Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 199, p /9/11-8$./ Copyright 199, American Society for Microbiology Vol. 56, No. 1 Mn(II) Regulation of Lignin Peroxidases and Manganese-Dependent Peroxidases from Lignin-Degrading White Rot Fungi P. BONNARMEt AND T. W. JEFFRIES* Institute for Microbial and Biochemical Technology, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, Wisconsin 5375 Received 4 July 1989/Accepted 6 October 1989 Two families of peroxidases-lignin peroxidase (LiP) and manganese-dependent lignin peroxidase (MnP)- are formed by the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium and other white rot fungi. Isoenzymes of these enzyme families carry out reactions important to the biodegradation of lignin. This research investigated the regulation of LiP and MnP production by Mn(II). In liquid culture, LiP titers varied as an inverse function of and MnP titers varied as a direct function of the Mn(II) concentration. The extracellular isoenzyme profiles differed radically at low and high Mn(II) levels, whereas other fermentation parameters, including extracellular protein concentrations, the glucose consumption rate, and the accumulation of cell dry weight, did not change significantly with the Mn(II) concentration. In the absence of Mn(II), extracellular LiP isoenzymes predominated, whereas in the presence of Mn(II), MnP isoenzymes were dominant. The release of "'Co from "4C-labeled dehydrogenative polymerizate lignin was likewise affected by Mn(II). The rate of "'CO release increased at low Mn(II) and decreased at high Mn(II) concentrations. This regulatory effect of Mn(II) occurred with five strains of P. chrysosporium, two other species of Phanerochaete, three species of Phkbia, Lentinula edodes, and Phellinus pini. Phanerochaete chrysosporium is the most extensively studied lignin-degrading white rot basidiomycete. It has been used to elucidate lignin biodegradation with model compounds (17, 37), and it was the first organism for which lignin peroxidases (LiPs) were described (8, 1, 33, 35). More recently, P. chrysosporium has been shown to produce two other types of extracellular enzymes, Mn(II)-dependent peroxidases (, 3, 11, 9), and glyoxal oxidase, an enzyme involved in extracellular H production (14). Both LiPs and manganese-dependent lignin peroxidases (MnPs) are glycosylated heme proteins containing protoporphyrin IX, and both are believed to play roles in the biodegradation of lignin (3). LiP and MnP differ in their catalytic mechanisms. LiP acts by abstracting single electrons from aromatic rings of lignin and lignin model compounds, leading to the formation of a cation radical (15) and subsequent cleavage reactions (1). MnP acts by generating Mn (III), a highly reactive intermediate, which, when stabilized by chelators (7), can diffuse from the enzyme active site to attack and oxidize the lignin structure in situ (9, 3). LiP and MnP have been demonstrated in a number of white rot fungi (7,, 7, 36). Previous studies on the lignin-degrading basidiomycete P. chrysosporium have shown the importance of nutritional factors in the appearance of ligninolytic activity (13, 19). Using assays based on the release of 14CO, Keyser et al. (16) demonstrated that the ligninolytic enzyme system of P. chrysosporium is synthesized under nitrogen starvation. Nitrogen repression of ligninolytic activity has been well studied in P. chrysosporium (5, 6, 31), and this regulatory effect has been observed in other white rot fungi as well (). Carbon source and sulfur limitations likewise induce ligninolytic activity in P. chrysosporium (13). Jager et al. (1) first reported the production of lignin peroxidases in agitated * Corresponding author. t Present address: Laboratoire de Microbiologie, INRA-PG, 7885 Thiverval-Grignon, France. 1 submerged cultures with various detergents, and Leisola and Fiechter (3) induced lignin peroxidase production in agitated cultures by the addition of veratryl alcohol. Trace elements are important in the production of lignin peroxidases by P. chrysosporium (18). An earlier study seeking an optimum mineral nutrient balance showed that ligninolytic activity increases with decreasing Mn(II) concentrations (13). In reassessing the micronutrient balance required to obtain an optimum medium for enzyme production, we noted that Linko (4) used unusually low Mn(II) concentrations in achieving high LiP activity. We therefore investigated the effects of Mn(II) on LiP and MnP production in P. chrysosporium and in fungi that had been shown to be lignin degraders (1, 8, 36). MATERIALS AND METHODS Organisms. The following white rot fungi were obtained from the culture collection at the Center for Forest Mycology Research at the Forest Products Laboratory, Forest Service, U.S. Department of Agriculture, Madison, Wis.: Phanerochaete chrysosporium BKM-F-1767 (ATCC 475); Phanerochaete chrysosporium SC6 (a mutant of BKM- F-1767); Phanerochaete chrysosporium ME 446 (ATCC 34541); Phanerochaete chrysosporium K3; Phanerochaete chrysosporium HHB-651; Phanerochaete flavido alba FP 1657; Phanerochaete magnoliae JHG-366-sp; Phlebia radiata MJL-1198-sp; Phlebia tremellosa PRL-845; Phlebia subserialis RLG-674-sp; Lentinula (Lentinus) edodes RA- 3-E (ATCC 4885); Phellinus pini FP-5336-sp. Unless otherwise noted, P. chrysosporium BKM-F-1767 was used. Culture conditions. P. chrysosporium strains were cultivated at 39 C, and all other fungi were cultivated at 3 C. All the organisms were maintained on yeast extract-malt extract-peptone-glucose (YMPG) slants at 3 C. YMPG contained (per liter): 1 g of glucose, 1 g of malt extract, g of peptone, g of yeast extract, 1 g of asparagine, g of KHPO4, 1 g of MgSO4 7H, 1 mg of thiamine hydrochloride, and g of agar. Agitated cultures were grown in -liter

2 VOL. 56, 199 Erlenmeyer flasks containing 75 ml of medium or in 15-ml Erlenmeyer flasks containing 4 ml of medium. When a mycelial inoculum was used, 45 ml of medium was used in a 15-ml flask. The -liter flasks were shaken at 1 rpm (.5-cm-diameter stroke), and the 15-ml flasks were shaken at 18 rpm. The basal medium contained (per liter) 1 g of glucose,. g of D-diammonium tartrate, mm sodium tartrate (ph 4.5) (5), g of KHPO4,.5 g of MgSO4 7H,.1 g of CaCl.H, 1 mg of thiamine hydrochloride,.5 g of Tween,.5 mm veratryl alcohol (4), and 7 ml of a trace elements solution without MnSO4. This trace element solution contained (per liter) 1.5 g of nitriloacetic acid, 3. g of MgSO4 7H, 1. g of NaCl,.1 g of FeSO4 7H,.1 g of CoSO4,.1 g of CaCl- H,.1 g of ZnSO4-7H,.1 g of CuSO4 5H,.1 g of AlK(SO4). 1H,.1 g of H3BO3, and.1 g of NaMoO4- H. Mn(II) was added separately as MnSO4. H or MnCl- H to obtain the specified concentrations of Mn(II). Unless stated otherwise, Mn(II) was added as its sulfate form. All Mn(II) concentrations given in parts per million (milligrams per liter) are for the free ion and not the salt. Unless stated otherwise, cultures of P. chrysosporium BKM 1767 were inoculated with conidiospores from a 7- to 1-day-old slant (5.5 x 14 conidiospores per ml final concentration). For cultivation of the other fungi, the inoculum was prepared from 5- to 15-day-old mycelial mats from nonagitated cultures (75 ml volume) by blending them twice for 15 s in a Waring blender. Mats were not washed prior to blending. From 1 to 4 ml of this mycelial homogenate was used as the inoculum. In the experiment designed to test the effect of Mn(II) on various fungi, high Mn(II) corresponded to 39.8 ppm and low Mn(II) to.3 ppm. The basal level of Mn(II) used was ppm, as given by Kirk et al. (18). Mn(II) was added after 48 h of growth. At the same time, cultures were flushed for 1 min with pure oxygen (gas flow rate, 6.5 liters/min for 15-ml flasks and 1.5 liters/min for -liter flasks, 76 mm Hg, 1 C) and then for 1 min every day thereafter. Each experiment was done in triplicate and repeated at least once. To determine the isoenzyme profile, 75-ml cultures were grown either without Mn(II) or with the basal level, and triplicate cultures were harvested at 4 and at 6 days. Mycelial pellets were separated from the extracellular fluid by filtration, and the culture fluid was concentrated by ultrafiltration with an Amicon membrane (Amicon YM 1; 1, Mr cut-off). Fast protein liquid chromatography (FPLC) analyses of LiP and MnP isoenzymes were performed by the method of Kirk et al. (18). Enzymatic assays. Lignin peroxidase activity was determined spectrophotometrically at room temperature by the method of Tien and Kirk (34) with veratryl alcohol as a substrate. Mn(II)-dependent peroxidase activity was determined spectrophotometrically at room temperature by the method of Paszczynski et al. (3) with vanillylacetone as a substrate. Reaction mixtures contained 755 j.l of diluted culture fluid (5 to 1 [ul of enzyme sample plus water),.1 mm vanillylacetone, 1 mm sodium tartrate (ph 5),.1 mm MnSO4, and.5 mm H in a final volume of 1 ml. The reaction was started by adding H, and the rate of disappearance of vanillylacetone was monitored at 334 nm. The extinction coefficient used for vanillylacetone was 18,3 M1 cm-1. LiP and MnP assays were performed daily. In the experiment designed to test the effect of Mn(II) on LIGNIN-DEGRADING WHITE ROT FUNGI 11 { qb ~ 'rue fh) FIG. 1. Regulation of LiP and MnP production by Mn(II). (a) LiP activity and (b) MnP activity with basal level of Mn(II) (O) and without Mn(II) (U). production of MnP by various fungi, possible interference from laccase was checked by using the protocol for MnP as described, but without the addition of hydrogen peroxide. Analytical. Glucose analyses were performed on a highpressure liquid chromatography (HPLC) carbohydrate analysis column with Aminex resin HPX-87C (Bio-Rad Laboratories, Richmond, Calif.). Water was used as the eluant. Protein concentrations were determined by the Quantigold method as described in the supplier's guidelines (Diversified Biotech, Newton Center, Mass.). Residual nitrogen concentration was determined with the Kjeldahl procedure (3a). Dry weight was measured by filtering and drying the pellets overnight at 15 C. ['4C]DHP degradation. In the studies of 14C-labeled dehydrogenative polymerizate (DHP) degradation, experiments were done in six replicate cultures per condition. After 48 h of cultivation, [14C]DHP was added (5,6 dpm per flask). Flushing with pure oxygen was started at this time (38 mi/mn, 15 min, 76 mm Hg, 1 C). This flushing was performed every day thereafter, and the 14CO released was trapped in a scintillation cocktail (16). RESULTS Effect of basal Mn(II) concentration on enzymatic activity. Initial studies examined the effect of removing Mn(II) from the basal medium. Titers of LiP (Fig. la) and MnP (Fig. lb) were measured. In Mn(II)-free cultures, LiP activity appeared earlier and was.5 times higher than in cultures grown under the basal conditions [11.15 ppm Mn(II)]. On the other hand, cultures grown in a medium containing no Mn(II) produced only very low MnP activity (Fig. lb). At the basal Mn(II) concentration, MnP activity was 4 times higher. Nutrient nitrogen and protein concentration. With or without Mn(II) in the culture medium, nutrient nitrogen was consumed at the same rate, and essentially all nitrogen was

3 1 BONNARME AND JEFFRIES t Sw-S.b.. vv v w Time (h) 3 _ 5 Go r. to PS 1a FIG.. Residual nitrogen and extracellular protein during enzyme production. Residual nitrogen was assayed with basal level of Mn(II) ([1) and without Mn(II) (-); extracellular protein was assayed with basal level of Mn(II) () and without Mn(II) (). taken up within 5 h (Fig. ). Small amounts of extracellular protein were present prior to 5 h, but once nutrient nitrogen was depleted, protein concentration in the extracellular culture fluid increased dramatically. This increase corresponded with the appearance of LiP and MnP activities (Fig. 1). However, the maximum protein concentration did not correspond to the maximum peak for enzymatic activity. Glucose consumption and cell growth. The presence or absence of Mn(II) had no significant effect on the rate of glucose consumption, nor did it have a significant effect on growth as measured by cell dry weight (Fig. 3). Under the conditions used here, cultures never became carbon limited (Fig. 3). The medium of cultures receiving the basal level of Mn(II) turned brown after about 48 h, whereas it turned yellowish without added Mn(II). Isoenzyme profiles. When P. chrysosporium was grown in the absence of Mn(II), seven separate isoenzymes of LiP activity were apparent (Fig. 4a and b). These were designated P1, P, P5, P6, P7, P8, and P9. Each of these protein peaks (as determined by A8 measurements) was associated with a heme (as indicated by absorption at 49 nm). The relative abundances of these seven isoenzymes were observed to shift when isoenzymes profiles were observed on day 4 (Fig. 4a and c) and day 6 (Fig. 4b and d). Notably, P5 and P7 decreased relative to P, but no major new proteins appeared. The amount of P8 decreased so that LiP activity.9 D 1 8 j w Time (h) FIG. 3. Consumption of glucose and accumulation of mycelial dry weight during enzyme production. Symbols:, dry weight with basal Mn(II); *, dry weight without Mn(II);, glucose concentration with basal Mn(II);, glucose concentration without Mn(II)..H APPL. ENVIRON. MICROBIOL. was no longer detectable in this fraction. Two small protein peaks (P3 and P4) had some MnP but no LiP activity. One protein peak without LiP or MnP activity (eluted in the region of P3) was observed. This peak did not appear to have an associated 49-nm absorption spectrum. When the basal concentration of Mn(II) was used, LiP isoenzymes P5 and P7 were greatly reduced relative to P, and five MnP isoenzymes were present (Fig. 4c and d). These are designated P3a, P3b, P4a, P4b, and P4c. Not all of the MnP isoenzymes were fully resolved by the analytical technique used. Essentially the same isoenzymes were present, albeit in different ratios, after 6 days of incubation (Fig. 4c and d). Effect of the time of Mn(II) addition. To know whether or not Mn(II) could induce MnP activity or repress LiP activity, we added 8.5 ppm Mn(II) to Mn(II)-free cultures at various times. The highest levels of LiP activity were attained when no Mn(II) was present (Fig. 5a). Mn(II) repressed LiP most when it was added 4 h after inoculation. Additions after 48, 7, and 96 h also repressed LiP activity, but in no instance did the time of Mn(II) addition affect the time of appearance of LiP activity. The time of Mn(II) addition had quite a different effect on the appearance of MnP activity (Fig. 5b). Very little activity was apparent in the absence of Mn(II), and the time of appearance was delayed until after Mn(II) addition. When Mn(II) was added after 7 or 96 h, MnP activity appeared and accumulated shortly thereafter. Regardless of the time of addition, however, cultures receiving the same concentration of Mn(II) all attained approximately the same titer of MnP activity. Effect of Mn(II) concentration. We examined the effect of adding various concentrations of Mn(II) to actively growing (48-h-old) cultures of P. chrysosporium. In these experiments, Mn(II) was added as MnSO4, but cultures in which Mn(II) was added as MnCl gave the same results (data not shown). Low concentrations of Mn(II) (<1.6 ppm) tended to stimulate production of LiP activity, but as the concentration of Mn(II) was increased beyond 8. ppm, the appearance of LiP activity was completely repressed (Fig. 6a). In contrast, essentially no MnP activity was present unless Mn(II) was added. MnP activity increased steadily with increasing Mn(II) concentration and reached a peak at about 4 ppm. A higher concentration of Mn(II) appeared to be slightly inhibitory (Fig. 6b). Effect of Mn(II) on other white rot fungi. In an attempt to examine the generality of the regulatory effect of Mn(II) on lignin peroxidases and Mn(II)-dependent peroxidases, we cultivated 13 different white rot fungi (including several strains of P. chrysosporium) under low or high concentrations of Mn(II). Most of these fungi do not sporulate well in laboratory culture, so to enable direct comparison of the results, a mycelial inoculum was used for all of them. Mn(II) had an inductive effect on MnP activity and a repressive effect on LiP activity when a high Mn(II) concentration was used. Five different strains of P. chrysosporium demonstrated this response, all to similar extents (Table 1). The highest LiP activity was obtained with P. chrysosporium BKM 1767 and the highest MnP activity was obtained with strain SC6, but these differences might not be significant. Some of these fungi grew better than others, so the rankings shifted when activities were normalized to the mycelial dry weights (Table ). The highest LiP specific activity was obtained with K3, and the highest MnP specific activity was obtained with SC6. A similar effect of Mn(II) was observed with the other white rot fungi examined. A low concentra-

4 VOL. 56, 199 LIGNIN-DEGRADING WHITE ROT FUNGI 13 (b) ). co D o en Co ) co 1) (C) (d) Downloaded from p 7 on April 13, 19 by guest Elution time (minutes) FIG. 4. Extracellular isoenzymes formed in the presence and absence of Mn(II) after 4 or 6 days of cultivation. Fractions associated with lignin peroxidase activity are shaded; fractions containing Mn(II)-dependent peroxidase are unshaded (see text for description of activities). Solid line, A49; broken line, A8. tion of Mn(II) resulted in better production of LiP, whereas at a high Mn(II) concentration this activity was lower or absent. MnP activity was detected in all the fungi we studied. In some instances, it was much higher in the presence of high Mn(II); in other instances, it was only marginally higher. When hydrogen peroxide was left out of the reaction mixture, no oxidation of vanillylacetone was observed, indicating that laccase did not interfere with our assay for MnP activity.

5 14 BONNARME AND JEFFRIES APPL. ENVIRON. MICROBIOL. I.1 I ' E. I bo.5 -J U, U)3 EUW 'E.5 U, U)4 U, EU EU Time (h) FIG. 5. Effect of the time of Mn(II) addition on the production of LiP activity (a) and MnP activity (b) at 4 h (U), 48 h (O), 7 h (), and 96 h () or after no addition (A). Effect of Mn(II) on 14Co release. We investigated the effect of Mn(II) on ligninolytic activity as determined by the release of 14CO from 14C-labeled DHP. As shown in Fig. 7, Mn(II) strongly repressed ligninolytic activity in agitated cultures of P. chrysosporium grown under nitrogen limitation. Ligninolytic activity of P. chrysosporium was more than seven times higher with low Mn(II) than with high Mn(II). High LiP and low MnP activities were measured in cultures grown under low Mn(II). High MnP activity but no LiP activity was detected in the high-mn(ii) cultures. Therefore, low Mn(II) correlated with low MnP, high LiP, and a high rate of 14CO release, while high Mn(II) correlated with high MnP, low LiP, and a low rate of 14CO release. DISCUSSION These results show that in nitrogen-limited cultures, Mn(II) plays an important role in regulating the appearance of LiP and MnP activity in several different white rot fungi and that Mn(II) can also regulate the overall mineralization of [14C]DHP lignin. Because Mn(II) is a substrate for MnP, the effect could be substrate-level induction, but this needs further study. Unlike the previously reported effects of nitrogen, carbon, and sulfur, Mn(II) affects the cell in a relatively specific manner. The production of mycelial dry weight and extracellular protein is not affected, nor are the rates of consumption of carbon and nitrogen sources. The effect of Mn(II) is also relatively easy to demonstrate. Sulfur limitation, for example, is effective only over a.c 'au E or U, la EU4 Go Time (h) FIG. 6. Effect of the concentration of Mn(II) on the production of LiP activity (a) and MnP activity (b) with no Mn(II) (U),.3 ppm (O), 1.6 ppm (), 8. ppm (), 4. ppm (A), and 199. ppm (A). narrow range and only advances the time of onset of ligninolytic activity. Carbon limitation effects a rapid onset, but the resulting mycelial mat is so frail that the burst of activity lasts only a few days and the mycelia appear to be autocatabolized (13). Nitrogen limitation has often been used to induce ligninolytic activity. The onset of lignin biodegradation under nitrogen limitation is delayed by about 4 h following nitrogen depletion (13). During this time, a rapid turnover of nitrogen occurs (5). Lignin biodegradation can be sustained as long as carbohydrate is present, but it is very difficult to maximize extracellular enzyme production while restricting available nitrogen. Both carbohydrate and nitrogen regulation of lignin biodegradation by white rot fungi are generalized metabolic responses that might be mediated through a secondary effector. The regulation by Mn(II) appears to be more specific. No loss of mycelial dry weight occurs, and different effects are observed with different components of the lignindegrading enzyme system. Indeed, Mn(II) could be a powerful tool to study LiP and MnP regulation. Our results show that when Mn(II) is low, mineralization of DHP proceeds rapidly, whereas when Mn(II) is high, mineralization of DHP is slower. The results suggest that MnP activity is not rate limiting in DHP mineralization, because 14CO production was highest when MnP was lowest and vice versa. Previous studies have shown that LiP activity and ligninolytic activity (DHP degradation) are simultaneous (), and our results are in agreement.

6 VOL. 56, 199 TABLE 1. Effect of Mn(II) on the titer of extracellular LiPs and MnPs produced by selected white rot fungia Activity (nmol/min per ml) Species and Lip MnP strain Low High Low High Mn(II) Mn(II) Mn(II) Mn(II) P. chrysosporium BKM 51 (7)b 86 (8) 1,789 (6) SC6 33 (7) 95 (7) 1,915 (6) ME446 8 (7) 157 (7) 1,69 (7) K3 197 (6) 3 (6) 1,548 (5) HHB (8) 9 (7) 696 (7) Lentinula edodes 17 (9) 4 (9) 1,146 (9) Phanerochaeteflavido alba 5 (9) 34 (6) 781 (6) Phanerochaete magnoliae (9) 46 (9) 79 (9) Phellinus pini 7 (8) 5 (8) 9 (9) Phlebia radiata 33 (9) 167 (7) 835 (7) Phlebia subserialis 6 (9) 146 (9) Phlebia tremellosa (1) 56 (1) a All experiments were performed in triplicate, and the standard deviation was less than 1%o of the mean. b Numbers in parentheses indicate the time of maximum activity (in days). We noted in comparing the time courses for extracellular protein accumulation with those for enzymatic activity that enzyme activity lagged behind protein accumulation. The delayed appearance of enzymatic activity might mean that once excreted, the enzymes need a "maturation period" to become active. We do not know whether extracellular processing occurs. Our observations on extracellular isoenzymes agree fairly well with those of other researchers. In cultures of BKM grown with Mn(II), we separated five MnPs and six to seven LiPs. These results are in good accordance with previous studies by Kirk et al. (18) (Table 3). In cultures grown without Mn(II) we observed one to two minor MnPs and seven LiPs. These results are consistent with recent work TABLE. Effect of Mn(II) on the specific activity of extracellular LiPs and MnPs produced by selected white rot fungi Sp acta (nmol/min per mg [dry wt] of mycelia) Species and LiP MnP strain Low Mn(II) High Mn(II) Low Mn(II) High Mn(II) P. chrysosporium BKM 15 ndb 51 1,38 SC6 114 nd 47 1,348 ME nd K3 173 nd 1,144 HHB651 6 nd 1 67 Lentinula edodes 18 nd Phanerochaeteflavido alba 31 nd Phanerochaete magnoliae nd 41 5 Phellinus pini 4 nd 73 Phlebia radiata 7 nd Phlebia subserialis nd nd Phlebia tremellosa nd nd 16 5 a Specific activity was determined on the day of maximum activity (see Table 1). b nd, Activity not detected. LIGNIN-DEGRADING WHITE ROT FUNGI 15 LI ~ rtme ) FIG. 7. Time course of '4CO release by P. chrysosporium at low (U) and high (l) Mn(II) levels. published by Linko (4), in which she reported seven LiPs and two MnPs in the extracellular fluid of P. chrysosporium grown under glucose limitation instead of nitrogen limitation. As noted above, Linko also used relatively low levels of Mn(II). Our results demonstrate that Mn(II) plays a key role in the regulation of LiP and MnP of P. chrysosporium and other white rot fungi that are effective lignin degraders in wood (8). All studies reported here were performed under conditions optimized for P. chrysosporium. MnP activity was produced by all the fungi studied. LiP was detected in most but not all other organisms tested. It is possible that the conditions we used were simply not appropriate for LiP production by these other white rot fungi. Manganese could regulate the appearance of these enzymes when they are growing on wood. Because of the complexity of native lignin and its inaccessibility in wood, it is difficult to discern which enzyme systems actually function in lignin biodegradation. Our results clearly show that LiP is formed in liquid cultures of P. chrysosporium when Mn(II) is present at less than 8 ppm. The concentration of Mn varies greatly with the wood species (6, 3, 38), but generally it is present in trunk wood at concentrations ranging from to ppm. It can be even higher in bark. We do not know whether this Mn is bound or otherwise inaccessible or if it is available to the organism, but it is possible that it could play a role in suppressing LiP activity. At such Mn concentrations, MnP would be induced. MnP acts by oxidizing Mn(II) to Mn(III), which then diffuses into the wood. After a period of time, Mn might be depleted in the region of the fungal hypha, and LiP would be derepressed. LiP acts directly on lignin substructures, and fol- TABLE 3. Comparison of heme proteins from extracellular fluid of P. chrysosporium BKM with isozymes identified in previous work Peak no. LiP activity MnP activity Tentative (49 nm) assignment' P1 + - Hl P + - H P3a,b - + H3 P4a,b - + H4 P4c - + H5 P5 + - H6 P6 + - H7 P7 + - H8 P8 + - H9 P9 + - H1 a See reference 18.

7 16 BONNARME AND JEFFRIES lowing the activity of MnP, the lignin might be more susceptible to LiP attack. Wood decayed by white rot fungi contain black regions and flecks with large concentration of Mn (4), indicating that these organisms mobilize manganese in wood, so we would expect Mn to play a role in regulation in situ. In conclusion, our results show that Mn(II) regulates production of LiPs and MnPs. More work must be done to demonstrate the role of these enzymes in the actual in vivo degradation of lignin. This finding provides a very simple means of control over the production of either LiPs or MnPs, over the ligninolytic ability of P. chrysosporium, and over the enzymatic activity of various other white rot fungi as well. It might also provide a way to produce high titers of these enzymes in liquid culture. Our results also indicate that Mn(II) might regulate ligninolytic activity in wood. ACKNOWLEDGMENTS We are grateful to others at the Forest Products Laboratory (Madison), J. Popp, P. Kersten, and T.K. Kirk, for valuable discussions, and M. Wesolowski for nitrogen analyses. P.B. thanks T. K. Kirk, Forest Products Laboratory (Madison), and G. Goma, Centre de Transfert en Biotechnologie Microbiologie (Toulouse, France), for the support necessary to complete these experiments. LITERATURE CITED 1. Agosin, E., J.-J. Daudin, and E. Odier Screening of white-rot fungi on (14C) whole-labelled wheat straw. Appl. Microbiol. Biotechnol. : Anderson, L. A., V. Renganatham, A. A. Chiu, T. M. Loehr, and M. H. Gold Spectral characterization of dairylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme. J. Biol. Chem. 6: Asada, Y., M. Miyabe, M. Kikkawa, and M. Kuwahara An extracellular NADH-oxidizing peroxidase produced by a lignin-degrading basidiomycete, Phanerochaete chrysosporium. J. Ferment. Technol. 65: a.Association of Official Analytical Chemists Total nitrogen, p Sections Official methods of analyis, 13th ed. Association of Official Analytical Chemists, Washington, D.C. 4. Blanchette, R. A Manganese accumulation in wood decayed by white-rot fungi. Phytopathology 6: Fenn, P., S. Choi, and T. K. Kirk Ligninolytic activity of Phanerochaete chrysosporium: physiology of suppression by NH4' and L-glutamate. Arch. Microbiol. 13: Fenn, P., and T. K. Kirk Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium. Arch. Microbiol. 13: Forrester, I. T., A. C. Grabski, R. R. Burgess, and G. F. Leatham Manganese, Mn-dependent peroxidases and the biodegradation of lignin. Biochem. Biophys. Res. Commun. 157: Glenn, J. K., M. A. Morgan, M. B. Mayfield, M. Kuwahara, and M. H. Gold An extracellular H-requiring enzyme preparation involved in lignin biodegradation by the white-rot fungi basidiomycete Phanerochaete chrysosporium. Biochem. Biophys. Res. Commun. 114: Gold, M. H., H. Wariishi, L. Akileswaran, Y. Mino, and T. M. Loehr Spectral characterization of Mn-peroxidase, an extracellular heme enzyme from Phanerochaete chrysosporium, p In E. Odier (ed.), Lignin enzymic and microbial degradation. INRA Publications, Versailles, France. 1. Hammel, K. E., M. Tien, B. Kalyanaraman, and T. K. Kirk Mechanism of oxidative CQ,-C, cleavage of a lignin model dimer by Phanerochaete chrysosporium linginase. J. Biol. Chem. 6: Huynh, V.-B., and R. L. Crawford Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium. FEMS Microbiol. Lett. 8: APPL. ENVIRON. MICROBIOL. 1. Jager, A., S. Croan, and T. K. Kirk Production of ligninases and degradation of lignin in agitated submerged cultures of Phanerochaete chrysosporium. Appl. Environ. Microbiol. 5: Jeifries, T. W., S. Choi, and T. K. Kirk Nutritional regulation of lignin degradation by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 4: Kersten, P. J., and T. K. Kirk Involvement of a new enzyme, glyoxal oxidase, in extracellular H production by Phanerochaete chrysosporium. J. Bacteriol. 169: Kersten, P. J., M. Tien, B. Kalyanaraman, and T. K. Kirk The ligninase of Phanerochaete chrysosporium generates cation radicals from methoxybenzenes. J. Biol. Chem. 6: Keyser, P., T. K. Kirk, and J. G. Zeikus Ligninolytic enzyme system of Phanerochaete chrysosporium synthesized in the absence of lignin in response to nitrogen starvation. J. Bacteriol. 135: Kirk, T. K., W. J. Connors, R. D. Bleam, W. F. Hackett, and J. G. Zeikus Preparation and microbial decomposition of synthetic ['4C]lignins. Proc. Natl. Acad. Sci. USA 7: Kirk, T. K., S. Croan, M. Tien, K. E. Murtagh, and R. L. Farrel Production of multiple ligninases by Phanerochaete chrysosporium: effect of selected growth conditions and use of a mutant strain. Enzyme Microb. Technol. 8: Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G. Zeikus Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117: Kirk, T. K., M. Tien, S. C. Johnsrud, and K.-E. Eriksson Lignin-degrading activity of Phanerochaete chrysosporium Burds.: comparison of cellulase-negative and other strains. Enzyme Microb. Technol. 8: Kirk, T. K., M. Tien, P. J. Kersten, M. D. Mozuch, and B. Kalyanaraman Ligninase of Phanerochaete chrysosporium: mechanism of the degradation of the non-phenolic arylglycerol 3-aryl ether substructure of lignin. Biochem. J. 36: Leatham, G. F., and T. K. Kirk Regulation of ligninolytic activity by nutrient nitrogen in white-rot basidiomycetes. FEMS Microbiol. Lett. 16: Leisola, M. S. A., and A. Fiechter Ligninase production in agitated conditions by Phanerochaete chrysosporium. FEMS Microbiol. Lett. 9: Linko, S Production and characterization of extracellular lignin peroxidase from immobilized Phanerochaete chrysosporium in a 1-L bioreactor. Enzyme Microb. Technol. 1: Linko, S., L. C. Zhong, M. S. A. Leisola, Y.-Y. Linko, A. Fiechter, and P. Linko Lignin peroxidase production by immobilized Phanerochaete chrysosporium in repeated batch shake cultures, p In E. Odier (ed.), Lignin enzymic and microbial degradation. INRA Publications, Versailles, France. 6. Matusiewicz, H., and R. M. Barnes Tree ring wood analysis after hydrogen peroxide pressure decomposition with inductively coupled plasma atomic emission spectrometry and electrothermal vaporization. Anal. Chem. 57: Niku-Paavola, M.-L., E. Karhunen, P. Salola, and V. Raunio Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem. J. 54: Otjen, L., R. Blanchette, M. Effland, and G. Leatham Assessment of 3 white-rot basidiomycetes for selective lignin degradation. Holzforschung 41: Paszczynski, A., V.-B. Huynh, and R. Crawford Enzymatic activities of an extracellular manganese-dependent peroxidase from Phanerochaete chrysosporium. FEMS Microbiol. Lett. 9: Paszczynski, A., V.-B. Huynh, and R. Crawford Comparison of ligninase-1 and peroxidase-m from the white-rot fungus Phanerochaete chrysosporium. Arch. Biochem. Biophys. 44: Reid, I. D Effects of nitrogen supplements on degradation

8 VOL. 56, 199 LIGNIN-DEGRADING WHITE ROT FUNGI 17 of aspen wood lignin and carbohydrate components by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 45: Taneda, K., M. Ota, and M. Nagashima The radial distribution and concentration of several chemical elements in woods of five Japanese species. Mokuzai Gakkaishi 3: Tien, M., and T. K. Kirk Lignin-degrading enzyme from hymenomycete Phanerochaete chrysosporium Burds. Science 1: Tien, M., and T. K. Kirk Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization and catalytic properties of a unique H-requiring oxygenase. Proc. Natl. Acad. Sci. USA 81: Tien, M., T. K. Kirk, C. Bull, and J. A. Fee Steady-state and transient-state kinetic studies on the oxidation of 3,4- dimethoxybenzyl alcohol catalyzed by the ligninase of Phanerochaete chrysosporium Burds. J. Biol. Chem. 61: Waldner, R., M. S. A. Leisola, and A. Fiechter Comparison of ligninolytic activities of selected white-rot fungi. Appl. Microbiol. Biotechnol. 9: Weinstein, D. A., K. Krisnangkura, M. B. Mayfield, and M. D. Gold Metabolism of radiolabeled P-guaiacyl ether-linked lignin dimeric compounds by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 39: Young, H. E., and V. P. Guinn Chemical elements in complete mature trees of seven species in Maine. Tappi 49: