chrysosporium: Synthesized in the Absence of Lignin in Response to Nitrogen Starvation

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1 JouRNAL OF BACTEOLOGY, Sept. 1978, p /78/ $02.00/0 Copyright 1978 American Society for Microbiology Vol. 135, No. 3 Printed in U.S.A. Ligninolytic Enzyme System of Phanerochaete chrysosporium: Synthesized in the Absence of Lignin in Response to Nitrogen Starvation PAUL KEYSER,f T. KENT KIRK,` AND J. G. ZEIKUS2 Forest Products Laboratory, Forest Service-U.S. Department ofagriculture, Madison, Wisconsin 53705,' and Department of Bacteriology, University of Wisconsin, Madison, Wisconsin Received for publication 13 March 1978 The relationship between growth, nutrient nitrogen assimilation, and the appearance of ligninolytic activity was examined in stationary batch cultures of the wood-destroying hymenomycete Phanerochaete chrysosporium Burds. grown under conditions optimized for lignin metabolism. A reproducible sequence of events followed inoculation: 0 to 24 h, germination, linear growth, and depletion of nutrient nitrogen; 24 to 48 h, cessation of linear growth and derepression of ammonium pennease activity (demonstrating nitrogen starvation); 72 to 96 h, appearance of ligninolytic activity (synthetic 14C-lignin -. '4CO2). Experiments with cycloheximide demonstrated that appearance of ligninolytic activity occurs irrespective of the presence of lignin; lignin did not induce additional activity. Addition of NH4' to cultures immediately prior to the time of appearance of the ligninolytic system delayed its appearance, suggesting that the NH4' led to interference with synthesis of the enzyme system. Addition of NH4' to ligninolytic cultures resulted in an eventual, temporary decrease in ligninolytic activity. The results suggest that all or essential protein components of the ligninolytic enzyme system are synthesized as part of a series of physiological ("secondary metabolic") events that are initiated by nutrient nitrogen starvation. Recent investigations of lignin metabolism by the wood-rotting hymenomycete Phanerochaete chrysosporium have demonstrated the critical importance of several culture parameters. The studies revealed, inter alia, that metabolisn of this complex and heterogeneous (1) aromatic polymer is maximal at low concentrations (approximately 2 mm) of nutrient nitrogen (15), and that lignin is metabolized only in the presence of a suitable growth substrate such as glucose (13). The onset of lignin metabolism _ (synthetic 4C0-lignin 14CO2) in cultures contaiing glucose and 2 mm nitrogen occurred only after an initial linear growth phase. Cessation of linear growth and the onset of lignin metabolism did not reflect depletion of glucose, but instead appeared to be a response to nitrogen limitation. The present investigation examined more closely the relationship between growth, nutrient nitrogen utilization, and the onset of lignin metabolism under conditions optimized for lignin metabolism. In the course of the studies it was discovered that ligninolytic activity appears irrespective of the presence of lignin in the cultures, and that its appearance is influenced by nitrogen metabolism. t Present addres: Corporate Research and Development, International Paper Co., Tuxedo Park, NY MATERIALS AND METHODS Lignins. Synthetic U-ring-, methoxyl-, and side chain (f,y)-labeled "C-lignins with specific activities of 9.0 x 106, 9.1 x 104, and 2.2 x 10i dpm/mg were prepared as described earlier (12). Identically prepared unlabeled synthetic lifnin was used in certain experiments to dilute the 'C-linins. Further reference to lgin refers to these synthetic model polymers. All ligins were stored at -30 C in aqueous suspension. These lignins were thawed, dried on a rotary film evaporator at <50 C, dissolved in N,N-dimethylformamide to give 10% solutions, and held at -30 C until used. Lignin suspensions for addition to cultures were prepared by slowly adding the dimethylformamide solutions aseptically to vigorously stirred sterile water (>1 ml of water per 50 j1 of dimethylformamide solution). No contamination of cultures resulted from addition of lignin. Fungi and inoculum. P. chrysosporium Burds. (ME446) was obtained from the Center for Forest Mycology Research, Forest Products Laboratory, U.S. Department of Agriculture, Madison, Wis., and was maintained at room temperature on 2% malt agar slants. Inoculum consisted of conidial suspensions, 790 filtered through glass wool and diluted so that the absorbancy at 650 nm equaled 0.5 in a 1-cm-pathlength cuvette (-2.5 x 106 spores per ml); conidia were taken from 3- to 4-week-old slants and exhibited >90% viability. Experimental cultures. Experimental culture conditions have been described (15). Experiments

2 VOL. 135, 1978 were conducted with replicated cultures in 125-ml Erlenmeyer flas, which were fitted with ports that permitted periodic flushing to exchange gases and allow trapping of evolved "CO2. Each flask contained mineral salts and vitamins (15), 0.01 M sodium o- phthalate buffer (ph 4.5), 56 mm glucose, and nutrient nitrogen (-2.4 mm N; sources indicated in text). Inoculation was with 1 ml of spore suspension per flask. Cultures were incubated without agitation at 39 C and under an atmosphere of = 100% 02. Determination of growth, glucose, nitrogen and DNA. Mycelium dry weights were determined after collecting and drying mycelia on tared 47-mmdiameter Metricel filters (0.20 pm; Gelman Instrument Co., Ann Arbor, Mich.). Total DNA was determined on the mycelia obtained by centrifugation of three combined replicate cultures. Mycelial pellets were extracted once with 5 ml of acetone, once with 5 ml of diethyl ether, and then once with 2 ml of 0.5 M HCl04 (0.50C for 20 min with each solvent). One milliliter of 0.5 M HCl04 was added, and the pellets were held at 700C for 50 min. Supernatants were recovered by centrifugation, and the pellets were extracted again with 1 ml of hot HC104, recovered by centrifugation, and finally washed with 1 ml of cold 0.5 M HC10O. (Extraction procedure was adapted from ref. 19.) Total volume of the combined extracts and washing was determined, and DNA was measured using the diphenylamine method (11). Blanks and known standards (calf thymus DNA, Sigma Chemical Co., St. Louis, Mo.) in the same volume as the samples were included with each determination. Glucose in culture filtrates was determined by the method of Nelson (18). Total extracellular nitrogen was determined in the dried residues of culture filtrates by the micro-kjeldahl procedure (using cultures which contained no dimethylformamide). Ammonium ion was determined with a glutamate dehydrogenase assay (20). Ammoum permeam assay. A procedure based on that of Hackette et al. (9) as descibed by Gold et al (8) was used. Six replicate cultures were combined, and the mycelia were fagmented in their residual medium for 10 at low speed in a blender. An aqueous solution of ["4C]methylamine hydrochloride (ICN, Irvine, Calif.) was added (final concentration, 10-4 M, 10' dpm/pmol) to the resulting suspension of mycelial fragments at 25 C. Samples (10 ml) were removed after 1, 2, 3, and 4 min, immediately filtered through glass wool, and washed with ice water. The wet gls wool with trapped mycelium was transferred to a scintillation vial containing 5 ml of an organic base (NCS tissue solubilizer, Amersham Corp., Arlington Heights, IIL). After 8 to 12 h, 5 ml of a scintillation fluid (1.2 g of 2,5-diphenyloxazole (PPO) and 15 mg of 1,4-bis-[2]-(4-methyl-5-phenyloxazolyl)benzene (PO- POP) per 100 ml of toluene) was added, and the radioactivity was determined (15). Permease activity was CalCUlated as rate of uptake of ["4C]methylamine per gram (dry weight) of mycelium. (Mycelium weight was determined on a separate 10-ml sample of the suspenion being asayed) Phenol oxidase asay. Three qualitative procedures were used. Two of the procedures involved color reactions with phenolic substrates (syringaldazine and gum guaiac), and were used with and without H LIGNINOLYTIC ENZYME SYSTEM OF P. CHRYSOSPORIUM 791 that peroxidase (EC ) as well as laccase (EC ) could be assayed (10). The third procedure did not distinguish between the two types of fungal phenol oxidase, and involved dimerzation of 4-tbutyl guaiacol following addition of the phenol to cultures. A suspension of the phenol in water (1 mg/ml) was prepared by aseptically adding a 5% solution in dimethylformamide to sterile water. One milliliter of the suspension was added aseptically to each test culture, which was then incubated for 24 h under 100% 02 at 390C. A chloroform extract of the cultures was then examined by thin-layer chromatography (silica gel; benzene-dioxane-acetic acid, 200:25:4, as solvent). Phenol oxidase activity was detected by the presence of the dehydrodimer of 4-t-butyl guaiacol (Rf of dimer ; Rf of 4-t-butyl guaiacol ). The dimer exhibited a characteristic high fluorescence under a shortwave (254 pm) UV lamp. (Both 4-t-butyl guaiacol and a reference sample of the dimer were obtained from W. J. Connors, U.S. Forest Products Laboratory.) Assay for ligninolytic activity. Assays were based on short-term "4C02 evolution from 14c-lignins. In a typical assay, a suspension of '4C-lignin (ring-, side chain-, or methoxyl-labeled) in 1 ml of sterile water (>5 x 104 dpm/ml) was added to each test culture, and the cultures were immediately flushed with 100% 02. After various periods of incubation at 390C, the cultures were again flushed with 100% 02 to remove 14CO2, which was trapped directly in a scintillation fluid (12). Activity was calculated as disintegrations per minute per hour per culture. Figure 1 shows the rate of 14C2 evolution at hourly intervals following addition of ring-labeled 14C-lignin to 6-day cultures (grown without lignin). The rate of 14CO2 evolution increased for 3 h, continued at a maximum for an additional 3 to 4 h, and then decreased to a relatively constant value, which was maintained for several days. Similar results were obtained with methoxyl- and side chain-labeled lignins, but the ring-labeled material, which had the highest specific radioactivity, was used to obtain the data presented. illation spectrometry. Radioactivity was measured in a Packard (Downer's Grove, Ill.) Tri-Carb model 3300 spectrometer. Data are corrected for background radioactivity and for counting efficiency. The latter was monitored routinely with an automatic external standard using a 'Ra source (Packard) and periodically with an internal standard of ["4C]toluene. Counting efficiency was always 65% or greater. Counting of all samples was continued at least for sufficient time to give values accurate within ±4% (95% confidence limits). Cycloheximide inhibition of protein synthesis. Experiments demonstrated that cycloheximide (Sigma) at concentrations greater than approximately 20 pg/ml (a7 x 10-2 pmol/mg of dry mycelium) blocked incorporation of ["4C]leucine into P. chrysosporium protein (trichloroacetic acid insolubles [6]). As indicated in the text, somewhat higher concentrations (50 to 100 pg/ml) were used to assure effectiveness in the experiments reported here. A fresh aqueous solution of the antibiotic was added (1 ml per culture) 1 h before addition of"c-lignin for ligninolytic enzyme assays.

3 792 KEYSER, KIRK, AND ZEIKUS 120 T T T T J. BACTERIOL. /00 _ (:3 (:3 -.u B0 _ 60 _ 40 _ 20 _ Downloaded from TIME (hours) FIG. 1. Kinetics of '4C"2 production immediately following addition of '4C-lignin to ligninolytic (6-dayold) cultures. At the time of inoculation, each culture contained, in a total of 10 ml of basal medium: 0.6 mm L-asparagine, 0.6 mm NH4NO3, 0.01 M sodium o-phthalate buffer (ph4.5), and 56 mm glucose. Cultures were grown without agitation, under an atmosphere of =100K% O: at 39 C. Data points (means ± 1 standard deviation) are radioactivity (14C02) recovered hourly fouowing addition of 5 x 10' dpm of ring-labeled "Clignin in 1 ml of water to each of six replicate cultures. Killed controls showed no activity. REiSULTS Constitutive nature of ligninolytic activity. Experiments were conducted to examine the ligninolytic activity of cultures ediately after treatment with the protein synthesis inhibitor cycloheximide; growth was in a lignin-free medium. Addition of the inhibitor to cultures approximately 3.5 days old or younger prevented subsequent appearance of activity (Fig. 2). When added to lignin-free cultures 5 days old or older, however, the inhibitor had no qualitative effect on the lininolytic activity (although there was some attenuation of existing activity during the 6-h assay) (Table 1). Similar results were obtained with ring-, methoxyl-, and side chain-labeled lignins. Competent (ligninolytic) cultures (5 to 7 days old) to which cycloheximide was added lost approximately 60, 40, and 25% of their activities against ring-, methoxyl-, and side chain-labeled lignins, respectively, within 24 h after addition of the inhibitor. Experiments were conducted to determine whether lignin-induced ligninolytic activity could be demonstrated. These studies revealed that the presence or absence of lignin in the cultures had no influence on the level or time of appearance of activity. Activity was first detectable in cultures after 3.5 days, as in the experiment described above, both in cultures containing no lignin and in cultures containing up to 10 mg of lignin from the time of inoculation. Cultures containing lignin from the time of inoculation exhibited a level of ligninolytic activity that was similar to that of cultures grown in the absence of lignin (Table 2). Relationship between nutrient nitrogen on March 17, 2019 by guest

4 VOL. 135, /20 /00 _ o80 60 CONTROLS LIGNINOLYTIC ENZYME SYSTEM OF P. CHRYSOSPORIUM 793 CYCLONEXIMIDE CULrURE AGE (days) FIG. 2. Effect of cycloheximide on the appearance of lgninolytic activity in cultures. Experimntal conditions are described in Fig. 1. W7hen the cultures were 85 h old (arrow), cycloheximide (5(X pg in I ml of water per culure) was added to 6 of 12 cultures and water only was added to the other 6 (controls). AU cultures contained 5 x 10 dpm of ring-labeled 4C-lignin from time of inoculation. Data are means I standard deviaton for six replicate cultes in each treatment. concentration and appearance of the ligninolytic ystem Figure 3 shows the relationship between growth (mycelial weight), residual medium glucose, total extracellular nitrogen, and the ligninolytic activity of cultures. Mycelial weight incrased in an essentially linear manner for 3 days following inoculation and then remained relatively stationary through day 7. Glucose decreased progressively, but over 60% of the original amount still remained in the 7-dayold cultures. In contrast, total extracellular nitrogen decreased by 90% to a minimum within 48 h. A low level of ligninolytic activity was detected in 4-day-old cultures, and the activity reached mimal levels by 5 to 6 days. Maximal activity was maintained for at least 10 days (cf. 15). Culture ph remained constant at ph 4.4 to 4.5 during these experiments. Microscopic examination of cultures during growth revealed that no ignificant numbers of any spore type were produced in cultures through day 8. Cultures usually sporulated (aerial conidia [5]) after 10 to 15 days. Experiments such as those summarized in Fig. 3 indicated synthesis of the ligninolytic enzyme system or essential protein component following linear growth cessation and a decrease in nutrient nitrogen to a minimum level. Linear growth apparently was nitrogen limited. Experiments demonstrated that other medium components were, like glucose, not limitng; increasing the concentration of the basal medium components (i.e., all medium components except N and glucose) by 10-fold had no influence on the time of appearance, or on the level of ligninolytic activity Ḟurther investigation of the relationship between nitrogen depletion and appearance of ligninolytic activity was made using a simple nitrogen source, ammonium tartrate, instead of the mixture of NH4NO3 and L-asparagine used above, which included ammonium-, nitrate-, and TABLz 1. Effect of culture age and cycloheximide on ligninolytic actiity Ligninolytic activity Culture age ('4CO2, dpm/h per culture) (days) Cycloheximidetreatedc ControLs ±2 13±6 5 83±17 92± ±13 93±18 'aexperimental culture conditions are described in Fi4.1. Assay was based on '4C02 evolved during 6 h under 100% 02 and 390C following addition of 5 x 104 dpm of ring-labeled 14C-ligin per culture. Values are means i±1 standard deviation for six replicate cultures. Cycloheximide (1 mg in 1 ml of water per culture) was added 1 h before ligninolytic assay was begun. TABLz 2. Effect ofprior incubation with lignin on the ligninolytic activity in culturee Ligninolytic activitye ('4C02, dpm/h per cul- Treatmentb ture) 0-6 h 6-24 h Lignin from start 57 ± 36 No lignin prior to assay 73 ± ± ± 5 Experimental culture conditions described in Fig. 1. b SiX of 12 replicate cultures contained 500 pg each of unlabeled lignin from time of inoculation. 'After 5.5 days, cycloheximide (500 pg per culture) was added to all cultures. One hour later, 3,000 pg of lignin, contning 3 x 0l dpm of ring-labeled 14Clinin, was added to each culture. 14C02, evolved during incubation under 100% 02 and 39'C, was removed after an additional 6 and 24 h. Data for cultures that oiginally contained lignin are corrected for dilution by that lignin (x7/6). Data are means ± 1 standard deviation for six replicate cultures per treatment.

5 794 KEYSER, KIRK, AND ZEIKUS J. BACTERIOL _ (3 /0 r 25 r 50 F I.: k I _ 0 t,j ( z 1.5 R M LO0.5 2 _ (I) 0.)1 -:j 40 _ 30 _ 20 _ /0 _ O L (Zi CUL TURE AGE (doys) FIG. 3. Relationship between culture parameters and ligninolytic activity during 7 days ofgrowth. Experimental culture conditions are described in Fig. 1. Ligninolytic activity is based on 14CO2 evolved during 6 h under = 10(f 02 and 39 C after addition of5 x 10' dpm of ring-labeled "4C-lignin per culture; activity values are means + 1 standard deviation for six replicate cultures. All other values are means 1 standard deviation for three replicate cultures. TABLE 3. Characteristics of cultures during 6 days ofgrowth with aihmonium tartrate as nitrogen source Culture age Mycelium dry wt Glucose DNA Extracellular m per- tivilytid Phenol (days) (mg)b UMO/nl)b Wture) (Nrnol/ml) (umol/min ("CO2, dpm/h oxidase' ture)(il011mi)ii per g)c per culture) o ± ± ± ± NDf ±0.5 44± < ± < ± ± ± ± ±0.9 25± ±21 + a'each culture contained, in a total of 10 ml of basal medium: 1.2 mm ammonium tartrate, 0.1 M sodium o- phthalate buffer (ph 4.5), and 56 mm glucose. They were grown under an atmosphere of =100% 02, at 390C and without agitation. Culture acidity remained constant at ph 4.5. b Mean ± 1 standard deviation for three replicate cultures. C Each value was obtained with six combined cultures (see the text). d Assay was based on `C02 evolved during 6 h under 100% 02 and 39 C following addition of 5 x 104 dpm of ring-labeled 14C-lignin per culture. Values are means ± 1 standard deviation for six replicate cultures. ' Qualitative assay based on dimerization of 4-t-butyl guaiacol (see the text). f ND, Not detected. I4 s

6 VOL. 135, 1978 amino-n. (Tartrate does not serve as a growth substrate.) Experiments summarized in Table 3 demonstrated that extracellular NH4' was depleted from cultures that contained ammonium tartrate 24 h after inoculation. Growth began between 12 and 24 h after inoculation, and the growth rate wasm l prior to day 2. Mycelial weight increased throughout the 6-day experiment, despite depletion of nitrogen by the first day, and in contrast to the pattern observed on the other medium (Fig. 3). Primary growth (DNA synthesis) began between 0 and 12 h, and ceased after approximately 30 h. iagninolytic activity was present in 4-day-old and older cultures. Thus the activity appeared between 2 and 3 days after depletion of NH,+ from the medium and approximately 2 days after the maximum (presumed linear) growth rate. Measurement of extracellular NH4+ does not indicate total available nutrient nitrogen, because internal pools must also be considered. Therefore, the enzyme ammonium permease was analyzed to estimate the actual degree of /20 l /00 LIGNINOLYTIC ENZYME SYSTEM OF P. CHRYSOSPORIUM 795 nitrogen starvation of the cultures. This enzyme becomes derepressed under conditions of N-starvation (9). Results showed that permease activity was not detectable in 24-h cultures, but was maximal in 48-h and older cultures (Table 3). Thus appearance of ligninolytic activity occurred between 1 and 2 days after the cultures became starved for nitrogen. Addition of 24!unol of NH4' (equivalent to original amount present) to 3-day ammonium tartrate cultures, which exhibited no ligninolytic activity, delayed appearance of activity for approximately 48 h (Fig. 4). Addition of NHF+ to 6-day-old ligninolytic cultures caused a slight but not reproducible decrease in ligninolytic activity within 6 h. However, within 16 h a substantial suppression of activity was evident, and this suppression persisted for 40 to 50 h (Table 4). Analyses disclosed that the newly added NH4+ was depleted within 16 h of addition to 6- day-old cultures. Phenol oxidase activity was detected in cultures after 4 to 5 days (Table 3). The activity Downloaded from CONTROLS on March 17, 2019 by guest CUL TURE AGE (doys) FIG. 4. Effect ofnh4' addition on appearance of ligninolytic activity. Experimental culture conditions are described in Table 3. All cultures contained 5 x 10f dpm of ring-labeled '4C-lignin from time of inoculation. After 78 h (arrow), 12 &mol of ammonium tartrate in 1 ml of 0.2 M sodium o-phthalate buffer (ph 4.5) was added to 6 of 12 replicate cultures. The remaining six cultures (controls) received 12 mnol of sodium tartrate (ph 4.5) in the buffer.

7 796 KEYSER, KIRK, AND ZEIKUS TABLE 4. Effect of NH4+ addition on ligninolytic activity of 6-day-old cultures Time after NH4+ ad- Ligninolytic ('4C02, activityt dpm/h per CUltur) dliltion (h)b NW{' added Controls ±7 57± ± ± ±8 27± ± ± aexperimental culture conditions are described in Table 3. b Ammonium tartrate addition to 10 of 20 replicate cultures and sodium tartrate addition to the other 10 (control) cultures are described in Fig. 4. 'One hour after addition of NHW+, 5 x 104 dpm of ring-labeled 14C- in 1 ml of water was added to each of the 20 cultures. Evolved 14CO2 during incubation under 100% 02 and 39 C was removed after 16, 28, 40, etc., h as indicated. Values reported are means ± 1 standard deviation for 10 replicate cultures in each treatment. was weak; neither laccase- nor peroxidase-type phenol oxidase was detectable with gum guaiac or syringaldazine assays (10). However, dimnerization of the phenol 4-t-butyl guaiacol over a 24- h period after addition to cultures indicated that oxidase activity was present. DISCUSSION The existence of a ligninolytic enzyme system that is synthesized irrespective of the presence of lignin has been demonstrated in P. chrysosporium. Additional lignin-induced activity could not be demonstrated. Preliminary experiments with Coriolus versicolor, another lignindegrading hymenomycete (white-rot fungus) taxonomically distinct from P. chrysosporium, have given similar results, suggesting that this finding might have broad relevance among the ligninolytic basidiomycetes. The fact that ligninolytic activity is not induced by lignin suggests that the ligninolytic system may be relatively nonspecific. Low specificity also seems apparent when one simply considers the diversity of lignin subunit structures (1) metabolized by the ligninolytic basidiomycetes. Even industrial lignins, the heavily modified by-products of the kraft and sulfite pulping processes, are still substantially metabolizable by P. chrysosporium (17). Thus the relatively low rates of ligin decomposition (in comparison to cellulose, for example) observed in the lignin-degrading basidiomycetes, indicating a low activity of the ligninolytic system, might indirectly reflect low specificity. Perhaps J. BACTERIOL. the fact that lignin alone does not serve as a growth substrate for these fungi (13) also is a consequence of low activity of the ligninolytic system, which in turn results from low specificity Ṫhe ligninolytic system or essential protein component apparently is synthesized in response to nitrogen starvation. The length of elapsed time between nitrogen starvation and the appearance of the lignin-degrading activity, however, suggests an indirect rather than a direct connection between lignin metabolism and nitrogen metabolism. The effect of nitrogen on the ligninolytic system is evidenced by the delayed appearance of activity caused by excess NH4' and by the diminution in activity within 16 h after addition of NH4+ to competent cultures. In many fungi, NH4+ is known to repress synthesis of some enzymes, a phenomenon termed "am-' monia metabolite repression" (21). Synthesis of the ligninolytic system (or essential component) in response to nitrogen starvation suggests that the activity is associated with a shift in physiology to "secondary metabolic" or "idiophasic" metabolism (3). Two other events also appear to be associated with this metabolic shift in P. chrysosporium cultured as described here: appearance of phenol oxidase activity (demonstrated in the present work) and de novo synthesis of 3,4-dimethoxybenzyl alcohol (veratryl alcohol), reported elsewhere (16). Whether these events are related to lignin metabolism is not clear. However, the coincidental appearance of ligninolytic and phenol oxidase activities is interesting, because the possible role of phenol oxidases in lignin metabolism has been investigated and debated for many years (2, 14). Certain recent evidence suggests an indirect but necessary role (2). A role of nitrogen metabolism in the initiation of secondary metabolism in other fungal systems has been demonstrated. For example, bikarevin in GibereUa fujukuroi (4), the extracellular glucan nigeran in Aspergillus aculeatus (8), and two phenolic compounds in Aspergillus fumigatus (22), are synthesized in response to N- starvation. In any event, these results suggest that decomposition of lignin in nature by P. chrysosporiur, and perhaps by white-rot fungi in general, will occur efficiently only in a nitrogen-limiting environment. This is precisely the kind of environment-namely, within nitrogen-poor woody tissues (7)-in which these organism commonly operate. The findings reported here have obvious practical implications for any scheme purporting to produce ligninolytic enzymes or to use ligninolytic fungi.

8 VOL. 135, 1978 ACKNOWLEDGMENIS We appreciate valuable discussions with R. Metzenberg and the excellent laboratory assistance of Kathleen Moore and L F. Lorenz. This research was supported in part by National Science Foundation grant PCM and in part by the College of Agiculture and Life Sciences, University of Wisconsin, Madison. Paul Keyser was supported through the Department of Bacteriology of the University of Wisconsin, Madison. LITERATURE CITED 1. Adler, E Lignin chemistry-past, present, and future. Wood Sci. Technol. 11: Ander, P., and KI-E. Erikon Lignin degradation and utilization by microorganisms. Prog. Ind. Microbiol. 14: Bu'Lock, J. D Secondary metabolism in fungi and its relationship to growth and development. p In J. E. Smith and D. R. Berry (ed.), The filamentous fung, vol 1, Industial mycology. Wiley, New York. 4. Bu'Lock, J. D., R. W. Detroy, A. Hostalek, and A. Munum-Al-Shakarchi Regulation of secondary biosynthesis in GibbereUa fujikuroi. Trans. Br. Mycol. Soc. 62: Burdsall, H. IL, and W. E. Eslyn A new Phanerochaete with a chrysosporium imperfect state. Mycotaxon 1: Clark, C., and E. L. Schmidt Growth response of Nitrosomonas europaea to amino acids. J. Bacteriol. 93: Cowling, E. B., and W. Merrill Nitrogen in wood and its role in wood deterioration. Can. J. Bot. 44: Gold, M. H., D. L Mitzel, and I H. Segel Regulation of nigeran accumulation by Aspergillus aculeatus. J. Bacteriol. 113: Hackette, S. L., G. E. Skye, C. Burton, and L H. Segel Characterization of an ammonium transport system in filamentous fungi with methyl-ammonium-'4c as the substrate. J. Biol. Chem. 245: Harkin, J. AL, and J. R. Obst Syringaldazine, an effective reagent for detecting laccase and peroxidase in fungi. Experientia 29: LIGNINOLYTIC ENZYME SYSTEM OF P. CHRYSOSPORIUM Herbert, D., P. J. Phipps, and R. E. Strange Chemical analysis of microbiol cells, p In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 5B. Academic Press Inc., New York. 12. 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. U.S.A. 72: Kirk, T. K., W. J. Connors, and J. G. Zeikus Requirement for a growth substrate during lignin decomposition by two wood-rotting fungi. Appl. Environ. Microbiol. 32: Kirk, T. K., W. J. Connors, and J. G. Zeikus Advances in understanding the microbiological degradation of lignin, p In F. A. Loewus and V. C. Runeckles (ed.), Recent advances in phytochemistry, vol. 11. Plenum Press, New York. 15. Kirk, T. K., E. Schultz, W. J. Connors, L F. Lorenz, and J. G. Zeikus Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporjum. Arch. Microbiol. 117: Lundquit, K., and T. K. Kirk De novo synthesis and decomposition of veratryl alcohol by a lignin-degrading Basidiomycete. Phytochemistry, vol. 17, in press. 17. Lundquist, K., T. K. Kirk, and W. J. Connors Fungal degradation of kraft lignin and lignin sulfonates prepared from synthetic "C-lignins. Arch. Microbiol. 112: Nelson, N A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153: Nickerson, K. W., B. K. McCune, and J. L Van Etten Polyamine and macromolecule levels during spore germination in Rhizopus stolonifer and Botryodiplodia theobromae. Exp. Mycol. 1: Robbins, J., and S. C. Weber An enzymic assay for ammonia in waste matter. J. Agric. Food Chem. 25: Rose, A. H Chemical microbiology: an introduction to microbial physiology. Plenum Press, New York. 22. Ward, A. C., and N. M. Packter Relationship between fatty-acid and phenol synthesis in Aspergillus fumigatus. Eur. J. Biochem. 46: