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A new polyene pyrone, aurovertin E (2), was isolated along with aurovertin B (1) from the culture mycelia of the basidiomycete Albatrellus confluens. Their structures were elucidated on the basis of spectroscopic studies including 2D NMR experiments. This is the first example of the occurrence of aurovertins in basidiomycetes.
The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150-year-old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100-m2 sampling plots located along a 120-m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered approximately 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.
A new exocellular laccase was purified from the basidiomycete C30. LAC2 is an acidic protein (pI = 3.2) preferentially produced upon a combined induction by copper and p-hydroxybenzoate. The spectroscopic signature (UV/visible and EPR) of this isoform is typical of multicopper oxidases, but its enzymatic and physico-chemical properties proved to be markedly different from those of LAC1, the constitutive laccase previously purified from the same organism. In particular, the LAC2 kcat values observed for the oxidation of the substrates syringaldazine (kcat = 65 600 min-1), ABTS (2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (kcat = 41 000 min-1) and guaiacol (kcat = 75 680 min-1) are 10-40 times those obtained with LAC1 and the redox potential of its T1 copper is 0.17 V lower than that of LAC1 (E degrees = 0.73 V). This is the first report on a single organism producing simultaneously both a high and a low redox potential laccase. The cDNA, clac2, was cloned and sequenced. It encodes a protein of 528 amino acids that shares 69% identity (79% similarity) with LAC1 and 81% identity (95% similarity) with Lcc3-2 from Polyporus ciliatus (AF176321-1), its nearest neighbor in database. Possible reasons for why this basidiomycete produces, in vivo, enzyme forms with such different behaviors are discussed.
A metabolic mechanism for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris has been proposed on the basis of biochemical analyses of glucose metabolism. There was a strong correlation between glucose consumption and oxalate production. Oxalic acid was found to accumulate in the culture fluid in about 80% of the theoretical yield or about 5-fold, on the basis of the fungal biomass harvested. The results clearly indicate that glucose was not completely oxidized to CO(2) by the tricarboxylic acid (TCA) cycle but converted mainly to oxalate. The determination of the 12 enzymes concerned has revealed the occurrence of the unprecedented metabolic coupling of the TCA and glyoxylate cycles that support oxalate biosynthesis. In this metabolic system, isocitrate lyase (EC ), together with oxaloacetase (EC ), was found to play a pivotal role in yielding oxalate from oxaloacetate via the acetate-recycling routes. Moreover, malate dehydrogenase (EC ), with an extraordinarily high activity among the enzymes tested, was shown to play an important role in generating NADH by oxidation of malate to oxaloacetate. Thus, it is proposed that the wood-rotting basidiomycete acquires biochemical energy by oxidizing glucose to oxalate.
Patterns of mating and mitochondrial DNA (mtDNA) inheritance were investigated for the Basidiomycete, Coprinus cinereus in order to better understand the relationship of reproductive biology and mtDNA evolution in fungi. Results showed that the unique mating system of basidiomycetes can lead to the formation of mitochondrial mosaics (i.e., colonies composed of sectors differing in mtDNA). Mitochondria do not migrate along with nuclei during mating. Intracellular mixed or recombinant mtDNA molecules were not observed. Interestingly, it was found that mating asymmetry, caused by nonreciprocal nuclear migration, may be an important part of the reproductive biology of C. cinereus.
The 5S rRNA sequences from the basidiomycetes or fungi imperfecti Rhizoctonia crocorum, Rhizoctonia hiemalis, Exobasidium vaccinii, Trichosporon oryzae, Tilletia controversa, Tilletiaria anomala, Dacrymyces deliquescens and Coprinus radiatus were determined. With the exception of Exobasidium, these sequences conform to the association previously found between septal pore type and sequence. The sequence from the supposed ascomycete anamorph Rhizotonia hiemalis clearly is allied with basidiomycete sequences.
Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.
The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.
The phenotypic and genetic heterogeneity of the basidiomycetous yeast species Rhodosporidium kratochvilovae was investigated in a group of recent isolates and collection strains. A polyphasic taxonomic approach was followed which included micromorphological studies, nuclear staining, determination of sexual compatibility, physiological characterization, comparison of electrophoretic isoenzyme patterns, PCR fingerprinting, determination of mol% G+C, DNA-DNA reassociation experiments and 26S and ITS rDNA sequence analysis. The results allowed a more natural circumscription of the species, both from the genetic and phenotypic perspectives. The relationships with anamorphic species of the genus Rhodotorula were studied and isolates previously identified as Rhodotorula glutinis were found to belong to Rhodosporidium kratochvilovae. Other isolates included in the study were found to represent members of Rhodotorula glutinis var. dairenensis. Rhodosporidium kratochvilovae was found to include heterothallic strains, besides those already known to be self-sporulating. A total of 17 isolates, which were found to belong to this species, were heterothallic, self-sporulating and anamorphic strains. It is anticipated that integrated polyphasic studies of basidiomycetous yeasts will provide a more coherent classification system and the basis for accurate identification schemes, which in turn are essential for detailed ecological studies.
The sequence of the small subunit nuclear ribosomal DNA (18S rDNA) was determined for seven selected species of the teliospore-forming yeasts and Filobasidiaceae in the basidiomycetous yeasts. A phylogenetic tree, including published reference sequences, was inferred from 1623 sites which could be unambiguously aligned. The molecular phylogeny, using a chytridiomycete as an outgroup, divided the eight basidiomycetous yeasts into two groups which correlated well with both septal ultrastructure (simple pore or dolipore) and cellular xylose (present or absent). The first group included the teliospore-forming yeasts Rhodosporidium toruloides and Leucosporidium scottii, and Erythrobasidium hasegawianum, which is currently a member of the Filobasidiaceae. The second group was formed by the filobasidiaceous yeasts, comprising a Cystofilobasidium capitatum/Leucosporidium lari-marini/Mrakia frigida branch, and a Filobasidium floriforme/Filobasidiella neoformans branch. Our molecular data support the principal chemotaxonomic and ultrastructural evidence, which indicates a very close affinity between C. capitatum and L. lari-marini.
Benzaldehyde and its metabolic intermediates were effectively degraded by the brown-rot basidiomycetes Tyromyces palustris and Gloeophyllum trabeum. The pathway of benzaldehyde degradation was elucidated by the identification of fungal metabolites produced upon the addition of benzaldehyde and its metabolic intermediates. The oxidation and reduction occurred simultaneously, forming benzyl alcohol and benzoic acid as major products. Hydroxylation reactions, which seemed to be a key step, occurred on benzaldehyde and benzoic acid, but not on benzyl alcohol, to form corresponding 4-hydroxyl and 3,4-dihydroxyl derivatives. 1-Formyl derivatives were oxidized to 1-carboxyl derivatives at several metabolic stages. All of these reactions resulted in the formation of 3,4-dihydroxybenzoic acid. This was further metabolized via the decarboxylation reaction to yield 1,2,4-trihydroxybenzene, which may be susceptible to the ring-fission reaction. Ring-U-14C-labelled benzaldehyde and benzoic acid were effectively mineralized, clearly indicating that the brown-rot basidiomycetes are capable of metabolizing certain aromatic compounds to CO2 and H2O, despite the fact that brown-rot fungi cannot degrade polymeric lignin. Inhibitor experiments, using hydroxyl radical scavengers, catalase and cytochrome P450 inhibitors, strongly suggested that the aromatic hydroxylation reactions found in the brown-rot fungi are catalysed by intracellular enzyme(s), but not by Fenton-reaction-derived hydroxyl radicals.
The small subunit nuclear ribosomal DNA (18S rDNA) sequence was determined for twelve species of basidiomycetous anamorphic yeasts, i.e. three species of Udeniomyces, seven species of Bullera, Cryptococcus albidus and Phaffia rhodozyma. For phylogentic analysis, these sequences were aligned with published sequences for 36 other fungal species. Molecular phylogenetic analysis of maximum likelihood and parsimony showed that the 44 species of basidiomycetes analysed were divided into three major lineages. The ballistosporous yeast genera Udeniomyces and Bullera were clearly separated. On the phylogenetic tree, Udeniomyces megalosporus, U. puniceus and U. piricola showed a very close relationship with one another, and composed a lineage with Mrakia frigida, P. rhodozyma and Cystofilobasidium capitatum at high bootstrap confidence level. On the other hand, eight species of Bullera made lineages with selected species of Tremella (Tremellaceae), Filobasidium and Filobasidiella (Filobasidiaceae), Cryptococcus albidus and Trichosporon cutaneum. The molecular phylogeny deduced from the 18S rDNA sequence showed a possibility of heterogeneity among the species of Bullera at the generic level.
Relative carbon isotope ratio ([delta]13C values) of primary and secondary products from different compartments of annual plants, pine needles, wood, and decomposing Basidiomycetes have been determined. An enrichment in 13C was found for storage tissues of annual plants, because of the high level of the primary storage products sucrose and starch; however, the enrichment was even greater in leaf starch. All of these compounds had the same relative 13C enrichment in positions 3 and 4 of glucose. Secondary products in conifer needles (lignin, lipids) were depleted in 13C by 1 to 2 [per mille (thousand) sign] relative to carbohydrates from the same origin. Air pollution caused a small decrease in [delta]13C values; however, the relative content of plant products, especially of the soluble polar compounds, was also affected. Decomposing fungi showed a global accumulation of 13C by 4[per mille (thousand) sign] relative to their substrates in wood. Their chitin was enriched by 2[per mille (thousand) sign] relative to the cellulose of the wood. Hence, Basidiomycetes preferentially metabolize "light" molecules, whereas "heavy" molecules are preferentially polymerized. Our results are discussed on the basis of a kinetic isotope effect on the fructose-1,6-bisphosphate aldolase reaction and of metabolic branching on the level of the triose phosphates with varying substrate fluxes.
Aqueous extracts of select members of the Basidiomycetes and Deuteromycetes (Fungi Imperfecti) were evaluated for the presence of shared allergenic determinants using skin prick and radio-allergosorbent test (RAST) inhibition. Twenty adults with perennial symptoms of rhinitis, with or without asthma, were skin-prick tested with six species of Deuteromycetes and seven species of Basidomycetes. Positive weal-and-flare reactivity to Pleurotus ostreatus was associated with Alternaria alternata, Fusarium solani and Epicoccum purpurescens. Positive skin reactivity to Calvatia cyathiformis was also associated with A. alternata and F. solani. Coprinus quadrifidus was associated only with F. solani, and Psilocybe cubensis was only associated with Aspergillus fumigatus. No other skin test associations were demonstrated. For every allergen tested by RAST inhibition, significant dose-dependent homologous inhibition was demonstrated. Although the ability of an individual heterologous extract to inhibit the direct RAST varied, inhibition was generally minimal. In the most extreme example, no heterologous allergen inhibited the A. alternata RAST. However, the Armillaria tabescens RAST was inhibited 52.6%, 38.1% and 25.1% by A. fumigatus, E. purpurescens, and Penicillium notatum, respectively, suggesting significant cross-reactivity. These results suggest that, although shared allergenic determinants exist between select species of Basidiomycetes and Deuteromycetes, crossreactivity is minimal and its clinical significance is not clear. These data confirm that for reliable diagnosis of fungal allergy, representatives of both major groups must be used.
A fruiting body-specific cDNA mfbAc, derived from the basidiomycete Lentinus edodes, has been shown to encode a high-molecular-mass (2157 amino acids) cell-adhesion protein MFBA containing the Arg-Gly-Asp (RGD) motif. A 425-amino-acid fragment containing the RGD motif of MFBA (designated MFBA(582-1006) peptide) produced in Escherichia coli exhibited cell-adhesion and spreading activity toward mammalian cells and cell-aggregation activity toward basidiomycetous hyphal cells via the RGD sequence. Here we investigated the biological activity of MFBA(582-1006) peptide in Saccharomyces cerevisiae. The DNA sequence encoding MFBA(582-1006) peptide, introduced into the yeast using an expression vector, resulted in a marked aggregation of the yeast cells. The aggregation was almost completely abolished by replacement of the RGD motif by an RGE motif in the peptide sequence.
Basidiomycetous fungus Flammulina velutipes R15 strain had two linear plasmids in its mitochondria designated pFV1 and pFV2. They were double-stranded DNAs, whose sizes were 8.3 and 8.9 kb, respectively. Sequencing analysis of 7364 bases of the pFV1 and 6861 bases of the pFV2 revealed that the both plasmids had one set of two open reading frames (ORFs) each of that encoded putative DNA and RNA polymerases similar to those of mitochondrial plasmids in other filamentous fungi. In phylogenetic analysis of deduced amino acid sequences of the ORFs and counterparts of other filamentous fungi, the pFV2 was expectedly clustered with plasmids of basidiomycetous fungi. whereas the pFV1 with kalilo plasmid of ascomycetous fungus Neurospora intermedia.
The in vitro activity of amphotericin B, fluconazole, flucytosine, itraconazole, voriconazole, and posaconazole was evaluated against 44 clinical isolates of filamentous basidiomycetous fungi. No statistically significant differences were noted between Schizophyllum commune (n = 5), Coprinus species (n = 8), Bjerkandera adusta (n = 14), and sterile, uncharacterized basidiomycetes (n = 17).
A new lignin-degrading basidiomycete, strain PM1 (= CECT 2971), was isolated from the wastewater of a paper factory. The major ligninolytic activity detected in the basidiomycete PM1 culture supernatant was a phenoloxidase (laccase). This activity was produced constitutively in defined or complex media and appeared as two protein bands in native gel electrophoresis preparations. No enzyme induction was found after treatment with certain potential laccase inducers. Laccase I was purified to homogeneity by gel filtration chromatography, anion-exchange chromatography, and hydrophobicity chromatography. The enzyme is a monomeric glycoprotein containing 6.5% carbohydrate and having a molecular weight of 64,000. It has an isoelectric point of 3.6, it is stable in a pH range from 3 to 9, and its optimum pH is 4.5. The laccase optimal reaction temperature is 80 degrees C, the laccase is stable for 1 h at 60 degrees C, and its activity increases with temperature. Spectroscopic analysis revealed that the enzyme has four bound copper atoms, a type I copper, a type II copper, and a type III binuclear copper. The amino-terminal sequence of the protein is very similar to the amino-terminal sequences of laccases from Coriolus hirsutus and Phlebia radiata.