30% H202 at 4°C overnight, sawdust obtained from cultures after 1 month of incubation at 25°C with P. chrysosporium or. C. versicolor, normal poplar sawdust, ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1987, p. 2384-2387 0099-2240/87/102384-04$02.00/0 Copyright C) 1987, American Society for Microbiology
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Wood Degradation by White Rot Fungi: Cytochemical Studies Using Lignin Peroxidase-Immunoglobulin-Gold Complexes GARCIA,'* JEAN PAUL LATGE,' MARIE CHRISTINE PREVOST,2 AND MATTI LEISOLA3 Unite de Mycologiel and Station Centrale de Microscopie Electronique,2 Institut Pasteur, 75724 Paris Cedex 15, France, SUSANA
and Institut fur Biotechnologie, Eidgenosische Technische Holzschule-Honggerberg, CH-8093 Zurich, Switzerland3 Received 17 February 1987/Accepted 26 June 1987
Using an anti-lignin peroxidase antiserum-protein A-gold complex, we found lignin peroxidase mainly intracellularly in several white rot fungi colonizing sawdust under laboratory conditions. This enzyme was also present in fungi found in naturally decayed wood. However, in all cases, lignin peroxidase was located mainly inside the fungal cells. Labeled lignin peroxidase did not bind to the lignocellulosic samples tested, with the exception of poplar milled-wood lignin. These results are discussed in relation to the role of lignin peroxidase during wood degradation. Electron microscopy has been a major technique in the study of ultrastructural aspects of wood (4, 6, 18) and wood decay (2, 8). Localization of lignin in the different layers of the cell wall has been studied by scanning electron microscopy coupled with energy-dispersive X-ray analysis of brominated wood (23, 24) and, more recently, after mercurization of the lignin molecule (U. Westermark, I. Eriksson, and 0. Lidbrandt, Abstr. Proc. 4th Cell Wall Meet., 1986, p. 281-283). Lignin has also been detected by transmission electron microscopy of wood tissue stained mainly with KMnO4 (2, 21) or with Coppick and Fowler reagent (19). Although KMnO4 is used extensively, doubt exists concerning its specificity for lignin (12). Binding specific markers to lignin is difficult owing to the heterogeneity and lack of stereoregularity of this molecule (7). In cytochemistry, substrates may be visualized by using specific enzymes coupled to an electron-dense marker (1). This technique has been used for hemicellulose localization with mannanase and xylanase coupled to colloidal gold (22, 27). Lignin peroxidase, an enzyme involved in the degradation of lignin, has recently been isolated from a ligninolytic fungus (26). Production of this enzyme can be enhanced by aromatic compounds such as veratryl alcohol (15). However, the role of lignin peroxidase in the degradation of natural lignocellulosic substrates has not yet been ascertained. The aim of this study was to label wood lignin with lignin peroxidase coupled to colloidal gold. Moreover, an indirect labeling technique involving the use of an antiserum directed against this enzyme has allowed us to localize the lignin peroxidase in fungal cells during lignin degradation in natural substrates. These observations are discussed in connection with the present understanding of the mechanism of lignin peroxidase action. MATERIALS AND METHODS Strains and culture conditions. White rot basidiomycetes used were Phanerochaete chrysosporium 2843, Coriolus versicolor 2276, and Pleurotus ostreatus 2722, from the Plant Biotechnology Institute, Saskatoon, Saskatchewan, Canada, and Dichomitus squalens 4332-34, from Centraalbureau voor *
Corresponding author. 2384
Schimmelcultures, Baarn, The Netherlands. The fungi were for at least 1 month at 25°C in petri dishes (diameter, 6 cm), each containing 1 g of poplar (Populus tremuloides) sawdust and 3 ml of water. Lignin samples. Several poplar sawdust samples were used for lignin labeling: sawdust treated with saturated chlorine water for 10 min at room temperature, sawdust treated with 30% H202 at 4°C overnight, sawdust obtained from cultures after 1 month of incubation at 25°C with P. chrysosporium or C. versicolor, normal poplar sawdust, and milled-wood lignin (MWL) obtained by a dioxane extraction of poplar wood by the method of Wegener and Fengel (28) (a gift from E. Odier, Institut National Agronomique, Paris, France). In addition, a piece of naturally degraded pine wood collected from a sandy forest soil on the Atlantic coast of France was used. Lignin peroxidase and anti-lignin peroxidase. Lignin peroxidase was purified from the extracellular enzyme solution of a carbon-limited agitated liquid culture of P. chrysosporium ATCC 24725 by the method of Leisola et al. (17) and dialyzed against water for 48 h at 4°C. Anti-lignin peroxidase rabbit antiserum was obtained by injecting a rabbit with 1 ml of a 0.05% lignin peroxidase solution in an equal volume of Freund incomplete adjuvant. Two inoculations were made at 1-month intervals. At 2 months after the last inoculation, a positive reaction was observed by the fused rocket immunoelectrophoresis technique (16). The rabbit was bled, and the serum was recovered by centrifugation. Enzyme-colloidal gold complexes. Colloidal gold (diameter, 5 nm) was prepared by reducing HAuCl4 with white phosphorus by the method of Horisberger (13). The stability of the gold granules in relation to pH was tested between pH 4.0 and 6.5, and a microtitration assay for the determination of optimal enzyme-gold coupling was done by the method of Hodges et al. (11) with a 0.1% lignin peroxidase solution. After coupling, the resulting enzyme-colloidal gold complex was centrifuged at 63,000 x g for 1 h, and the pellet was recovered with 500 ,ul of 0.1 M Na2HPO4-KH2PO4 buffer0.15 M NaCl (PBS) solution (pH 7.2) containing 1% bovine serum albumin (PBS-1% BSA) and 0.02% NaN3. This solution (lignin peroxidase-gold) was stored at 4°C. A protein A-gold complex was prepared similarly with a 0.1% protein A solution. Electron microscopy. Samples were fixed with 2.5% pformaldehyde-0.1% glutaraldehyde in 0.1 M sodium cacogrown
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dylate buffer (pH 7.4) at room temperature for 1 h. After several buffer washes, residual free aldehydes were quenched with 10 mM NH4Cl in the same buffer at 4°C overnight. Samples were embedded in the hydrophilic resin Lowicryl K4M (3). Ultrathin sections were recovered on Formvar (Ladd Research Industries, Inc., Burlington, Vt.)coated grids and inverted over PBS. Lignin labeling in wood. Sections of the lignin samples were incubated directly with lignin peroxidase-gold (pH 5.5) or lignin peroxidase-gold (pH 7.0), diluted 1:10, 1:50, or 1:100 in PBS-1% BSA for 1 h at room temperature, and washed successively with PBS-0.1% BSA, PBS, and water. A control experiment was performed with a protein-gold complex which did not have any lignin peroxidase activity, such as protein A-gold (pH 7.0). An indirect labeling technique was also used. Sections were incubated for 1 h at room temperature on a (drop of) solution containing 1 ml of unlabeled lignin peroxidase in PBS-1% BSA, 0.5 ml of 0.33 M sodium tartrate buffer (pH 3.0), 0.17 ml of 4 mM veratryl alcohol, and 70 ,ul of 10.8 mM H202. These conditions have been reported to be optimal for lignin peroxidase activity (26). Sections were then washed *with PBS-0.1% BSA and incubated for 1 h at room temperature on anti-lignin peroxidase rabbit antiserum diluted 1:100 or 1:300 in PBS-1% BSA. Sections were then washed, incubated on protein A-gold diluted 1:100 in PBS-1% BSA, and washed successively with PBS-0.1% BSA, PBS, and water. Protein A-marker conjugates provide valuable visual tags based on the ability of protein A to bind in a specific manner to rabbit immunoglobulin G molecules (20). To demonstrate the specificity of the labeling, we performed several control experiments: (i) lignin peroxidase without H202, veratryl alcohol, or both H202 and veratryl alcohol; (ii) denatured lignin peroxidase after being heated at 70°C for 1 h; (iii) normal rabbit serum diluted 1:100 or 1:300; and (iv) protein A-gold. Lignin peroxidase labeling. Lignin peroxidase was localized in situ by incubating ultrathin sections of fungi grown on sawdust for 1 h at room temperature on an anti-lignin peroxidase antiserum diluted 1:100 or 1:300 in PBS-1% BSA and then washing them with PBS-0.1% BSA. Sections were then incubated for 1 h on protein A-gold diluted 1:100 in PBS-1% BSA and washed successively with PBS-0.1% BSA, PBS, and water. Controls included sections treated with normal rabbit serum or only protein A-gold. To test the specificity of the lignin peroxidase labeling in wooddestroying fungi, we performed the same immunocytochemical reactions on thin sections of two nonligninolytic fungi: Candida albicans 3153-A (Institut Pasteur) and Aspergillus fumigatus 1028 (Institut Pasteur), embedded in Lowicryl K4M as described above.
rotted pine that were partially hydrolyzed by ligninolytic fungi were not labeled with the enzyme probe. However, isolated MWL could be labeled by lignin peroxidase and detected by anti-lignin peroxidase antiserum-protein A-gold complex (Fig. lb), although lignin peroxidase-gold did not label MWL. Lignin peroxidase labeling. All the tested ligninolytic fungi except Pleurotus ostreatus could be labeled with the antilignin peroxidase antiserum-protein A-gold complex (Fig. lc to f). In P. chrysosporium and D. squalens, gold granules were found mainly intracellularly in the cytoplasmic area close to the plasmalemma (Fig. lc and d). Coriolus versicolor was only slightly labeled (only intracellularly) (Fig. le). No extracellular lignin peroxidase was detected in the proximity of the fungi or associated with the wood. Similarly to in vitro experiments, fungi degrading pine wood in nature were labeled with the anti-lignin peroxidase antiserum-protein A-gold complex (Fig. lg to i). Lignin peroxidase was concentrated mainly in the plasma membrane region (Fig. lg and h). Some lignin peroxidase-gold complexes were visualized extracellularly, sometimes trapped in the extracellular fungal slime (Fig. li), but never in direct contact with the wood. Wall channels possibly responsible for the transport of the enzyme to the exterior were detected (Fig. lh and i). No labeling of C. albicans or A. fumigatus was observed, indicating that the anti-lignin peroxidase-protein A-gold complex reaction was specific to lignin peroxidase produced by ligninolytic fungi.
RESULTS Lignin visualization. No labeling was observed after incubation of ultrathin sections of poplar cell walls with lignin peroxidase-gold complexes. Incubating the poplar sections with lignin peroxidase under the optimal conditions for in vitro activity did not improve the binding of the enzyme to the wood lignin (Fig. la). Similarly, treatments of the wood with H202 and chlorine water to produce reactive free radicals that would bind to the enzyme have also failed. The absence of labeling of all wood sawdust samples in the presence or absence of H202 and veratryl alcohol demonstrated that lignin peroxidase was unable to bind to the lignin of natural substrates. Also, poplar sawdust and the naturally
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DISCUSSION Lignin peroxidase production in vitro has been observed for several ligninolytic fungi (M. Leisola, unpublished data). The cross-reactivity observed between an anti-P. chrysosporium lignin peroxidase antiserum and the different fungi growing either under laboratory conditions or in naturally biodegraded wood suggests that other ligninolytic fungi are also able to produce similar or highly related lignin peroxidase complexes, which are stored near the plasmalemma. The absence of labeling in Pleurotus ostreatus indicates that this fungus produces an enzyme different from that produced by P. chrysosporium. Our recent results show that Pleurotus ostreatus produces extracellular lignin peroxidases which are not recognized by the P. chrysosporium antibodies by immunoblotting methods (M. Leisola and R. Waldner, unpublished results). Lignin peroxidase was located mainly intracellularly in naturally decayed wood, indicating that lignin peroxidase was not released, at least in high amounts, extracellularly. Moreover, no extracellular lignin peroxidase was detected when unfixed samples of P. chrysosporium grown on poplar sawdust were successively incubated with anti-lignin peroxidase antiserum and anti-rabbit immunoglobulin G-fluorescein isothyocyanate conjugate (S. Garcia and J. P. Latge, unpublished results). This experiment confirmed our transmission electron microscopy studies and indicated that the absence of external labeling was not due to the fixation and embedding treatments. However, our studies have demonstrated that the enzyme can be transported through wall channels to the external medium. More work is necessary to understand the kinetics of synthesis and release of the lignin peroxidase under natural conditions. The structure of the channels allowing the transport of proteins through the wall is presently unknown. However, several studies on zoopathogenic fungi have suggested that similar channels are involved in the secretion of antigens (25).
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FIG. 1. (a) Absence of labeling in poplar wood with lignin peroxidase (detected with anti-lignin peroxidase rabbit antiserum-protein A-gold). Magnification, x30,OOO. (b) Poplar MWL labeled with lignin peroxidase and detected with anti-lignin peroxidase-protein A-gold. Magnification, x60,000. (c to i) Localization of lignin peroxidase in different fungi by using an anti-lignin peroxidase from P. chrysosporium ATCC 24725 antiserum detected with protein A-gold. (c) P. chrysosporium (magnification, x40,000); (d) D. squalens (magnification, x60,000); (e) Coriolus versicolor (magnification, x40,000); (f) Pleurotus ostreatus (absence of labeling) (magnification, X40,000); (g to i) pine wood decayed in nature by unidentified fungi (arrows in panels h and i indicate possible wall channels involved in the liberation of the enzyme) (magnification, x60,000). Abbreviations: C, cytoplasm; W, fungal wall; Pp, primary wall; Sl, outer layer of secondary wall; S2, middle layer of secondary wall; S, slime layer. Bar, 0.2 ,um.
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The localization of lignin peroxidase inside the cell wall during the degradation of natural substrates may also indicate that in nature, lignin peroxidase is not necessary directly in contact with lignin. Harvey et al. (10) have suggested and Haemmerli et al. (9) demonstrated that the lignin peroxidase can oxidize lignin through one-electron transfer mediators such as veratryl alcohol. This means that no direct contact is necessary between the enzyme and the lignin. Nevertheless, the ability of the enzyme to recognize lignin is demonstrated, since it is able to bind to an isolated lignin, MWL. The absence of labeling of wood with lignin peroxidase-anti-lignin peroxidase-protein A-gold could be due to the inaccessibility of the lignin in wood to the active sites of the enzyme. Janshekar et al. (14) and Chua et al. (5) have shown that isolated lignins are tightly bound to the fungal mycelia during degradation by P. chrysosporium. We suggest that the lignin degradation in such a case needs a direct contact with the fungal cell wall and is initiated by lignin peroxidase and mediated by veratryl alcohol cation radicals. On the other hand, there are results based on electron microscopy (2) that show that lignin is being degraded at some distance from the fungal hyphae. With the present level of understanding of the lignin biodegradation mechanisms, these observations are difficult to explain. The enzymes such as lignin peroxidase and laccase, which are known to attack lignin, do not degrade lignin in vitro but, rather, polymerize it (9). Thus at some distance from the fungal hyphae, lignin could be modified by the action of veratryl alcohol cation radicals, activated oxygen species, or Mn(III). This kind of modifica-
8. Eriksson, K.-E., A. Grunewald, T. Nilsson, and L. Wallander. 1980. A scanning electron microscopy study of the growth and attack on wood by three white-rot fungi and their cellulaseless mutants. Holzforschung 34:207-213. 9. Haemmerli, S. D., M. Leisola, and A. Fiechter. 1986. Polymerisation of lignins by ligninase from Phanerochaete chrysosporium. FEMS Microbiol. Lett. 35:33-36. 10. Harvey, P. J., H. E. Schoemaker, and J. M. Palmer. 1986. Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Lett. 195:242-246. 11. Hodges, G. M., M. A. Smolira, and D. C. Livingston. 1984. Scanning electron microscopy immunocytochemistry in practice, p. 189-233. In J. M. Polak and I. M. Varndell (ed.), Immunolabelling for electron microscopy. Elsevier Science Publishers B.V., Amsterdam. 12. Hoffmann, P., and N. Parameswaran. 1976. On the ultrastructural localization of hemicelluloses within delignified tracheids of spruce. Holzforschung 30:62-70. 13. Horisberger, M. 1979. Evaluation of colloidal gold as a cytochemical marker for transmission and scanning electron microscopy. Biol. Cell. 36:253-258. 14. Janshekar, H., C. Brown, T. Haltmeier, M. Leisola, and A. Fiechter. 1982. Bioalteration of Kraft pine lignin by Phanerochaete chrysosporium. Arch. Microbiol. 132:14-21. 15. Kirk, T. K., S. Croan, M. Tien, K. E. Murtagh, and R. L. Farrell. 1986. Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8:27-32. 16. Laurell, C. B. 1965. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 10:358-361. 17. Leisola, M., U. Thanei-Wyss, and A. Fiechter. 1985. Strategies for production of high ligninase activities by Phanerochaete chrysosporium. J. Biotechnol. 3:97-107. 18. Liberman-Maxe, M. 1982. La differenciation des tracheides du polypode. Etude cytologique et cytochimique. Ann. Sci. Nat. Bot. Biol. Veg. 13(4):91-111. 19. Monties, B., E. Odier, G. Janin, and I. Czaninski. 1981. Ultrastructural evidence of bacterial and chemical delignification of poplar wood. Holzforschung 35:217-222. 20. Roth, J. 1982. The protein A-gold (pAg) technique-a qualitative and quantitative approach for antigen localization on thin sections, p. 108-133. In G. R. Bullock and P. Petrusz (ed.), Techniques in immunocytochemistry, vol. 1. Academic Press, Inc. (London), Ltd., London. 21. Ruel, K., F. Barnoud, and K.-E. Eriksson. 1981. Micromorphological and ultrastructural aspect of spruce wood degradation by wild-type Sporotrichum pulverulentum and its cellulaseless mutant cel 44. Holzforschung 35:157-171. 22. Ruel, K., and J. P. Joseleau. 1984. Use of enzyme-gold complexes for the ultrastructural localization of hemicelluloses in the plant cell wall. Histochemistry 81:573-580. 23. Saka, S., and R. J. Thomas. 1982. A study of lignification in loblolly pine tracheids by the SEM-EDXA technique. Wood Sci. Technol. 16:167-179. 24. Saka, S., R. J. Thomas, and J. S. Gratzl. 1978. Lignin distribution. Determination by energy-dispersive analysis of X rays. Tappi 61:473-476. 25. Takamiya, H., A. Vogt, S. Batsford, E. S. Kuttin, and J. Muller. 1985. Further studies on the immunoelectron microscopic localization of polysaccharide antigens on ultrathin sections of Candida albicans. Mykosen 28:17-22. 26. Tien, M., and T. K. Kirk. 1984. Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H202-requiring oxygenase. Proc. Natl. Acad. Sci. USA 81:2280-2284. 27. Vian, B., J. M. Brillouet, and B. Satiat-Jeunemaitre. 1983. Ultrastructural visualization of xylans in cell walls of hardwood by means of xylanase-gold complex. Biol. Cell. 49:179-182. 28. Wegener, G., and D. Fengel. 1979. Rapid ultrasonic isolation of milled wood lignins. Fractionation and degradation experiments. Tappi 62:97-100.
tion is expected to lead to aromatic ring opening and incorporation of oxygen into the lignin structure. Further demethoxylation and acid and quinone formation will probably occur (M. S. A. Leisola, S. D. Haemmerli, J. D. G. Smit, J. Troller, R. Waldner, H. E. Schoemaker, and H. Schmidt, I Int. Congr. Lignin Enzymic Microbial Degradation, Paris, 1987, p. 6). However, for real depolymerization of lignin, the equilibrium which favors spontaneous polymerization must be shifted by some means. This might occur by reductive reactions and by immediate incorporation of the degraded material through the fungal cell wall. For all of these processes the lignin must be located near the hyphae. To understand all the observations made so far on lignin degradation in vitro and in vivo, more basic research must be carried out. LITERATURE CITED 1. Bendayan, M. 1981. Electron microscopical localization of nucleic acids by means of nuclease-gold complexes. Histochem. J. 13:699-710.
2. Blanchette, R. A., and I. D. Reid. 1986. Ultrastructural aspects of wood delignification by Phlebia (Merulius) tremellosus. Appl. Environ. Microbiol. 52:239-245. 3. Carlemalm, E., R. M. Garavito, and W. Villiger. 1982. Resin development for electron microscopy and an analysis of embedding at low temperature. J. Microsc. (Oxford) 126:123-143. 4. Catesson, A. M. 1983. A cytochemical investigation of the lateral walls of Dianthus vessels. Differentiation and PITmembrane formation. Int. Assoc. Wood Anat. Bull. 4:89-101. 5. Chua, M. G. S., S. Choi, and T. K. Kirk. 1983. Mycelium
binding and depolymerization of synthetic 14C-labeled lignin during decomposition by Phanerochaete chrysosporium. Holzforschung 37:55-61. 6. Czaninski, I. 1979. Cytochimie ultrastructurale des parois du xyleme secondaire. Biol. Cell. 35:97-102. 7. Eggeling, L. 1983. Lignin-an exceptional biopolymer and a rich resource .? Trends Biotechnol. 1:123-127.
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