Manganese Deficiency Can Replace High Oxygen Levels Needed for Lignin Peroxidase Formation by Phanerochaete chrysosporium

Abstract

The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.

The basidiomycete Phanerochaete chrysosporium is the most extensively characterized white rot fungus. The major components of the lignin-degrading enzyme system of this organism are members of two families of extracellular glycosylated heme peroxidases, lignin peroxidase (LIP) and manganese peroxidase (MNP) (21, 40). Expression of the ligninolytic enzymes by P. chrysosporium is an idiophasic event triggered by nitrogen or carbon limitation and is highly dependent on culture conditions and medium composition (9, 12, 38, 43). The formation of one of the two enzymes, LIP, is particularly dependent on exposure of cultures to high oxygen tensions (8, 12). It has been proposed that oxygen transfer into stationary cultures is restricted, and consequently a high partial pressure of oxygen in the culture headspace is needed to make sufficient oxygen available to the submerged hyphae (22, 26). Indeed, LIP formation was recently observed in a culture that was exposed to air by immobilizing the fungus on a porous support in a nonimmersed liquid culture, which improved the availability of oxygen to the fungus (8, 32). The concentration of Mn²⁺ in the medium has been reported to affect the formation of LIP and MNP. The two families of enzymes are regulated differently by Mn; while the formation of MNP is dependent on Mn, which enhances transcription of the enzyme (2, 4, 14, 30), LIP formation is inhibited by Mn. However, LIP gene transcription reportedly is not directly repressed by Mn (2, 14, 23, 25, 30). Mn also affects lignin biodegradation; the rate of synthetic lignin mineralization decreases in the presence of high levels of Mn (29). Although the suppressive effect of Mn on LIP formation in cultures of P. chrysosporium has been widely described, the mechanism of the regulatory effect of Mn on LIP formation is not clear. Mn is known to possess antioxidant properties. As a free metal ion, Mn²⁺ has the ability to act in the reverse mode of the Fenton reaction, thus destroying free radicals (5, 6 39). In addition, Mn serves as a cofactor of Mn superoxide dismutase (Mn-SOD), which plays a dominant role in protecting cells against oxidative stress by converting superoxide radicals into H₂O₂, a less reactive oxygen species. Hence, the regulatory effect of Mn on LIP formation may be mediated by the effect of Mn on the level of oxygen radicals in the fungus. In this work, we found that in both N-limited and N-excess cultures of P. chrysosporium the high oxygen level required for LIP formation could be replaced by Mn deficiency. In addition, Mn deficiency correlated with suppression of intracellular Mn-SOD activity.

MATERIALS AND METHODS

Strain and culture conditions.

P. chrysosporium Burds BKM-F-1767 (=ATCC 24725) was maintained at 4°C on 2% malt extract agar slants. The growth medium was based on the medium described by Tien and Kirk (41) but contained 20 mM acetate buffer (pH 4.5) instead of dimethyl succinate buffer, as previously reported (7). The initial glucose concentration was 56 mM (10 g/liter), and the initial concentrations of nitrogen (as diammonium tartrate) were 2.4 and 45 mM for nitrogen-limited and nitrogen-excess cultures, respectively. The culture medium contained 0.2 mM veratryl alcohol unless indicated otherwise. To monitor the effect of Mn deficiency on LIP formation, the shallow stationary cultures used for inoculum preparation were grown without added Mn. To monitor the effect of the Mn level on LIP formation, various Mn concentrations ranging from 0 to 1,500 mg/liter were added as MnSO₄ at the inoculation stage. The background level of Mn in the Mn-deficient medium was 0.03 mg/liter. The shallow stationary cultures were incubated at 37°C in 250-ml Erlenmeyer flasks containing 20 ml of culture medium. The flasks were sealed with rubber stoppers and were flushed daily for 2 min with pure oxygen by using 30-gauge hypodermic needles (length, 3 cm 23 gauge) and a flow rate of 500 ml/min. The oxygen gas used was medical grade. Flasks containing cultures grown under free air exchange conditions were sealed with dense-paper plugs (Heinz Herenz, Hamburg, Germany). Nonimmersed liquid culture system experiments were conducted as described by Dosoretz et al. (10). Three or more replicates were performed for all experiments.

Enzymatic activities.

LIP activities were measured in the extracellular fluids of the fungal cultures as described by Tien and Kirk (42); 1 U of activity was defined as the amount of activity that oxidized 1 μmol of veratryl alcohol to veratraldehyde per min. MNP activity was measured as described by Kuwahara et al. (21); phenol red was used as the substrate, and 1 U of activity was defined as the amount of activity that oxidized 1 μmol of veratryl alcohol to veratraldehyde per min. Mn-SOD activity was measured in cell extracts prepared from fungal mycelium by using the polyacrylamide gel electrophoresis (PAGE) staining procedure (1). Mycelium samples were washed with double-distilled water and extracted by extruding the mycelium by several passages through two syringes connected by a small-orifice valve. The homogenate was centrifuged for 5 min at 10,000 × g, and the supernatant was used for the Mn-SOD activity assay. The assay was performed by electrophoresing 10-μg protein samples on native slab gels containing 8% polyacrylamide by using the Bio-Rad Mini Protean II gel electrophoresis system. The gels were electrophoresed at 10 mA for 14 h at 4°C. After electrophoresis, the gels were incubated at room temperature for 30 min in the dark in 80 ml of 0.1 M potassium phosphate buffer (pH 7.8) containing 1 mM EDTA, 1 mg of riboflavin, 16 mg of nitroblue tetrazolium, 200 μl of N,N,N′,N′-tetramethylethylenediamine, and 2 mM cyanide. The gels were then exposed for about 30 min to visible light on a light table, until the background color turned dark blue upon reduction of the nitroblue tetrazolium (1).

Heme protein detection.

Equal volumes of extracellular fluid were concentrated 25-fold by ultrafiltration with a 10-kDa-cutoff type PM-10 membrane (Amicon, Danvers, Mass.), centrifuged for 10 min at 25,000 × g, and then dialyzed against 10 mM sodium acetate (pH 6.0). Samples were analyzed for heme protein by anion-exchange high-performance liquid chromatography by using a MonoQ column (Pharmacia, Piscataway, N.J.) and a flow rate of 1 ml/min and monitoring the absorbance at 409 nm (19). Heme proteins (isozymes H1 to H10) were identified on the basis of elution properties and the results of activity tests, as reported previously (7, 37).

Analytical techniques. (i) Protein and glucose contents.

Protein contents were determined with a Bio-Rad protein determination kit as recommended by the manufacturer; crystalline bovine serum albumin (fraction V) was used as the standard. The glucose level was determined by the dinitrosalicylic acid method, as described by Ghose (13).

(ii) Dry weight.

The mycelium dry weight was determined on day 4 of cultivation by washing the mycelium twice in distilled water and lyophilizing the samples to complete dryness.

(iii) Soluble Mn concentrations.

The concentrations of soluble Mn in the cultures were determined after the colloidal Mn was removed by centrifuging the extracellular fluid at 20,000 × g for 20 min. The extracellular fluid was acidified with 5% (wt/vol) HNO₃, and the preparation was analyzed with an inductively coupled plasma-atomic emission spectrometer (Atomscan 16; Thermo Jorrel Ash, Franklin, Mass.)

RESULTS

Effects of Mn and O₂ on growth, LIP and MNP activities, and isozyme composition.

The combined effects of Mn and oxygen levels on the formation of LIP by P. chrysosporium were studied by using shallow stationary cultures grown under both N-limited and N-excess conditions and exposed to air or oxygen (Fig. 1). Similar trends in LIP activity were observed in the N-limited and N-excess cultures which were exposed to oxygen; under these conditions the highest levels of activity (200 to 250 U/liter) were found in cultures grown with Mn. The levels of activity in cultures grown without added Mn were only about one-half the levels of activity in cultures grown with added Mn. No LIP activity at all was detected in cultures exposed to air and grown with Mn. Unexpectedly, significant levels of LIP activity were found in both N-limited (120 U/liter) and N-excess (180 U/liter) cultures grown in the presence of air without Mn, although the onset of LIP activity was delayed in N-excess cultures (LIP activity appeared on day 7). The typical darkening of the cultures, which was attributed to precipitation of MnO₂ (17), was not observed in Mn-deficient cultures and in cultures grown in the presence of air.

FIG. 1.

LIP activity profiles in shallow stationary cultures of P. chrysosporium grown under N-limited (A) and N-excess (B) conditions and exposed to air or to oxygen with and without added Mn²⁺. The concentrations of Mn²⁺ added were 30 and 300 mg/liter for N-limited and N-excess cultures, respectively. The error bars show the standard deviations. oxy, oxygen.

It was evident from our monitoring of the effects of oxygen and Mn levels on the heme protein profiles in the extracellular fluid (Fig. 2) that H1 and H2 were the predominant LIP isozymes in N-excess cultures, whereas H2 and (to some extent) H6 and H8 were the predominant isozymes in N-limited cultures. In addition to the dominant effect that N concentration had on the ratio of H1 to H2, this ratio was affected by Mn, and higher ratios were obtained with cultures containing high Mn concentrations than with Mn-deficient cultures. MNP isozyme H4 was detected in N-limited cultures supplemented with Mn (Fig. 2b), whereas in N-excess cultures (Fig. 2a) only trace amounts of MNP isozyme H4 appeared. This finding is consistent with previous reports which described the repression of MNP by nitrogen (10, 14).

FIG. 2.

Heme protein profiles of the extracellular fluids of cultures of P. chrysosporium grown with and without added Mn²⁺. Samples of extracellular fluid were removed at the times corresponding to peak LIP activity. (a) N-excess culture containing Mn grown in the presence of oxygen. (b) N-limited culture containing Mn grown in the presence of oxygen. (c) N-excess culture not containing Mn grown in the presence of oxygen. (d) N-limited culture not containing Mn grown in the presence of oxygen. (e) N-excess culture not containing Mn grown in the presence of air. (f) N-limited culture not containing Mn grown in the presence of air. AU, absorbance units.

In order to study the combined effects of oxygen and various Mn concentrations on LIP formation, N-excess cultures of P. chrysosporium were grown either in the presence of air or in the presence of oxygen with Mn at concentrations ranging from 0 to 1500 mg/liter. In cultures exposed to oxygen, the LIP activity remained at a consistently high level regardless of the Mn concentration, except in cultures without added Mn, which formed low levels of LIP. However, it should be noted that the appearance of LIP activity was delayed by high Mn concentrations. In cultures exposed to air, LIP activity was highest when the medium did not contain Mn, was 14 and 80% lower in the presence of 3 and 13 mg of Mn per liter, respectively, and was completely absent at an Mn concentration of 60 mg/liter or higher (Fig. 3). The MNP activity was affected by the Mn concentration. For cultures grown in the presence of oxygen, the level of MNP activity increased as the Mn concentration increased up to 1,500 mg/liter. Lower levels of MNP activity were observed in cultures grown in the presence of air, and the MNP activity was less affected by increases in the Mn concentration (Fig. 4). Fungal growth was affected by both high Mn levels and high oxygen levels, both of which increased uptake of glucose in the N-excess cultures. In cultures grown in the presence of oxygen and 300 mg of Mn per liter the glucose level on day 2 was 1.2 mg/ml, compared with 4.7 mg/ml for a culture grown in the presence of oxygen without Mn and 6.5 and 7.5 mg/ml for cultures grown in the presence of air with and without Mn, respectively. A similar trend was observed for N-limited cultures, although the rate of glucose uptake was slower. The Mn concentration affected the fungal biomass, and the mycelium dry weight determined on day 4 was 50% higher for cultures grown in the presence of 300 mg of Mn per liter than for cultures grown without sufficient Mn.

FIG. 3.

Peak LIP activity as a function of the initial Mn²⁺ concentration in the medium. Shallow stationary cultures of P. chrysosporium were grown in N-excess medium in the presence of either air or oxygen. The numbers above the columns indicate the days on which the LIP activity peaks occurred. The error bars show the standard deviations.

FIG. 4.

Peak MNP activity as a function of the initial Mn²⁺ concentration in the medium. Shallow stationary cultures of P. chrysosporium were grown in N-excess medium in the presence of either air or oxygen. The numbers above the columns indicate the days on which the MNP activity peaks occurred. The error bars show the standard deviations.

Effect of Mn concentration on LIP formation in cultures grown in a nonimmersed system.

Since the inhibitory effect of Mn on LIP formation was observed in cultures exposed to air but not in cultures exposed to oxygen, it is possible that the suppressive effect of Mn occurs when the availability of oxygen is low and can be overcome by increasing the availability of oxygen. In order to examine this hypothesis, the effects of various Mn concentrations on LIP formation were determined in a nonimmersed liquid culture system which had previously been shown to increase oxygen availability (11, 36). The results (Fig. 5) indicated that in the case of immobilized cultures exposed to air, 13 mg of Mn per liter did not suppress LIP formation, whereas in shallow stationary cultures exposed to air, 13 mg of Mn per liter clearly repressed LIP production (Fig. 3). These results indicate that LIP repression by Mn can be influenced by increasing the availability of oxygen.

FIG. 5.

Effect of Mn of LIP activity in N-excess cultures of P. chrysosporium immobilized on polyurethane foam cubes in a nonimmersed liquid culture system. Cultures containing various Mn²⁺ concentrations (0, 13, 30, 300, and 1,500 mg/liter) were grown in the presence of air. The error bars show the standard deviations.

Effect of veratryl alcohol on LIP formation.

Mn deficiency has been reported to induce veratryl alcohol formation by Bjerkandera sp. and P. chrysosporium (25). Since veratryl alcohol has a protective effect on LIP activity (42), it has been suggested that Mn deficiency indirectly enhances LIP titers by increasing the production of veratryl alcohol (25). In order to examine this hypothesis, the effect of adding exogenous veratryl alcohol on LIP formation was tested in the presence of 30 mg of Mn per liter in N-excess cultures exposed to either air or oxygen (Fig. 6). Veratryl alcohol was added at a concentration of 2 mM at the time of inoculation. The control cultures did not contain veratryl alcohol. In cultures exposed to air, no LIP was formed, regardless of the presence of veratryl alcohol. In cultures exposed to oxygen, LIP was formed both in the presence and in the absence of veratryl alcohol, but higher levels of LIP activity were observed in the presence of veratryl alcohol.

FIG. 6.

Effect of veratryl alcohol on LIP activity in N-excess shallow stationary cultures of P. chrysosporium containing 30 mg of Mn²⁺ in the presence of air or oxygen. The error bars show the standard deviations. VA, veratryl alcohol.

Effect of oxygen on soluble Mn concentration.

The formation of LIP regardless of Mn the concentration, as observed in N-excess cultures exposed to oxygen in the present work, is not consistent with the results of previous reports which described the repressive effect of high Mn levels on LIP formation in N-limited cultures. It has been reported that LIP repression by high levels of Mn is relieved by dismutation reactions, which result in the formation and precipitation of Mn⁴⁺ as MnO₂ (31). Therefore, in order to determine whether the formation of LIP in cultures containing high levels of Mn was a result of precipitation of MnO₂, we determined the levels of soluble Mn in N-excess cultures exposed to air or oxygen and supplemented with 300 mg of Mn per liter (Fig. 7). In cultures exposed to oxygen, the level of soluble Mn began to decline on day 4, whereas in cultures exposed to air, the levels of soluble Mn remained unchanged throughout the cultivation period.

FIG. 7.

Soluble Mn²⁺ concentrations in N-excess shallow stationary cultures of P. chrysosporium containing 300 mg of Mn²⁺ per liter grown in the presence of air or oxygen. The error bars show the standard deviations.

Effect of Mn on the formation of Mn-SOD.

Since the results described above indicated that Mn deficiency can apparently replace the requirement for oxygen during LIP formation, it is possible that this effect of Mn may be related to the properties of Mn as a regulator of Mn-SOD antioxitive activity. The level of Mn-SOD activity was therefore determined as a function of culture age with and without added Mn in the medium. In N-excess cultures grown in the presence of oxygen (Fig. 8), no Mn-SOD activity was detected in cultures grown without added Mn, whereas substantial activity, which reached a maximum level on day 3, was detected in cultures grown with added Mn. The decrease in Mn-SOD activity from day 3 onward was accompanied by the onset of LIP activity on day 4 (Fig. 1). No Cu/Zn-SOD activity was detected when cyanide, which has an inhibitory effect on Cu/Zn-SOD, was omitted from the gel staining procedure (data not shown). This last finding is evidence of the importance of Mn-SOD in scavenging superoxide radicals in P. chrysosporium. The influence of various Mn concentrations on the level of Mn-SOD activity was determined with 3-day-old cultures (Fig. 9). There was a correlation between the level of Mn in the medium and Mn-SOD activity; Mn-SOD activity was nearly undetectable at low Mn levels (3 mg/liter or less), whereas at Mn concentrations greater than 13 mg/liter the Mn-SOD activity remained at an almost constant high level.

FIG. 8.

Mn-SOD activity as a function of culture age in the presence (+) or in the absence (−) of Mn²⁺ in the medium. The numbers at the top indicate the number of days of incubation. The Mn²⁺ concentration was 300 mg/liter. Samples of cell extracts from N-excess cultures were loaded (10 μg of protein per lane) onto a native PAGE gel and stained for Mn-SOD activity.

FIG. 9.

Peak Mn-SOD activity as a function of Mn²⁺ concentration in the growth medium. N-excess cultures were grown in the presence of air and were harvested on day 3. Samples of cell extracts were loaded (10 μg of protein per lane) onto a native PAGE gel and stained for Mn-SOD activity.

DISCUSSION

The results of this study show that the trigger for LIP formation normally provided by a high oxygen concentration can be replaced by Mn deficiency and that this substitution is effective under both N-limited and N-excess conditions. The suppressive effect of Mn on LIP formation in N-limited cultures of P. chrysosporium has been described previously (2, 17, 30), but the mechanism for the regulatory effect of Mn is not clear. Mester et al. (25) reported that in both Bjerkandera sp. and P. chrysosporium, Mn deficiency enhanced endogenous production of veratryl alcohol and increased LIP titers. These authors suggested that Mn deficiency indirectly increases LIP titers by increasing the concentration of veratryl alcohol, which protects LIP from inactivation by peroxide. The higher LIP activity observed in this work when veratryl alcohol was added to cultures exposed to oxygen could indeed be attributed to the protective role of veratryl alcohol (42). Exogenous addition of veratryl alcohol did not, however, overcome the suppressive effect of Mn on LIP formation in cultures exposed to air. This suggests that in addition to its role in the formation of veratryl alcohol, which has a protective effect on LIP, Mn deficiency regulates LIP formation by another mechanism. Li et al. (23) showed that no LIP expression occurred in an N-sufficient culture grown with Mn concentrations ranging from 0 to 180 μM, in contrast to our results. However, the high glucose concentration and the oxygenation regime used by Li et al. may well have doubled the biomass produced and decreased oxygen availability, thus suppressing LIP formation (9). Indeed, by using immobilized cultures with improved oxygen availability (36), we demonstrated that in cultures exposed to air, the suppressive effect of Mn on LIP formation occurs at higher concentrations of Mn than the Mn concentrations necessary in shallow stationary cultures. Since the suppressive effect of Mn on LIP formation can be overcome by a high level of oxygen, it is possible that the two factors (Mn deficiency and a high level of oxygen) regulate LIP formation by similar mechanisms. High levels of oxygen can play a role in the removal of soluble Mn from a culture, since the formation of MNP, which catalyzes the oxidation of Mn²⁺ to Mn³⁺, is induced by oxygen, as well as by Mn (24). In this regard, it is important to note that the transcription of MNP is activated by oxygen and Mn, which shows that the two elements do not necessarily conflict (24). It has been suggested that LIP synthesis begins only after soluble Mn is completely removed by precipitation of Mn⁴⁺ as MnO₂ (31). However, the present work showed that in N-excess cultures, the removal of soluble Mn started only after LIP formation began. Several workers have shown that LIP can oxidize Mn in the presence of veratryl alcohol and oxalate (18, 32). Since it has been determined that oxalate is formed in N-excess cultures, as well as in N-limited cultures (11), it is possible that LIP plays a crucial role in the removal of soluble Mn.

In addition to its effect on LIP accumulation, Mn also affected the heme protein profiles in the extracellular fluid. In fact, the ratio of H1 to H2 was lower in cultures without Mn than in cultures containing Mn, especially in N-limited cultures. It has recently been reported that LIP dephosphorylation process catalyzed by an extracellular phosphatase results in the conversion of isozyme H2 to isozyme H1 (37). Since this phosphatase activity is reportedly enhanced by Mn²⁺ (35), it is possible that Mn affects LIP isozyme profiles enhancing the dephosphorylation of isozymes. In addition, the formation of MnO₂, which was evident from the darkening of the mycelia, especially at high Mn levels, can stabilize LIP. Indeed, the higher levels of LIP activity in cultures containing Mn in the presence of oxygen than in cultures lacking Mn can be explained by the high levels of MnO₂ produced in the former cultures. Since MnO₂ is a very good scavenger of H₂O₂, it can protect LIP from H₂O₂ inactivation (17).

The question arising from this work concerned the mechanism by which Mn deficiency can replace the high level of oxygen needed for LIP formation. A hypothetical explanation which may be inferred from our results is that Mn deficiency and a high level of oxygen can both increase the level of oxygen radicals generated within the fungal cell. The fact that no Mn-SOD activity was observed when there was an Mn deficiency is consistent with results reported for other organisms (3, 16). Because of the importance of Mn-SOD and high Mn²⁺ levels as antioxidants, especially in the absence of any Cu/Zn-SOD activity, it may be assumed that Mn-deficient cultures of P. chrysosporium are more susceptible to oxidative stress than nondeficient cultures are. Indeed, Saccharomyces mutants lacking mitochondrial Mn-SOD have been reported to be hypersensitive to oxygen (15, 44). In this regard it should be pointed out that exposure to a high level of oxygen reportedly increases the level of reactive oxygen species normally generated in cells of a wide range of organisms and induces the formation of antioxidative enzymes (28). The possibility that LIP plays an antioxidant role was previously proposed by Morpeth (27), who demonstrated that LIP formation was initiated only after a decrease in the levels of the antioxidant enzymes catalase and SOD. Previous studies demonstrated that high levels of oxygen did not affect the growth and respiration of several white rot fungi, including P. chrysosporium (33, 34). In addition, Mn can regulate cell functions other than Mn-SOD formation (20). In conclusion, the results of this work demonstrate that Mn deficiency can replace the high oxygen level required for LIP formation, but the mechanism governing this phenomenon is not clear and should be examined further.