Characteristics of Gloeophyllum trabeum alcohol oxidase, an extracellular source of H2O2 in brown rot decay of wood

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K(m) = 2.3 mM, k(cat) = 15.6 s(-1)). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H(2)O(2), a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H₂O₂-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw.     

Detection of Lignin Peroxidase and Xylanase by Immunocytochemical Labeling in Wood Decayed by Basidiomycetes

The white rot fungi used in this study caused two different forms of degradation. Phanerochaete chrysosporium, strain BKM-F-1767, and Phellinus pini caused a preferential removal of lignin from birch wood, whereas Trametes (Coriolus) versicolor caused a nonselective attack of all cell wall components. Use of polyclonal antisera to H8 lignin peroxidase and monoclonal antisera to H2 lignin peroxidase followed by immunogold labeling with protein A-gold or protein G-gold, respectively, showed lignin peroxidase extra-and intracellularly to fungal hyphae and within the delignified cell walls after 12 weeks of laboratory decay. Lignin peroxidase was localized at sites within the cell wall where electron-dense areas of the lignified cell wall layers remained. In wood decayed by Trametes versicolor, lignin peroxidase was located primarily along the surface of eroded cell walls. No lignin peroxidase was evident in brown-rotted wood, but slight labeling occurred within hyphal cells. Use of polyclonal antisera to xylanase followed by immunogold labeling showed intense labeling on fungal hyphae and surrounding slime layers and within the woody cell wall, where evidence of degradation was apparent. Colloidal-gold-labeled xylanase was prevalent in wood decayed by all fungi used in this study. Areas of the wood with early stages of cell wall decay had the greatest concentration of gold particles, while little labeling occurred in cells in advanced stages of decay by brown or white rot fungi.     

Ultrastructural and Immunocytochemical Studies on the H2O2-Producing Enzyme Pyranose Oxidase in Phanerochaete chrysosporium Grown under Liquid Culture Conditions

The ultrastructural distribution of the sugar-oxidizing enzyme pyranose 2-oxidase (POD) in hyphae of Phanerochaete chrysosporium K-3 grown under liquid culture conditions optimal for the enzyme's production was studied by transmission electron microscopy immunocytochemistry. Using the 3-dimethylaminobenzoic acid-3-methyl-2-benzothiazolinone hydrazone hydrochloride H₂O₂ peroxidase spectrophotometric assay, POD was detected in mycelial extracts from days 7 to 18, with maximum activity recorded on day 12. Onset of POD activity occurred in the secondary phase of hyphal development at a time of stationary growth, glucose limitation, and pH increase. POD was also detected extracellularly in the culture fluid from days 7 to 18, with maximum activity recorded on day 13. At early stages of development (3 to 4 days), using anti-POD antibodies and immunogold labeling, POD was localized in multivesicular and electron-dense bodies and in cell membrane regions. After 10 to 12 days of growth, at maximum POD activity, POD was concentrated within the periplasmic space where it was associated with membrane-bound vesicles and other membrane structures. At later stages of development (17 to 18 days), when the majority of hyphae were lysed, POD was observed associated with residual intracellular membrane systems and vesicles. Transmission electron microscopy immunocytochemical studies also demonstrated an extracellular distribution of the enzyme at the stationary growth phase, showing its association with fungal extracellular slime. In studies of ligninolytic cultures of the same fungus, POD was found to have a similar intracellular and extracellular distribution in slime as that recorded for cultures grown with cornsteep. POD's peripheral cytoplasmic distribution shows similarities to the cellular distribution of that reported previously for H₂O₂-dependent lignin and manganese peroxidases in P. chrysosporium.     

Pathways for Extracellular Fenton Chemistry in the Brown Rot Basidiomycete Gloeophyllum trabeum

The brown rot fungus Gloeophyllum trabeum uses an extracellular hydroquinone-quinone redox cycle to reduce Fe³⁺ and produce H₂O₂. These reactions generate extracellular Fenton reagent, which enables G. trabeum to degrade a wide variety of organic compounds. We found that G. trabeum secreted two quinones, 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and 4,5-dimethoxy-1,2-benzoquinone (4,5-DMBQ), that underwent iron-dependent redox cycling. Experiments that monitored the iron- and quinone-dependent cleavage of polyethylene glycol by G. trabeum showed that 2,5-DMBQ was more effective than 4,5-DMBQ in supporting extracellular Fenton chemistry. Two factors contributed to this result. First, G. trabeum reduced 2,5-DMBQ to 2,5-dimethoxyhydroquinone (2,5-DMHQ) much more rapidly than it reduced 4,5-DMBQ to 4,5-dimethoxycatechol (4,5-DMC). Second, although both hydroquinones reduced ferric oxalate complexes, the predominant form of Fe³⁺ in G. trabeum cultures, the 2,5-DMHQ-dependent reaction reduced O₂ more rapidly than the 4,5-DMC-dependent reaction. Nevertheless, both hydroquinones probably contribute to the extracellular Fenton chemistry of G. trabeum, because 2,5-DMHQ by itself is an efficient reductant of 4,5-DMBQ.     

Field Evaluations of Subterranean Termite Preference for Sap-Stain Inoculated Wood

Few studies have focused on interactions between subterranean termites and the ophiostomatoid fungal associates of pine bark beetles or root feeding weevils. Field stake tests were employed at four locations throughout Mississippi to determine the feeding preference of subterranean termites for blue-stained, unstained, and partially decayed southern pine sapwood stakes. This study also utilized wood decayed by Gloeophyllum trabeum, a fungus previously shown to elicit a positive subterranean termite feeding response, as a positive control. Stakes inoculated with G. trabeum received significantly more attacks than all other treatments after 16 weeks. Of the stakes attacked by subterranean termites, stakes inoculated with Ophiostoma minus were degraded faster than any other treatment. Subterranean termite preference for stakes treated with either of two Leptographium spp. and the untreated negative controls did not differ; however, each was fed upon less than all other treatments. The feeding rate on stakes inoculated with O. ips and G. trabeum being fed upon by subterranean termites was not significantly different. These results represent the first evidence of wood containing non-structurally degrading fungi (O. ips and O. minus) eliciting a feeding preference from subterranean termites greater than that of decayed wood. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, subterranean termite control, and the utilization of blue-stained southern pine building products in the southeastern U.S.     

Pyranose Oxidase, a Major Source of H(2)O(2) during Wood Degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida

The production of the H(2)O(2)-generating enzyme pyranose oxidase (POD) (EC 1.1.3.10) (synonym, glucose 2-oxidase), two ligninolytic peroxidases, and laccase in wood decayed by three white rot fungi was investigated by correlated biochemical, immunological, and transmission electron microscopic techniques. Enzyme activities were assayed in extracts from decayed birch wood blocks obtained by a novel extraction procedure. With the coupled peroxidase-chromogen (3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolinone hydrazone hydrochloride) spectrophotometric assay, the highest POD activities were detected in wood blocks degraded for 4 months and were for Phanerochaete chrysosporium (149 mU g [dry weight] of decayed wood), Trametes versicolor (45 mU g), and Oudemansiella mucida (1.2 mU g), corresponding to wood dry weight losses of 74, 58, and 13%, respectively. Mn-dependent peroxidase activities in the same extracts were comparable to those of POD, while lignin peroxidase activity was below the detection limit for all fungi with the veratryl alcohol assay. Laccase activity was high with T. versicolor (422 mU g after 4 months), in trace levels with O. mucida, and undetectable in P. chrysosporium extracts. Evidence for C-2 specificity of POD was shown by thin-layer chromatography detection of 2-keto-d-glucose as the reaction product. By transmission electron microscopy-immunocytochemistry, POD was found to be preferentially localized in the hyphal periplasmic space of P. chrysosporium and O. mucida and associated with membranous materials in hyphae growing within the cell lumina or cell walls of partially and highly degraded birch fibers. An extracellular distribution of POD associated with slime coating wood cell walls was also noted. The periplasmic distribution in hyphae and extracellular location of POD are consistent with the reported ultrastructural distribution of H(2)O(2)-dependent Mn-dependent peroxidases. This fact and the dominant presence of POD and Mn-dependent peroxidase in extracts from degraded wood suggest a cooperative role of the two enzymes during white rot decay by the test fungi.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine _COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.     

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.     

Efficient expression and purification of recombinant alcohol oxidase in Pichia pastoris

In order to improve the production of alcohol oxidase (AOX), a recombinant Pichia pastoris (P. pastoris) system was constructed by transformation of the plasmid pPIC9K-AOX into P. pastoris GS115. The effects of different expression conditions on alcohol oxidase activity in the culture supernatant were investigated in the shake flask scale. The results showed that the highest extracellular activity (562 U/L) of alcohol oxidase was obtained after 56 h induction with 4% methanol at OD₆₀₀ 1.0 in the medium containing 50 g/L maltose, which is about 4.2 folds higher than previously reported. High-purity functional recombinant AOX (>90%) was purified from the culture with the Ni-NTA affinity column and Sephadex G-100 chromatographical methods, with a total recovery rate of 68.9%. Further studies showed that the purified rAOX had similar enzymatic characteristics as the native enzyme, except that the thermal stability and resistance to H₂O₂ inhibition of rAOX were significantly greater compared to the previous report. The purified rAOX was well tolerant to various water-miscible organic solvents. This efficient expression and purification process will be promising for large-scale production of rAOX as an important diagnostic enzyme for alcohol detection in many areas.     

Anastomosis Formation and Nuclear and Protoplasmic Exchange in Arbuscular Mycorrhizal Fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 ± 0.06 μm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

The Characterization of the Endoglucanase Cel12A from Gloeophyllum trabeum Reveals an Enzyme Highly Active on β-Glucan

The basidiomycete fungus Gloeophyllum trabeum causes a typical brown rot and is known to use reactive oxygen species in the degradation of cellulose. The extracellular Cel12A is one of the few endo-1,4-β-glucanase produced by G. trabeum. Here we cloned cel12A and heterologously expressed it in Aspergillus niger. The identity of the resulting recombinant protein was confirmed by mass spectrometry. We used the purified GtCel12A to determine its substrate specificity and basic biochemical properties. The G. trabeum Cel12A showed highest activity on β-glucan, followed by lichenan, carboxymethylcellulose, phosphoric acid swollen cellulose, microcrystalline cellulose, and filter paper. The optimal pH and temperature for enzymatic activity were, respectively, 4.5 and 50°C on β-glucan. Under these conditions specific activity was 239.2±9.1 U mg⁻¹ and the half-life of the enzyme was 84.6±3.5 hours. Thermofluor studies revealed that the enzyme was most thermal stable at pH 3. Using β-glucan as a substrate, the K_m was 3.2±0.5 mg mL⁻¹ and the V_max was 0.41±0.02 µmol min⁻¹. Analysis of the effects of GtCel12A on oat spelt and filter paper by scanning electron microscopy revealed the morphological changes taking place during the process.     

Hbr1 Activates and Represses Hyphal Growth in Candida albicans and Regulates Fungal Morphogenesis under Embedded Conditions

Transitions between yeast and hyphae are essential for Candida albicans pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an HBR1/hbr1 strain formed constitutively filamentous colonies throughout the matrix, resembling EFG1 null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of HBR1/hbr1 cells producing cytokinesis-defective hyphae whereas farnesol treated EFG1 null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the HBR1 heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O₂. Consistent with these morphogenesis defects, the HBR1 heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.