APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1992, p.

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Vol. 58, No. 11

0099-2240/92/113667-10$02.00/0
Copyright 1992, American Society for Microbiology

Ultrastructural and Immunocytochemical Studies on the H202-Producing Enzyme Pyranose Oxidase in Phanerochaete chrysosporium Grown under Liquid Culture Conditions
GEOFFREY DANIEL,`* JINDRICH VOLC,2 ELENA KUBATOVA,2 AND THOMAS NILSSON' Department of Forest Products, Swedish University ofAgricultural Sciences, Box 7008, S-750-07, Uppsala, Sweden, 1 and Institute of Microbiology, Czechoslovakian Academy of Sciences, Prague 4, Czechoslovakia2
Received 25 March 1992/Accepted 18 August 1992

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 H202 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 extraceliular 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 H202-dependent lignin and manganese peroxidases in P. chrysosporium.

Hydrogen peroxide has been implicated in lignin- and cellulose-degrading systems of both white rot and brown rot basidiomycete fungi. With ligninolytic fungi, H202 acts as a cosubstrate for lignin peroxidase during oxidation of lignin (32) and for Mn(II) peroxidase during oxidation of Mn(II) (9, 25). With brown rot fungi, H202 is thought to play a role in the initial very rapid and destructive attack of cellulose by mediating the production of hydroxyl radicals. To have a complete understanding of fungal lignocellulose-degrading processes, it is therefore important to determine the source of H202. Presently, a range of oxidase enzymes including glucose 1-oxidase (18, 19), pyranose 2-oxidase (POD) (syn = glucose 2-oxidase [7]), glyoxal oxidase (20), methanol oxidase (24), and fatty-acyl coenzyme A oxidase (12) have been proposed as possible donors of H202 in basidiomycete fungi. Of these enzymes, perhaps the greatest evidence for involvement has been presented for the sugar oxidases and glyoxal oxidase. Glyoxal oxidase has been identified in culture fluids from ligninolytic cultures of Phanerochaete chrysosponum (20) and more recently in extracts from wood chips degraded by the same fungus (6). The enzyme is, however, only reported to utilize secondary fungal metabolites. Similarly, both of the sugar oxidases, glucose 1-oxidase and POD, have been identified in mycelial extracts from the white rot fungus P. chrysosporium (7, 26). In addition, POD has been reported produced from a range of other white rot and, to a
*

lesser extent, brown rot basidiomycete fungi (14, 15, 23, 37). Sugar oxidases have further been postulated to function in concert with laccase to maintain a laccase-glucose:quinone oxidoreductase cycle during which they are thought to hinder spontaneous repolymerization of quinone intermediates produced by laccase, thereby increasing the efficiency of depolymerization during lignin degradation (11, 31). The present work describes enzymatic, immunocytochemical, and ultrastructural studies to determine the intraand extracellular distribution of POD in cultures of P. chrysosporium K-3 grown under conditions optimal for the enzyme's production as well as in ligninolytic cultures. POD (,B-D-glucose:oxygen 2-oxidoreductase; EC 1.1.3.10.) was chosen instead of glucose 1-oxidase (EC 1.1.3.4.) for this work because of its reported occurrence in a range of white rot fungi and because of its reported activity on xylose in addition to D-glucose, both important wood sugars. Although generally considered as an intracellular enzyme, POD has been recorded in culture fluids of ligninolytic cultures of Panus tigrinus 144, suggesting a possible extracellular role during lignocellulose degradation (10). MATERIALS AND METHODS

Corresponding author.
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Fungal strains. P. chrysosporium K-3 (16) was used for enzyme production and for transmission electron microscopy (TEM) immunocytochemical studies. P. chrysosporinum K-3 was obtained from the Czechoslovakian Academy of Science

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Culture collection of basidiomycetes (strain CCBAS 571). The fungus is routinely maintained on 2.5% malt agar plates. Liquid culture conditions. Agitated fungal cultures (150 rpm) were set up as previously outlined (36), using 80 ml of medium per 500-ml Erlenmeyer flask. The culture medium contained (per liter of tap water) 20 g of D-glucose, 15 g of cornsteep syrup (Sigma Chemical Co., St. Louis, Mo.), and 1.5 g of MgSO4. 7H20. The medium was adjusted to pH 5.5 with 6 M NaOH before autoclaving. Samples for analysis were taken over a 18-day growth period by using the entire contents of single or duplicate flasks. Estimates of mycelial growth were determined gravimetrically. The presence of glucose in the growth medium was determined by using Clinistix indicator paper (Ames, Miles Laboratories, Slough, United Kingdom). Ligninolytic cultures. Nonagitated cultures of P. chrysosporium K-3 were set up by the method of Kirk et al. (21). Mycelial samples were removed after a 6-day growth period and processed for TEM immunocytochemistry as previously described (4). Under these conditions, loss of color of Poly B 411 (Sigma) occurred after 3 to 6 days indicating ligninolytic conditions. POD purification. Mycelia harvested from 12-day-old cultures grown with cornsteep were disrupted, and a crude extract was obtained. This extract was then subjected to purification by hydrophobic interaction on phenyl-Sepharose CL-4B, then anion-exchange chromatography on Mono Q HR 5/5, and finally by gel filtration on Superose 6 HR 10/30 columns (see reference 36 for further details). The purified enzyme formed a single band with the polyclonal POD antiserum as determined by immunoelectrophoresis with a PhastSystem (Pharmacia, Uppsala, Sweden). Purified POD produced in this way was used for subsequent antibody production and as a positive control for enzyme-linked immunosorbent assay (ELISA) studies. Enzyme assays. Pyranose oxidase activity was assayed by measuring production of H202 as described by Volc and Eriksson (35). Each mixture (2.0 ml) contained 200 ,umol of Na phosphate buffer (pH 6.5), 100 ,umol of D-glucose, 5.0 ,umol of DMAB (3-dimethylaminobenzoic acid; Fluka Chemie, Buchs, Switzerland), 0.1 ,umol of MBTH (3-methyl2-benzothiazolinone hydrazone hydrochloride; Fluka Chemie), 85 nkat of horseradish peroxidase (Sigma Chemical Co.), and enzyme extract to be assayed. The reaction was monitored at 590 nm at 25C. One unit (1 U = 16.67 nkat) of the enzyme was defined as the amount of activity that would produce 1 ,umol of H202 per min under the assay conditions. Enzyme assays were performed on the extracts from 2.0 g (fresh weight) as previously described (36). Polyclonal antibodies. Antiserum to POD was raised in New Zealand White rabbits immunized over a 6-month period after multiple intracutaneous injections of the purified enzyme immulsified in Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.). The immunoglobulin fraction (immunoglobulin G) was subsequently prepared by ammonium sulfate precipitation by standard techniques (17). In the ELISA, the POD antiserum produced a strong positive cross-reaction with the POD antigen (A405 = 1.72 versus control A405 = 0.01 at 1:100 dilution, 15-min development time). In control ELISA studies, the POD antibody did not show cross-reactivity with glucose 1-oxidase (,B-D-glucose: oxygen 1-oxidoreductase; EC 1.1.3.4) fromAspergillus niger (Sigma Chemical Co.) with an antibody dilution of 1:100 and in a dilution series (10-1 to 10-6) of the enzyme antigen. Light microscopy. For light microscopy, semi-thin sections (0.5 p,m) of resin embedded mycelial pellets were stained

Time (Days)

FIG. 1. POD production by P. chrysosporium K-3. Symbols: O, mycelial dry weight; *, pH values; POD activity in fungal extract (@) and culture filtrate (0).

with 0.5% (wt/vol) aqueous toluidine blue (pH 7.0) and examined by bright-field illumination with a Leitz Orthoplan microscope. TEM. Mycelial fractions for fixation were removed from cultures after 3, 4, 10, 12, 16, and 18 days of growth, which represented stages during initiation, at maximal POD production, and in scenescent stages of the hyphae (Fig. 1). Samples (normally 10 to 20 pellets of mycelium) for ultrastructural and immunological studies were fixed for 3 h in 3% (vol/vol) glutaraldehyde containing 2% paraformaldehyde in 0.1 M Na cacodylate (pH 7.2) buffer at room temperature. Samples were then washed three times in buffer and postfixed in 1% (wt/vol) osmium tetroxide in 0.1 M buffer for 1 h at room temperature. Additional samples were pre- and postfixed as described above but in the presence of ruthenium red (RR) (0.01% [wt/voll) to contrast extracellular polysaccharides. After further washes in buffer and distilled water, samples were dehydrated in an ethanol series (20 to 100%; 10% steps, 10 min each), infiltrated with London resin (London Resin Co., Basingstoke, United Kingdom), and polymerized overnight at 60C. Other samples were fixed for 3 h in 4% (vol/vol) paraformaldehyde containing 1% (vol/ vol) glutaraldehyde or 8% paraformaldehyde in the same buffer and thereafter dehydrated and infiltrated with resin as described above. Selected material was sectioned with a Reichert FC4 ultramicrotome, and sections were collected on nickel grids. Poststaining, when performed, was conducted with 4% aqueous uranyl acetate (5 min) and lead citrate (27) (15 min). Observations were made with a Philips 201 or CM/12 TEM operated at various accelerating voltages. ELISA conducted on the effects of glutaraldehyde-paraformaldehyde fixation on antigenicity of the POD antigen for its antibody showed A405 values of 1.53 (3% glutaraldehyde-2% paraformaldehyde) and 1.56 (4% paraformalde-

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hyde-1.0% glutaraldehyde). Postfixation in OS04 produced a ca. 50% loss in antigenicity (A405 = 0.76). Results reported here are for samples fixed in 3% glutaraldehyde-2% paraformaldehyde and then osmium, in which the ultrastructural preservation was superior to that obtained with the other fixatives used. TEM immunocytochemistry studies. Ultrathin sections for immunolabeling were processed as described previously (4). Sections were incubated overnight at 4C in anti-POD (1:100) containing 1% (wt/vol) bovine serum albumin and 0.05% Tween 20 and subsequently gold labeled with goat antirabbit IgG 15-nm gold probes (B-2340; Janssen Life Science Products, Beerse, Belgium). Poststaining when performed was done with uranyl acetate and lead citrate as described above. Controls included omission of the primary antiserum stage and use of anti-POD preadsorbed with POD.
RESULTS Enzyme production. POD production as measured by the DMAB-MBTH H202 peroxidase assay in cornsteep cultures is shown in Fig. 1. POD was first detected after 7 days during the stationary phase of growth as both the mycelial mass was decreasing and the glucose levels were becoming depleted. After 8 days of growth, no glucose was detected with the Clinistix indicator paper. Maximum mycelial POD activity was recorded after 12 days of growth (2.15 U g [dry weight]-1), when the pH had increased from pH 4.5 to 8.2. The enzyme was also detected in the extracellular culture fluid with maximum activity (8.87 mU/ml) recorded after a 12- to 13-day growth period (Fig. 1). During the experiment, the total pH increased from 4.5 to 8.5 (Fig. 1). Light microscopic observations on mycelial pellets. Under the present shake culture conditions, P. chrysosporium K-3 produced small yellow pellets of mycelia which increased to a maximum diameter of approximately 3 to 4.0 mm after 3 to 4 days of growth. Production of pellets is a well-known growth characteristic for this fungus under shake conditions and is not observed in stationary cultures. Light microscopic observations of smaller pellets (200 to 400 ,um) showed them to consist of an outer ring of mycelia surrounding a central region of loosely packed hyphae. During the culture period, the outer ring of mycelia became densely packed and intermeshed with slime. Observations on mycelial pellets after 3 to 4 days during the active growth phase showed hyphae to have a densely staining cytoplasm containing few vacuoles and considerable numbers of storage bodies. At this stage, few spores or lysed hyphae were present. Extracellular slime where present appeared to be closely applied to the hyphal cell walls. After 10 days of growth, many of the hyphae showed a less densely staining cytoplasm and vacuolation had increased. Extracellular slime was now prominent surrounding hyphae, forming a complex mycelium-slime network in both the inner and outer regions of the pellets. Two types of slime were noted, namely (i) a loosely packed material interspersed between cells and (ii) a more compact denser slime closely surrounding individual or small groups of hyphae. At this stage, the relative numbers of lysed hyphae and asexual spores had increased in comparison with those seen at 4 days. After 16 to 18 days of growth, during the scenescent phase, the majority of hyphae were lysed but were still held together in an extensive extracellular slime matrix. TEM and TEM immunocytochemistry. For ultrastructural and immunocytochemical observations, smaller pellets (200 to 400 ,um) were selected, and observations were made

randomly on hyphae from the peripheral regions of the pellets. (i) Three- to four-day culture period. Hyphae after 3 to 4 days of growth fixed in glutaraldehyde-paraformaldehyde displayed a compact cytoplasmic structure, a well-developed endomembranous system, and numerous mitochondria (Fig. 2a and b). Hyphae were packed with electron-lucent storage structures as well as lesser numbers of larger membrane-bound electron dense bodies (Fig. 2). These electrondense bodies contained amorphous materials at various stages of apparent degeneration (Fig. 2c and d). In addition to nuclei, hyphae contained a well-developed microtubular system, with bands often running along the length of the hyphae. Membrane structures and multivesicular bodies were also prominent and were often associated with the electron-dense bodies and outer cell membrane (Fig. 2b and d). Multiple projections of the outer cell membrane and other membranous materials were also observed in the periplasmic space (i.e., cell membrane-fungal cell wall interface) beneath the fungal cell wall (Fig. 2c). Using POD antibodies, POD was localized in the periplasmic space, and an overall positive labeling of electron-lucent regions was noted (Fig. 3a to e). The contents of the electron-lucent regions, particularly membrane structures, were specifically labeled, as well as multivesicular bodies (Fig. 3b, d, f, and g). Frequently, mycelial structures were observed in which one hypha had developed within the confines of an existing hypha (i.e., intrahyphae [301) (Fig. 3d). Both hyphae, however, showed typical fungal cell walls. Such intrahyphae showed peripheral and intracellular membrane POD labeling characteristics similar to that observed for normal hyphae (cf. Fig. 3a and d). In contrast, the disrupted intracellular contents of the outer encapsulating hyphae, particularly membrane structures, showed strong POD labeling similar to that of lysed cells (Fig. 3d). At this phase of development, some extracellular slime was noted, but generally this was closely pressed to the outer hyphal cell and specific labeling was not recognized. (ii) Ten- to 12-day culture period. After 10 to 12 days of culture, hyphae showed a tendency for an increase in cell vacuolation and both a degeneration as well as decrease in the numbers of electron-dense structures. A similar pattern of POD labeling, with the cell membrane and periplasmic space of hyphae reacting most strongly, as seen for 3- to 4-day-old hyphae, was noted (Fig. 4c and d). Intrahyphae were apparent, and a strong labeling for POD with membranous structures in the encapsulating hyphae was noted (Fig. 4a). The membranous contents within degenerated electrondense bodies also labeled strongly, indicating that POD was associated with these structures (Fig. 4b). A characteristic feature of many hyphae at this phase of maximum POD activity was the presence of a pronounced periplasmic space (Fig. 4c), apparently produced by contraction of the cell cytoplasm from the fungal cell wall. Membranous structures and vesicles within the periplasmic space labeled strongly for the presence of POD, as did similar structures in the contracted cytoplasm (Fig. 4c). The contents and membranous structures associated with individual multivesicular bodies showed labeling, as did, on occasion, membranes of the endoplasmic reticulum. Labeling of multivesicular body membranes were noted, suggesting that this may represent a route of POD secretion into the periplasmic space (Fig. 4e). Small involutions of the cell membrane were observed, producing a small localized periplasmic space containing membrane-bound vesicles positively labeled for POD (Fig. 4f and g). Partially lysed cells typically showed a

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FIG. 2. Transmission electron micrographs showing general ultrastructural features of hyphae from 4-day-old cultures. (a) Longitudinal section showing dense cytoplasm, amorphous electron-dense bodies (Ed), electron-lucent regions (El), nucleus (N), microtubule bundle (M), and mitochondria (Mt). (b) Transverse section showing features similar to those in panel a and also a central multivesicular body (arrow) and structures within a periplasmic space (Ps). (c) Oblique section showing membranous protruberances (Mp) from the outer cell membrane into the periplasmic space. Membranous materials appear to be produced within the cell cytoplasm (arrows). (d) Association of amorphous electron-dense (Ed) and multivesicular bodies (Mvb). The multivesicular body appears to have direct contact with the outer cell membrane (Cm) and periplasmic space (Ps) (arrow). Bars: a, 1.0 ,m; b, 0.5 Lm; c, 0.25 ,um; d, 0.1 p,m. Abbreviations: Cm, cell membrane; Cw, fungal cell wall; El, electron-lucent region; Ed, electron-dense body; M, microtubule bundle; Mt, mitochodria; Mp, membranous protruberances; Mvb, multivesicular body; N, nucleus; Ps, periplasmic space.

strong positive POD labeling of membranous structures and vesicles intermeshed in the disrupted cell cytoplasm or in the now enlarged periplasmic space (Fig. 5a and b). Extracellular slime projecting from hyphae labeled positively for POD, particularly that surrounding lysed hyphae. Inclusion of RR during fixation caused an overall pinkishred staining of the fungal pellets at all growth phases. A conspicuous feature of including RR seen in hyphae from both 12-day-old and, to a lesser extent, 4-day-old cultures was the development of characteristic electron-dense aggregates of RR-Os04 in the periplasmic space (Fig. Sc). In some hyphae, the aggregates were small and finely distributed at the cell wall interface, whereas in other examples, the

aggregates were large and appeared to project from the outer side of the cell membrane (Fig. 5c). (iii) Sixteen- to 18-day culture period. At this stage, the majority of hyphae were lysed and contained a disrupted cell cytoplasm in which vesicles and membrane structures were prominent. Hyphae were generally enmeshed in a complex network of extracellular slime of which a certain fibrillar fraction close to the external fungal cell wall stained strongly with RR. In lysed cells, POD was localized preferentially with vesicular structures, both with their surrounding membranes and intracellular contents (Fig. 6a and b). Very often, strong labeled whorled membranous structures and vesicles were the only remaining structures visible in totally lysed

FIG. 3. Transmission electron micrographs showing intracellular distribution of POD in P. chrysosporium from 4-day-old cultures. (a) Transverse section of a hypha giving an overview of the spatial distribution of POD in the peripheral cell cytoplasm in association with the cell membrane (arrows). (b) Transverse section of a hypha showing association of POD with the cell membrane (Cm) and intracellular electron-lucent regions (El). (c) Transverse section of a hypha with localization of POD in the periplasmic space (Ps) and cell membrane (Cm). (d) Hyphal transverse section showing a typical intrahyphal structure surrounded by the shell of an outer lysed hypha. The spatial distribution of POD in the intrahypha was similar to that observed in normal hyphae. Membranous structures (Ms) and vesicles contained in the lysed outer hypha were labeled strongly for POD. Both hyphae are surrounded by apparently normal cell walls. (e) Higher magnification showing labeling of the cell membrane (Cm) and an intracellular vesicle (v) structure. (f and g) POD associated with electron-lucent (El) regions and a multivesicular body (Mvb). The frequent association observed between membrane structures of multivesicular bodies and the cell membrane (Cm) may represent a possible route for POD secretion. Bars: a to d, 0.5 pm; e and g, 0.1 p,m; f, 0.2 p,m.

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FIG. 5. Transmission electron micrographs showing P. chrysosporium hyphae from 12-day-old cultures after immunocytochemistry or treatment with RR. (a) Oblique section showing a typical partially lysed hypha. POD was concentrated in the periplasmic space (Ps), associated with membrane-bound vesicles (v) and other structures. Labeling of membrane structures (Ms) in the disrupted cell cytoplasm was also apparent. (b) High magnification showing labeling of both vesicles (v) and vesicle membranes situated in the periplasmic space of a partially lysed cell. (c) Longitudinal section showing presence of electron-dense aggregates of RR-Os04 in hyphae from mycelial pellets treated with RR. Aggregates were localized in the periplasmic space where they appeared to project from the outer regions of the cell membrane (Cm) (arrows). Bars: a and c, 0.5 pLm; b, 0.25 pLm.

hyphae (Fig. 6b). The presence of POD in peripheral cell regions was variable, and while some hyphal ghost walls showed a strong presence of POD (Fig. 6c), other hyphae labeled only poorly, suggesting the absence of POD. Extracellular slime surrounding hyphae labeled most strongly for POD in samples fixed in paraformaldehyde only and embedded in London resin (Fig. 6d and e). The greatest labeling and evidence for POD was observed in slime surrounding lysed hyphae. Slime materials processed after paraformaldehyde fixation were, however, poorly defined, and it was not possible to visualize a fibrillar structure after POD labeling (Fig. 6e). Fibrillar structures after RR treatment were observed in only a minor portion of the extracellular slime, often in close contact with the hyphae. Both controls included in the labeling experiments, including omission of the POD stage and use of anti-POD preadsorbed with POD, produced negative results. Ligninolytic cultures. Studies done on mycelial from 6-dayold static ligninolytic cultures of P. chrysosponium K-3 showed a labeling pattern similar to that observed for cornsteep cultures. POD was observed associated with intracellular membrane structures and was also localized along the cell membrane (Fig. 6f and h). Examples of intrahyphae within autolysed hyphae were also noted (Fig.

6g). Only a weak indication for POD was noted in extracellular slime materials enmeshing fungal hyphae (Fig. 6f).
DISCUSSION With liquid cultures of P. chrysosporium K-3 grown with cornsteep, maximum POD production occurred after 12 days during the secondary phase of mycelial growth when glucose was depleted and the pH of culture medium had risen over 8.0. These culture characteristics are similar to that reported previously for POD production by Trametes (Coriolus) vesicolor and P. chrysosporium (7, 23). At the fine structural level, maximum POD production was correlated with an increase in hyphal vacuolation, cytoplasmic disruption, and cell lysis and occurred when hyphae were enrobed in extracellular slime. At this time, POD was localized preferentially within the periplasmic space and intracellularly associated with membrane structures. POD was also found associated with extracellular slime, particularly at later stages of development and especially surrounding autolysed cells. At earlier phases of hyphal development (i.e., 3 to 4 days), POD was preferentially localized within intracellular membrane structures associated with electron-dense bodies as well as along the cell membrane-fungal wall interface. Immunocyto-

FIG. 4. Transmission electron micrographs showing spatial distribution of POD in P. chrysosporium hyphae from 12-day-old cultures at the time of maximum POD activity. (a) Transverse section showing a characteristic intrahypha. Such hyphae were more frequently observed after 12 days than at 4 days. The spatial distribution of POD was similar to that seen at 4 days. Ms, membranous structure. (b) Association of POD with membranous structures in electron-dense (Ed) bodies from the more central cytoplasm of a hypha. (c) Hyphal longitudinal section showing a contracted cell cytoplasm and a pronounced periplasmic space (Ps). Membrane structures (Ms) and vesicles in the periplasmic space showed strong evidence for the presence of POD. Electron-lucent (El) regions in the contracted cell cytoplasm also showed strong labeling for POD. (d) High magnification showing concentration of POD along the cell membrane and inner fungal cell wall (Cw) where a periplasmic space is not evident. Frequently in hyphae also treated with RR, more intense labeling occurred in regions showing strong staining with the dye (arrows). (e) POD associated with a membrane whorled structure which is also in contact with the cell membrane and periplasmic space. (f and g) Frequently, POD was associated with membrane-bound vesicles (v) in the periplasmic space (Ps), where small involutions of the outer cell membrane (Cm) were evident. Bars: a and c, 0.5 p.m; b, d to g, 0.1 ,um.

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FIG. 6. Transmission electron micrographs showing spatial distribution of POD in P. chrysosporium hyphae from 17-day-old cornsteep cultures and from 6-day-old ligninolytic cultures. (a) Transverse section showing highly disrupted hypha with only membrane structures and vesicles (v) remaining. (b) Longitudinal hyphal section of a highly lysed cell showing strong labeling for POD on residual membrane structures (Ms). The membrane structures may represent remnants of disrupted multivesicular bodies. (c) Hyphal shell with POD associated with the cell wall and its residual materials. (d and e) Extracellular slime, both close to and at a distance from hyphae, showing labeling for the presence of POD. A slime fibrillar structure was not evident. (f to h) Longitudinal and transverse sections of hyphae from ligninolytic cultures. POD was localized along the cell membrane and with membrane structures (Ms) in the cell vacuoles (Cv) (arrows). Like cornsteep cultures, intrahyphae in autolysed hyphae were also noted. Sparse labeling was noted for POD in the extracellular slime. Bars: a to c and g, 0.5 pm; d, e, and h, 0.25 p.m; f, 1.0 pLm.

chemical labeling of hyphae after 3 to 4 days of development further indicated an earlier occurrence of POD than that recorded by the DMAB-MBTH H202 peroxidase method (i.e., 7 days), indicating the relative greater sensitivity of the immunological assay.

A significant feature of hyphae from 3- and 10-day-old cultures was the presence of multivesicular bodies. Internal vesicles in these structures were often interconnected and on occasions in contact with the cell membrane. Strong POD labeling indicates that such multivesicular bodies represent

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cytoplasmic sites of POD. Multivesicular bodies were also observed associated with the endomembranous system and cell membrane, where they may represent an important step in the pathway for the enzyme to the periplasmic space and secretion. Multivesicular bodies have been reported previously from other fungi, where their presence has been correlated with increased enzyme production and extracellular secretion (2). The secretory pathway of extracellular enzymes from fungal hyphae into their immediate surroundings is, however, presently poorly understood, and both molecular sieving of proteins through living cell walls (33) and release via cell autolysis (3) have been reported as likely processes. Since maximum mycelial POD production was correlated in the present study by an increase in hyphal cytoplasmic disruption, its release may have occurred primarily during cell autolysis. Hyphal autolysis is further thought to result in an increase in cell wall porosity (3), which would also be consistent with release of a relatively high-molecular-weight enzyme such as POD. During degradation of wood, lysed hyphae are frequently observed in close proximity to viable cells. Under these conditions of limited nitrogen, cell autolysis could facilitate recycling of the existing mycelial protein pool for further growth. Recently, hyphal autolysis has been also correlated with lignin peroxidase release from ligninolytic cultures of P. chrysosporium (22). One of the major criteria put forward for a role for H202producing enzymes in lignocellulose degradation is that the enzyme should be extracellular or at least have a subcellular location so that H202 may be freely released to the outside either to function together with H202-dependent enzymes (e.g., peroxidases) or to act directly on the substrate. In the present work, POD was localized preferentially on membrane structures in electron-lucent vesicles, multivesicular bodies, and in the periplasmic space of apparently healthy hyphae. A periplasmic distribution for sites of H202 production has been reported previously for other white rot fungi (e.g., T. versicolor [13]), including P. chrysosponum under ligninolytic conditions (8), as determined by using the 3,3'diaminobenzidine staining reaction. The periplasmic sites of H202 production reported by Forney et al. (8) are similar in distribution to that observed here with POD antibody labeling and RR staining. Both T. versicolor and P. chrysosponium are also well-known producers of both POD and peroxidase enzymes (23, 34). While the true chemical nature of the staining reaction which produced RR aggregates in the periplasmic space is not currently understood, RR is known to bind to electron-negative sites on a variety of proteins and polysaccharides. A similar periplasmic location of RR aggregates has also been noted in hyphae of T. versicolor and Oudemansiella mucida during the degradation of wood (5a). The specific localization of a sugar-oxidizing and H202producing enzyme in the periplasmic space has, however, not to our knowledge been reported previously. Kelley and Reddy (18) reported the H202 sugar-oxidizing enzyme in extracts from low-nitrogen-containing cultures of P. chrysosporium as glucose 1-oxidase. We consider that under our experimental conditions, the sugar-oxidizing enzyme localized was unlikely to be glucose 1-oxidase, since the present POD antibodies did not show cross-reactivity with glucose 1-oxidase from A. niger in an ELISA (see Materials and Methods). In addition, the Mrs of the two enzymes are also widely different (POD, 300,000; glucose 1-oxidase, 180,000). POD was also localized in P. chrysosporium K-3 hyphae from ligninolytic cultures, suggesting

that this enzyme is likely to be produced under conditions of lignin degradation. Hydrogen peroxide produced by oxidase enzymes in the periplasmic space of fungal hyphae or that released during hyphal autolysis may be able to diffuse into the extracellular surroundings and function in situ with lignin-degrading enzymes during lignocellulose degradation. Both lignin and Mn(II)-dependent peroxidases are known to have a periplasmic distribution and to occur extracellularly during wood degradation (1, 4, 5, 28, 29). Recent ELISA studies (3a) on wood degraded by T. versicolor and 0. mucida, both known effective producers of peroxidase and POD, have shown the presence of both enzymes in extracts. This evidence together with present ultrastructural findings for a similar periplasmic distribution of POD and extracellular release suggests that this sugar-oxidizing enzyme could represent an important donor of H202 for H202-dependent peroxidases during lignocellulose degradation by P. chrysosponum. Work is now in progress to determine the ultrastructural distribution of these three enzymes in situ during wood degradation.
ACKNOWLEDGMENTS This work was supported partly by the Swedish Council for Forestry and Agricultural Research and by the Czechoslovakian and
Swedish Academies of Sciences.

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