial culture collections and reported for use in biotechnological applications like enzyme production.

In this study, we describe the ORF of the gene (Nf mnp2) encoding the main MNP isozyme (MNP2) of isolate Nf b19. Molecular characterization of the Nf b19 MNP2, and fragments of its MNP3, three lignin peroxidase (LIP) and two laccase encoding genes, as well as of the ribosomal SSU 18S RNA and internal transcribed spacer ITS1+5.8S+ITS2 sequences used as phylogenetic markers, suggests that isolate Nf b19 in fact belongs to the white-rot corticioid genus Phlebia. According to the ITS phylogeny, Nf b19 is positioned close to the clade of the species Phlebia acerina, which is next to the P. radiata clade.

Our data imply a need for systematic reidentification of isolate Nf b19, in view of its evident molecular relatedness to Phlebia spp., which are classified in the family Corticiaceae (order Aphyllophorales, class Homobasidiomycetes) (Hibbett & Thorn, 2001) rather than in the previously proposed genus Nematoloma (Hypholoma) belonging to the family Strophariaceae (order Agaricales, class Homobasidiomycetes).

METHODS

RESULTS AND DISCUSSION

Here we describe the complete ORF of the MNP2-encoding gene (mnp2), and deduced amino acid sequences of genomic fragments of another MNP-encoding gene (mnp3), as well as three LIP-, two laccase- and one GAPDH-encoding gene of the basidiomycete isolate Nf b19.

The degenerate primer pairs that were targeted at conserved codon regions, and the genome-walking approach, aided in obtaining the genomic ORF clone of Nf b19 mnp2. The nucleotide sequence with description of intron–exon structure is deposited in GenBank (accession EF491855, Table 2⇓). The beginning of the predicted mature MNP2 was identical to the experimentally determined N-terminal peptide sequence of the Nf b19 MNP2 enzyme (M. Hofrichter and others, unpublished results) excluding the 23 aa leader peptide. Comparison of the primary structure of Nf b19 MNP2 reveals 96 % amino acid identity to the MNP2 of Phlebia radiata 79 (gene Pr mnp2, Table 2⇓, Uniprot accession Q70LM3) with differences within 15 amino acids (Fig. 1⇓) that are apparently not involved in enzyme catalysis or haem coordination.

The splicing positions of the seven introns were also identical to those detected within Pr mnp2 (Hildén et al., 2005). According to the gene and primary protein structure, Nf b19 mnp2 belongs to the classical long MNPs within group B of fungal haem peroxidases (Hildén et al., 2005; Martínez, 2002), where the so far cloned MNPs of the white-rot basidiomycetes Ceriporiopsis subvermispora (four mnp genes), Dichomitus squalens (two genes), Phanerochaete chrysosporium (four genes, five in the genome) and Phanerochaete sordida (three genes), and the long MNPs from Physisporinus (Ceriporiopsis) rivulosus (gene mnpA) (Hakala et al., 2006), Phlebia sp. MG60 (two genes) and Phlebia radiata 79 (gene mnp2) (Hildén et al., 2005) also belong (Fig. 2⇓). These fungal mnp genes contain six or seven highly conserved short introns at similar positions (Hildén et al., 2005; Hakala et al., 2006).

Phlebia radiata 79 (Pr 79, ATCC 64658) was isolated in Finland as an efficient lignin-degrading white-rot fungus, and was shown to produce three MNP and three LIP isoforms (Moilanen et al., 1996; T. K. Lundell. and others, unpublished results). Recently, we have cloned and characterized the Pr 79 mnp2, mnp3, lip1, lip3 and lip4 genes (Hildén et al., 2005, 2006). The five lignin-modifying-peroxidase-encoding genes as well as two laccases (Pr lac1 and lac2) are concomitantly expressed when the fungus is grown on its natural substrates, either hardwood or softwood (Hildén et al., 2006; Mäkelä et al., 2006).

In view of the high level of sequence identity between Nf b19 mnp2 and Pr mnp2, we looked for the presence of other peroxidase- and laccase-encoding genes in isolate Nf b19 as previously found in Pr 79. This approach led to partial cloning of mnp3, lip1, lip3, lip4, lac1, lac2 and gapdh gene fragments using PCR primer pairs designed for amplification of the respective genes from Pr 79. Within all these Nf b19 gene fragments (Table 2⇑), introns were at identical positions and similar in length as in the respective Pr 79 genes. Moreover, nucleotide sequence similarity was over 80 % between the Nf b19 gene fragment and the corresponding region in the Pr 79 gene (Table 2⇑).

Translated ORF regions of genomic Nf b19 mnp3, lip1, lip3, lip4 and gapdh fragments showed 98 % amino acid identity to the predicted Pr 79 peroxidases and GAPDH (Fig. 3⇓). The translated 51 aa of the lac1 fragment was 100 % identical to the respective region in Pr Lac1 (Uniprot accession Q01679) whereas the 68 aa sequence of the Nf b19 lac2 fragment was 89 % identical to Pr Lac2 (Uniprot accession Q0KHD1).

To more thoroughly investigate the phylogenetic relationship between these two seemingly unrelated basidiomycetous isolates, according to their original fruiting body morphology and taxonomic classification to two distinct orders and families under the class Homobasidiomycetes (Hibbett & Thorn, 2001), the nuclear ribosomal SSU 18S rDNA and more variable ITS (includes ITS1, 5.8S and ITS2) regions were amplified from Nf b19 and Pr 79 with the universal fungal primers NS1 and NS6, and ITS1 and ITS4 (Table 1⇑), respectively, and cloned and sequenced.

The SSU and LSU rRNA-encoding genes are highly conserved and universally present in all living organisms, permitting phylogenetic comparisons among distantly related species (Berbee & Taylor, 2001). The ITS1 and ITS2 regions, in contrast, can have more nucleotide variations since their transcripts are excised from the final rRNA fragments. Therefore, the ITS sequence including both ITS1 and ITS2, which are separated by the conserved short 5.8S rRNA, has been commonly used to infer phylogenetic relationships of closely related species as well as to assess the variability of a population, e.g. of geographically distant isolates (ecotypes).

The amplified and cloned partial rDNA 18S and ITS Nf b19 products (Table 1⇑), yielding GenBank accessions EF491865 for the 18S and EF491864 for the ITS sequence, respectively, showed the highest level of similarity to corresponding sequences from Phlebia species according to fasta search (). This notion was further confirmed by amplifying and cloning also the 18S and ITS regions of Pr 79: the resulting DNA fragments were of similar size to those obtained for Nf b19 (sequence accession EF491866 for Pr 79 18S, and EF491867 for Pr 79 ITS) (Table 1⇑). While submitting the sequences, we noticed recent, unpublished sequence deposits of the same Pr 79 (ATCC 64658) (accession AY946267 for 18S, and DQ056859 for ITS) that were 100 % and 99.9 % identical with our partial Pr 79 18S and ITS sequences, respectively.

The 18S and ITS sequences were further analysed by molecular evolutionary computing methods. Ribosomal SSU 18S partial (1324 nt) sequences of species belonging to the genera Phlebia, Phlebiopsis and Phanerochaete of the order Aphyllophorales, family Corticiaceae, and Hypholoma (Nematoloma) species of the order Agaricales, family Strophariaceae, were compared by applying both minimum-evolution and maximum-parsimony approaches (Fig. 4⇓). The tree topologies were similar with both methods. The 18S sequence of the heterobasidiomycete yeast-like Cryptococcus (Filobasidiella) neoformans was used as an outgroup. The 18S tree reveals close positioning of Nf b19 to the Phlebia radiata clade branch, nearest to Phlebia sp. strain DABAC9, which is not given any species-level identity. The latter fungus was recently isolated by plating aqueous extracts from a soil that was heavily contaminated with PAHs (D'Annibale et al., 2006).

However, the more extensive ITS trees constructed with all the available ITS1-5.8S-ITS2 sequences of good quality and 520–560 nt in length from Phlebia spp. and members of closely related genera (Fig. 5⇓) imply that Nf b19 may be either (i) another more diverged isolate belonging to the species Phlebia acerina, or (ii) a completely new Phlebia species positioned between the P. acerina and P. radiata clades. To finally clarify the taxonomic position of the Nf b19 isolate, it will be necessary to complement our molecular studies by classical fungal genetics, e.g. using mating experiments (Kauserud et al., 2006).

Our ITS trees indicated furthermore that Phlebia sp. DABAC9 (D'Annibale et al., 2006) is another isolate of the species P. acerina (Fig. 5⇑). Five of the six ITS sequences retrieved for P. radiata collapsed within one branch including our Pr 79 ITS, implying that also the P. radiata CBS, AFTOL and Japanese isolates are of the same origin as our P. radiata 79 (ATCC 64658).

It has recently been demonstrated that strains of Phlebia acerina together with Phlebia lindtneri and several Phlebia brevispora isolates are capable of degrading chlorinated dibenzo-p-dioxins (Kamei et al., 2005; Kamei & Kondo, 2005). Moreover, the same authors have reported the ability of the P. lindtneri isolate USDA GB1027 to biotransform chloronaphthalenes and other PAHs (Mori et al., 2003). Interestingly in this respect, the basidiomycete Nf b19, which was found and isolated in Bariloche (Argentina), the Phlebia sp. DABAC from Italy, and six strains of P. acerina from Japan, which all possess promising degradative capabilities concerning the removal of hazardous organopollutants, are most closely related to the P. radiata clade (Fig. 5⇑). Accordingly, the recently isolated saline-tolerant fungus Phlebia sp. MG-60 (Kamei et al., 2007) and P. lindtneri USDA GB1027, both Japanese isolates, branch next to the P. radiata and P. acerina clades. As noted before in an ITS-phylogenetic study on Phanerochaete spp. (De Koker et al., 2003), the genus Phlebia is noticeably polyphyletic, which is also evident from our ITS and 18S trees outside the P. acerina, P. radiata and P. brevispora clades (Figs 4⇑ and 5⇑).

According to the 18S and ITS sequence phylogeny, the only other representative of the genus Nematoloma, N. longisporum isolate AFTOL 1893, clusters with strains of the genus Hypholoma (Figs 4⇑ and 5⇑). In the class Homobasidiomycotina, the genus Hypholoma (Nematoloma) belongs to the order Agaricales, family Strophariaceae, whereas the genus Phlebia is classified to the order Aphyllophorales, family Corticiaceae (Hibbett & Thorn, 2001).

Previous reports on the efficiency of Pr 79 and Nf b19 in decomposition and mineralization of synthetic lignin (DHP) (Lundell et al., 1990; Moilanen et al., 1996; Hofrichter et al., 1999) and similarities between the catalytic properties of MNP2 of Nf b19 (Hofrichter et al., 1998), and MNP2 and MNP3 of Pr 79 (Karhunen et al., 1990; Hofrichter et al., 2001; T. K. Lundell and others, unpublished results) can now be explained by the evident taxonomic proximity of the fungal isolates, implying favourable features for environmental biotechnology. These enzymic data are furthermore supported by very recent cloning of two additional long MNP-encoding genes from another Phlebia isolate, Phlebia sp. MG-60 (Kamei et al., 2007). The latter MNP2 and MNP3 show high sequence identity and phylogenetic branching next to the Nf b19 MNP2 and P. radiata MNP2 (Fig. 2⇑).

Since the basidiomycete Nf b19 was originally isolated from a typical agaric fruiting body growing on wood (Hofrichter & Fritsche, 1997), it can be assumed that both N. frowardii and the b19 Phlebia sp. strain were present in the decaying log, but only the latter organism survived during the cultivation and preservation procedures. According to the molecular identification on protein-encoding genes (mnp, lip, lac, gapdh), and ribosomal 18S rRNA and ITS sequences, we recommend designating the basidiomycete isolate as Phlebia sp. Nf b19 until a systematic reidentification of the fungus has been accomplished.