As 51 was partially inhibited by 1,lO-phenanthroline, the enzyme was considered t o be a metalloproteinase although EDTA and phosphoramidon had no effect.
Microbiology (1995), 141, 1575-1 583
Printed in Great Britain
Proteolytic activities in two wood-decaying basidiomycete fungi, Serpula lacrymans and Coriolus versicolor Rekha V. Wadekar,’ Michael J. North’ and Sarah C. Watkinson’ Author for correspondence: Sarah C. Watkinson. Tel: +44 1865 275000. Fax: +44 1865 275074. e-mail : sarah.watkinson @ plant-sciences.oxford.ac.uk
1
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
2
Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, UK
Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula lacrymans and Coriolus versicolor, have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In S. /acryinans#intracellular and extracellular azocaseinase activity was optimal at pH 5.6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, 51 and 54 (apparent M, 65000 and 30000, respectively) and two less active enzymes, S2 and 53 (apparent M, 47000 and 43000, respectively). S1, the predominant intracellular proteinase, was present at all ages of the mycelium (tested up t o 3 months). It is active over a broad pH range, with highest activity around neutral pH. As 51 was partially inhibited by 1,lO-phenanthroline, the enzyme was considered t o be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical t o S 1 was also detected in the medium of older cultures. 54 is a pepstatinsensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 50 and above. S2 and 53 were identified as intracellular metalloproteinases, present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of S. lacrymans, proteinase 51 was the only enzyme present throughout (and the intracellular azocaseinase activity increased), which suggested a likely role of 51 in intrahyphal protein mobilization. 54 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of C. versicolor were optimal at pH 7-0 (intracellular) and pH 5 6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels, C1 (apparent M, 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases, there may have been overlapping bands due t o enzymes of both types. Extracellular samples gave a more complex pattern, (five bands, C2-C6, M, 50000-100000). C2 and C4 are PMSF-sensitive proteinases, C5 and C6 are probably metalloproteinases, while C3, which was most active at pH 4.0, was unaffected by any of the inhibitors tested, including pepstatin A. No aspartic proteinase equivalent t o 5. lacrymans 54 appeared t o be produced by C. versicolor. From the information gained about the intracellular or extracellular location of these enzymes, and the conditions under which they are active, an in vivo role may be tentatively ascribed to some of them. Keywords : Serptlla lacr_ymans,Coriolus versicolor, basidiomycetes, proteolytic activity, wood-decaying fungi
0001-9646 0 1995 SGM
1575
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
INTRODUCTION Wood-decaying basidiomycetes are able to develop very large colonies with wood, a carbon-rich, but nitrogenpoor material, as their sole source of nutrients. Their growth presumably requires sensitive control of their nitrogen economy, involving regulation of proteinase activity both for the extracellular digestion of the protein in wood, and for the intracellular turnover and spatial reallocation of nitrogen from mycelial protein. Secretion of extracellular proteinase from the hyphae could be necessary for acquiring nitrogen from wood protein, but represents a potential waste of a colony's limited nitrogen resources. Both positive and negative feedback controls of extracellular proteinase secretion are therefore likely. Nitrogen derived from wood is accumulated in the mycelium to levels well above those in surrounding wood, for example Serpzlla lacymans mycelium utilizing wood as a sole nutrient source contains 3.7% nitrogen compared with 0.07 % in the wood substrate (Watkinson e t al., 1981). Nitrogen is accumulated in the mycelium as free amino acids and an ethanol-insoluble protein fraction yielding amino acids on hydrolysis. In 1-week-old mycelium of S. lacymans grown in a synthetic culture medium with a nitrogen content comparable to that of wood, the total free amino acid was equivalent to 74 pM nitrogen, and the amino acid released by hydrolysis of the ethanol insoluble component of the mycelium to 2054 pM nitrogen, per g dried mycelium (Venables & Watkinson, 1989a). The mycelium in established parts of the colony can thus be regarded as a rich source of nitrogen, most of which is initially in the form of protein. Intracellular proteinases which hydrolyse mycelial protein and make it available for re-use, are likely to play a key role in the nitrogen economy of the whole colony. Regulation of proteinases of wood-decay basidiomycetes is likely to be a part of the physiological processes of morphogenesis, because of the close relationship between fungal nutrition and morphogenesis. The composition and spatial arrangment of the nutrient substrate affects not only the rate of growth of the colony but also the differentiation of structures such as mycelial strands. Such developmental responses probably result from evolutionary optimization of foraging. Fungi such as S. lacymans grow on discontinuous nutrient resources, the fungal colony behaving like a foraging individual, with new growth localized in parts of the colony where new resources are most abundant (Rayner etal., 1985;Dowson e t al., 1988). As their colonies grow, different parts of the same mycelium may be growing into freshly-encountered wood, or extending out from it supported by translocation of nutrients from the food base, or senescing and autolysing. The advance of the mycelium from one food resource to another has been shown by 15Nlabelling to be accompanied by a transfer of nitrogen into the new resource (Watkinson, 1984), a transfer which is expected because of the high carbon:nitrogen ratio of wood. It therefore seems likely that intracellular proteolysis to mobilize protein stores is induced by a requirement for nitrogen, and that sites of induction and proteolysis could be at separate points in the colony. Hedlund e t al. (1991) 1576
have shown that insect grazing can induce proteolysis at a distance in Mortierella isabellina colonies. Very little is known about the proteolytic enzymes of timber-decaying fungi. Before their role and regulation can be understood, it is necessary to establish the characteristic enzymes. In this study, two species have been used. S. lacymans, the wood dry rot fungus, was chosen for its economic significance and because it develops mycelial strands for exploitation of its resources. Corioltls versicalar was chosen as a timber-decaying basidiomycete with a very different physiology; it is a white rot and does not normally form strands.
METHODS Organisms and culture conditions. The isolates of S.lacymans (Wulf. Fr.) Schroet (culture no. 12C) and C. versicolor (L. ex Fr) (culture no. 28A) were supplied by the Forest Products laboratory of the Building Research Establishment, UK. They were subcultured and maintained on 2 YOmalt agar [2 % (w/v) malt extract and 1-5YO(w/v) Oxoid no. 3 agar]. Static liquid cultures were grown on peptone medium (20 ml), containing 1 YO (w/v) bactopeptone, 0.3 YOyeast extract and 0.2% D-glucose in 50 ml conical flasks, with each flask inoculated with a 10 mm disc cut submarginally from a 5-10-dold fungal colony growing on 2 YO(w/v) malt agar. The flasks were incubated at 22 "C and harvested at various ages as indicated. For autolysis experiments, cultures were grown initially on peptone medium. Two-week-old healthy cultures were then transferred on day 15 to salts only 'starvation' medium (gl-': KH,PO,, 1; MgSO,. 7H,O, 0.5; FeSO,. 7H,O, 0.01 ; KC1, 1). Cultures were harvested at the time of transfer to 'starvation' medium and periodically after transfer. Parallel sets of cultures were used for preparing samples for azocaseinase assays and gelatin-SDS-PAGE analysis. Azocasein hydrolysis. Samples used to measure proteinase activity with azocasein as substrate were prepared as follows. After washing with deionized water, all the mycelium from a single flask was ground in a mortar with purified sand (1 g) and 5 ml extraction buffer. The extraction buffer was 0.2 M acetate, pH 5.6, except when the pH dependence was to be determined when the concentration of the buffer was 0.05 M. The resultant homogenate was spun for 10 min at 1990g in an Econospin bench top centrifuge and the supernatant was used to analyse the intracellular activity. Extracellular samples were collected by separating culture fluid from mycelium by filtration. Freshly prepared samples were used directly for azocaseinase assays. Occasionally the extracellular samples were stored in a refrigerator at 4 "C, never for longer than 24 h, but storage had no effect on proteinase activity. The assay procedure was based on the method originally described by Prestidge e t al. (1971), and differed only in respect of the azocasein concentration used (0.5 YOinstead of 2 YO, w/v). Azocasein hydrolysis was measured at 25 "C in reaction mixture containing 0.1 ml buffer,0.1 ml deionized water, 0.3 ml intracellular sample or 0.05 ml extracellular sample, 0-5 ml 0.5% azocasein (Sigma) and water to a total volume of 1 ml. The buffers used were : 0-5 M citrate/phosphate (pH 4*0),0.2 M sodium acetate/acetic acid (pH 4-0-5.6) and 0.15 M sodium/ potassium phosphate (pH 5-6-7.0). The reaction was stopped by adding 2 ml of 7 Yo (v/v) perchloric acid. The tubes were spun at 1990g for 10 min and then 2.6 ml of supernatant was mixed
Proteolytic activities of wood-decaying fungi with 0.4 ml 10 M NaOH. In controls, the perchloric acid was added immediately before the azocasein. Activity was measured One A unit was equivalent to the hydrolysis as change in A436. of 1.33 mg azocasein. Activity was expressed as specific activity [mg azocasein hydrolysed h-' (unit dry weight fungus)-'] or as total activity (mg azocasein hydrolysed per h per flask, irrespective of mycelial weight).
0-07
Gelatin-SDSPAGE. Samples were prepared as follows. The protein in mycelial extracts or culture filtrates obtained as described above was concentrated by precipitation with ammonium sulphate. The sample was taken to 80% saturation by addition of solid ammonium sulphate and then centrifuged at 1990g for 30 min. Supernatant fluid was discarded and the pellet was resuspended in extraction buffer. The ammonium sulphate was removed by Amicon mini-concentrators (Centricon 3 or 10 ultrafilter units, cut off 3000 or 10000, respectively), by spinning for approximately 30 min at 5000 r.p.m. The exact time varied and depended on the starting volume of the samples.
PH
Electrophoresis was carried out as described by Lockwood e t al. (1987). Samples were mixed with an equal volume of electrophoresis buffer containing 0.0625 M Tris/HCl (pH 6-8), 2 YO (w/v) SDS, 5 YO(w/v) 2-mercaptoethanol, 20 YO(v/v) glycerol and 0.002 YObromophenol blue. They were then electrophoresed in 10 Yo (w/v) polyacrylamide gels containing 0.2 'YO gelatin using the SDS-discontinuous buffer system described by Hames (1981). After loading the samples (10-20 ml, approximately 12-25 mg protein) onto the stacking gel, they were electrophoresed at a constant current of 15 mA for about 45 min per gel. After electrophoresis the gels were treated with 2.5 % (v/v) Triton X-100 for 30 min. The proteinase bands were developed by incubating gels overnight in an appropriate incubation buffer in a shaking incubator at 25 "C. The gels were then stained with either 0.1 YOCoomassie Blue in 40 YO(v/v) methanol and 10 YO (v/v) acetic acid or 0.1 YOamido black in 7 % (v/v) acetic acid. They were destained with acetic acid. The apparent M, of the proteinases were estimated from their mobility in relation to that of standard molecular marker proteins from Sigma.
-
RESULTS T w o methods were selected for analysing the proteinase activities of S.lacymans a n d C. versicolor. Azocasein was used as a substrate t o measure relative proteinase activity
0
4 3.0
Protein determination. Protein concentration was determined by the method of Bradford (1976), using BSA as a standard. Determination of inhibitor sensitivities. The effect of inhibitors on azocaseinase activity was determined by incubating samples with inhibitor at room temperature for 1 h prior to assay. Control samples containing an equivalent volume of water or organic solvent were pre-incubated identically. For gelatin-SDS-PAGE analysis, samples were incubated with inhibitors, water, or organic solvent as above before loading. After electrophoresis the individual lanes were cut out into strips, treated with Triton X-100 for 30 min and incubated overnight ( 15-18 h) in the appropriate incubation buffer containing the same inhibitor or solvent. Stock solutions of EDTA, leupeptin, iodoacetic acid and phosphoramidon were prepared in water, pepstatin and PMSF were dissolved in ethanol, and 1,lo-phenanthroline solutions were prepared in either DMSO or methanol. Final concentrations of inhibitors were chosen as recommended in the literature (North, 1989; Salvesen & Nagase, 1989). All inhibitors were obtained from Sigma except for phosphoramidon which was supplied by Scientific Marketing Associates.
~
4.0
5-0
6.0
7.0
8.0
PH
...............................................................................................................,.,..................................,,.... Fig. 1. pH-dependence of azocaseinase activity of intracellular (closed symbols) and extracellular (open symbols) samples from (a) cultures of 3-week-old 5. lacrymans and (b) 2-week-old C. versicolor. Buffers: 0.5 M citratelphosphate 0.2 M acetate (H, 0.15 M phosphate (V,V). Each point is the mean of four t o six samples from three replicate cultures assayed by the sampling out method.
a),
i n unfractionated samples, while gelatin-SDS-PAGE was used in a n attempt to identify t h e individual enzymes which contribute to the overall activity.
Azocaseinase activity in S. lacrymans Azocaseinase activity could be detected in samples derived from mycelia a n d from culture medium (Fig. la). In both, activity was optimal a t pH 5.6 (Fig. l a ) which confirmed earlier findings for extracellular activity (Venables, 1987).
A s azocasein tends t o precipitate at l o w pH it was n o t possible to use it for comparable assays below pH 4.
Both intracellular a n d extracellular sample activity, 1577
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
Table 7. Effect of inhibitors and assay pH o n azocaseinase activity in 3-week-old S. lacrymans The pH values for inhibition assays were selected so as to optimize the activities of those enzymes previously found to be susceptible to the inhibitor being assayed. The values given are means f s of~ 4 to 6 readings of samples from two or more replicate cultures. ____
Inhibitor
Concn
(mM)
Pepstatin A
PMSF EDTA 10-Phenanthroline Iodoacetic acid Pepstatin A plus 1,lo-phenanthroline
0.036 0.036 1 1 5 5 10
t
Intracellular Assay
Extracellular
Inhibition
P*
Inhibition (Yo)
87( f1.4) 23( f11.8) 1 1 0 8*0(& 5.1)’ 0 100 11(f8.7)
5.6 7.0 6.2 6.2 6.2 7.0 6.2 5.6 7.0
86( f0.7) 45( f11.9) 5 0 25( f1.6) 15( f8*9)* 0 96( f 1.1) 24( & 23.8)
2
3
PH
(W
5-6 7.0 5.6 5.6 5.6 7.0 5.6 5.6 7.0
Assay
* Activity enhanced. t0.036 mM Pepstatin A and 5 mM phenanthroline.
(a)
1
(b)
2
kDa
65 47 43 -
1
4
5
6
7
8
9
10
kDa
- 51 - s2 - 53
65 -
- 51
30 -
- 54 pH 4.0
pH 5.0
pH 5.6
pH 6.2
pH 7.0
Fig. 2. Band patterns in gelatin-containing gels following electrophoretic separation o f proteinases from cultures o f 5. lacrymans. (a) Bands produced when samples were taken from a 2-week-old culture. Lanes: 1, intracellular sample; 2, extracellular sample (for details of sample preparation see Methods). Gels were incubated a t pH 6.2. (b) Bands from intracellular samples (lanes 1, 3, 5, 7 and 9) and extracellular samples (lanes 2, 4, 6, 8 and 10) incubated a t a range of pHs after separation. Cultures were 12 weeks old. M, values were estimated by comparison with standards (Sigma kits MW-SDS-70L and MW-SDS-200) run in parallel (not shown).
measured at pH 5.6, were reduced by more than 85% by pepstatin A, a specific inhibitor of aspartic proteinases (Table 1). For the extracellular activity, measured at pH 6.2, 1,lO-phenanthroline caused a reduction of approximately 25 %, suggesting a contribution from metalloproteinases. At pH 7.0, 1,lo-phenanthroline enhanced activity, and pepstatin had less effect than at pH 5-6, for both intracellular and extracellular samples. None of the other inhibitors tested had a significant effect. 1578
Gelatin-SDSPAGE of S. lacrymans proteinases
Analysis of mycelial extracts and culture filtrates showed a relatively simple proteinase pattern for both (Fig. 2a). Samples were examined from cultures of different ages from 10 d to 3 months old. In 10-d-old cultures, three to four faint intracellular proteinase bands were detected, with hardly any activity being found in extracellular samples (data not shown). Analysis of older cultures
Proteolytic activities of wood-decaying fungi Table 2. Effect of inhibitors on azocaseinase activity in 2-week-old C. versicolor ...........................................................................................................................................................................................................
........................................
See legend to Table 1 for details.
Inhibitor
Pepstatin A PMSF EDTA 1,lo-Phenanthroline Iodoacetic acid PMSF plus 1,lOphenanthrolinet
Concn (mM)
Intracellular Assay
0.072 2 5 5 10
Inhibition
PH
(%I
5-6 7.0 7-0 7-0 7-0 7-0
0 27( f1.4) 56( f1.87) 62( f0.53) 0 78( & 2.23)
Extracellular* Inhibition (%)
0
14( f1.8)
66( f0.67) 74( f066) 0 74( f2.53
* All extracellular assays were at pH 5.6.
t 2 mM PMSF and 5 mM 1,lO-phenanthroline. (2-10 weeks old) revealed a proteinase S1, with an apparent M, of 65 000, in intracellular samples of all ages, and an apparently identical enzyme in the medium. The extracellular form was at higher levels at later stages and was presumably released into the medium as the culture aged. Proteinase S1 was most active around neutral p H and was affected by only one of the proteinase inhibitors tested, 1,lo-phenanthroline, by which it was partially inhibited (Table 3). Some loss of S1 activity was observed with ethanol and methanol when used in controls. An attempt to concentrate samples by acetone precipitation resulted in inactivation. Two other intracellular proteinases, S2 and S3 (apparent Mr 47000 and 43000, respectively), were detected in younger cultures up to 4 weeks old (Fig. 2a) but were absent from mycelium of older cultures. Their activity was totally inhibited by all three of the metalloproteinase inhibitors tested, namely 1,lO-phenanthroline and, in contrast to S1, both EDTA and phosphoramidon. The other major proteinase of 5’.lacymans was S4 which had a lower apparent M, (about 30000). It was found predominantly in the medium and was not detected in the youngest cultures (10 d old), although it was detectable from 2 weeks onwards. Within the pH range tested, it was most active at pH 4.0 and was not detected on gels incubated at pH 5.0 and above (Fig. 2b). This proteinase was almost completely inhibited by pepstatin A (Table 3), identifying it as an aspartic proteinase. Proteinase S4 appeared to be modified, presumably by limited proteolysis, during storage at -20 OC, as additional bands were apparent after 4 weeks’ storage, but since the new bands were detectable on substrate-containing gels, these altered forms had clearly retained activity (data not shown). A comparison of the results of gelatin-SDS-PAGE analysis with those of azocaseinase assays suggested that proteinases S1, S2 and S3 could have been responsible for only a small proportion of the azocaseinase activity at pH 5.6. These intracellular metalloproteinases were
inhibited by 1,lo-phenanthroline but this inhibitor did not significantly affect the intracellular azocaseinase activity. The results are, however, consistent with proteinase S4 being responsible for most of the extracellular azocaseinase activity at low pH since both were inhibited by pepstatin. At pH 7.0 pepstatin inhibits less than half the azocaseinase activity in both intracellular and extracellular samples, so S4 presumably contributes less to overall activity at neutral pH. Phenanthroline enhanced azocaseinase activity at pH 7.0 rather than inhibiting it, and reduced the inhibition caused by pepstatin when the two inhibitors were used together (Table 1). The reason for this enhancement of azocaseinase activity at neutral pH is not clear. Proteinase S4 was not found in the gelatin-SDS-PAGE analysis of intracellular samples, and no pepstatin-sensitive proteinase was present to account for the pepstatinsensitive azocaseinase activity. A possible explanation for this was that the intracellular samples contained a different aspartic proteinase with very little activity towards gelatin. However, analysis of samples by SDS-PAGE using gels in which azocasein was substituted for gelatin failed to reveal any additional proteinases which could have been responsible for the intracellular azocaseinase. A second possibility considered was that an intracellular proteinase may have been inactivated during electrophoresis. As extracellular S4 was not inactivated under these conditions this would have required the presence of either a different, less stable enzyme, or an inactivating factor(s) in the mycelium. The latter possibility was tested as follows. Concentrated samples of mycelial extract, medium and a mixture of the two were compared by gelatin-SDS-PAGE. A significant amount of S4 activity was detected in both extracellular and mixed samples, but not the intracellular sample (data not shown). Thus, if an inhibitor or an inactivating factor had been present it could have been there only in limited amounts. All mixtures of intracellular and extracellular samples gave the expected levels of azocaseinase activity indicating 1579
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N 1
kDa 100 -
c150
-
2
3
4
- c2 - c3 -c4 - c5 - C6
10
Fig. 3. Patterns o f bands in gelatin-containing gels following electrophoretic separation o f proteinases from cultures o f C. versicolor. lntracellular (lanes 1 and 3) and extracellular (lanes 2 and 4) samples were incubated a t pH 4.0 (1 and 2) and pH 6-2 (3 and 4). Mrvalues were estimated by reference t o markers as in Fig. 2.
that cell extracts contained neither an inhibitor nor an activator of azocaseinase activity. The nature of the enzyme responsible for the pepstatin-sensitive azocaseinase of S. lacrymans mycelium therefore remains to be elucidated.
Effect of starvation-induced autolysis on the proteinase activities of S. lacrymans In fungi such as S. lacrymans, the breakdown of intracellular protein during starvation-induced autolysis may be of major importance for the relocation of nitrogen sources within the colony. T o examine the possible involvement in this process of the proteinases revealed here, cultures of S. lacvmans were starved by transferring them to salts-only medium, and proteinase activity was measured using azocasein and gelatin-SDS-PAGE.
30 50 Incubation time (d)
70
culture medium before transfer
mx
6-
EP :z;
4-
vEa LJ
g2
- 2
Rise in activity in fresh medium immediatelyafter transfer
10
30
50
1
..........................................................................................................................................................
Fig. 4. Azocaseinase activity in 5. lacrymans after transfer t o 'starvation' medium. Cultures were incubated for 2 weeks o n peptone medium before transfer and harvested periodically (48 h and 2, 4 and 7 weeks) after transfer. (a) lntracellular total (V)and specific (m) activity a t pH 6.2; (b) extracellular total activity a t pH 4.0. Each point represents the mean o f six samples from t w o replicate cultures.
When cultures were starved, the intracellular azocaseinase specific activity and total activity, measured at p H 6.2, showed a sharp increase during the 48 h after transfer (Fig. 4a). This increase accompanied the reduction in biomass and in total mycelial protein due to autolysis (Fig. 5). During subsequent incubation the total intracellular azocaseinase activity decreased. Extracellular total activity, measured at pH 4-0, accumulated significantly by the second week after transfer (Fig. 4b). Proteolytic activity was first detected in the medium 2 h after transfer. Of the individual proteinases detected by gelatin-SDSPAGE, the activity of the EDTA-sensitive proteinases S2 and S3 progressively decreased in activity after transfer, and were almost absent by week four (Fig. 6). The other metalloproteinase, S1, was still active within mycelium at weeks two and four, and also gradually appeared in the medium. Pepstatin-sensitive extracellular proteinase S4 was detected as a faint band on gels incubated at pH 4.0 with samples harvested at 2 and 4 weeks after transfer. Its activity was considerably less than that found in extracellular samples taken prior to transfer (data not shown). During starvation the pH of the culture medium changed. In the first 48 h after transfer, the pH dropped from 4.5 to 1580
0
Incubation time (d)
600
n
400
m
.-C
: aJ
CI
m
.200
20 40 Incubation time (d)
60
f8
.,
..........................................................................................................................................................
Fig. 5. Changes in dry weight and total protein content o f the fungal mycelium per flask during starvation o f 5. lacrymans. Dry weight, mean reading o f three replicate cultures; 0 , mycelial protein, based on the samples prepared from t w o replicate cultures for PAGE analysis.
Proteolytic activities of wood-decaying fungi Table 3. Effect of inhibitors on 5. lacrymans and C. versicolor proteinases detected by gelatin-PAGE
....................................... . ................. Inhibition of bands was detected at optimal pH conditions by incubating all gel portions at pH 4 0 and 6.2, chosen on the basis of the different observed enzymic pH optima in SDS-PAGE. .........................................................................................................
I
.....I......
.....................
Inhibition of bands*
Inhibitor Concn (mM) Pepstatin A 0.036 PMSF 1 Leupeptin 2 EDTA 1 1,lO5 Phenanthroline Phosphoramidon 0.01 10 Iodoacetic acid
S. Zacrymans
Intracellular
Extracellular
NE
S4JJ
NE
NE
NE
NE
S2JL s3.14 SlJ, S2JJ, S34J s244, s344
NE
NE
SI(extra)J NE NE
Concn (mM)
0.072 2 2 5
5
ND
10
C.versicoZor Intracellular NE
c14
Extracellular NE
( 3 . 1s4.1J.
NE
NE
c1
c1
C5$, C6$J C5$, C644
ND
ND
NE
NE
Not determined. * Effects observed: 4,partial inhibition; $4,complete inhibition; NE, no effect.
ND,
3.9, but subsequently increased again (Table 4). Despite
the reduced pH, the enzyme that would be most active under these conditions, the aspartic proteinase S4, was only detected as a faint band. Because no aspartic proteinases could be detected in intracellular samples using gelatin-SDS-PAGE, the contribution of these enzymes to the increased activity during autolysis was assessed by measuring the extent to which pepstatin inhibited the azocaseinase activity present before and 48 h after transfer of mycelia to starvation medium. Data obtained from at least six samples from two replicate cultures showed that while total azocaseinase activity (measured at pH 5.6) rose from 1-45kO.1 to 2-05kO-035mg azocasein hydrolysed per h per flask, the amount of activity inhibited by 0.044 mM pepstatin did not change significantly (0.885 & 0,025 mg azocasein hydrolysed per h per flask before transfer and 0-925& 0.085 mg azocasein hydrolysed per h per flask 48 h after transfer). Thus the increase in intracellular activity was not due to aspartic proteinases. However, most of the extracellular activity recovered 48 h after transfer was pepstatin-sensitive. The results suggest that it was likely to be proteinase S1 which was mainly responsible for the increase in intracellular azocaseinase activity, while the activity released immediately into the medium was due to the pepstatinsensitive aspartic proteinase, S4. The release of the latter probably resulted from autolysis rather than any increased production of enzyme.
Azocaseinase activity in C. versicolor The intracellular activity of C. versicolor azocaseinase was optimal at neutral pH while the extracellular activity was highest at pH 5.6 (Fig. lb). These latter results can be compared with those reported by Staszczak & Nowak
Table 4. Changes in pH of culture filtrates recovered a t each harvest during autolysis of 5. lacrymans Values are based on the readings obtained from two replicate cultures.
Culture mediumlharvest
PH
Original peptone medium Peptone medium after 2 weeks’ mycelial growth Original ‘starvation ’ medium 48 h* 2 weeks* 4 weeks* 7 weeks*
6.65 3-23 4-48 3.88 4.42 5.56 5.30
* Period after transfer to starvation medium. (1984) who determined two p H optima of 7.0 and 5.0-5-4,
respectively, for both intracellular and extracellular activities of C. versicolor (strain ATCC 44308) using haemoglobin as substrate. The azocaseinase activity in C. versicolor was inactivated by a different set of inhibitors from those effective in 5’. lacrymans samples, suggesting that other classes of proteinases were involved (Table 2). The intracellular activity at p H 6-2 was reduced by 27 %O by PMSF suggesting that a serine proteinase contributed. As activity was inhibited by more than 50% by EDTA and 1,lO-phenanthroline, a metalloproteinase was also present. Similar inhibitory effects were observed on the extracellular activity. A combination of PMSF and 1,lOphenanthroline gave slightly greater inhibition than that with 1,lo-phenanthroline alone. This was not apparent with the extracellular activity. 1581
R. V. W A D E K A R , M. J. N O R T H a n d S. C. W A T K I N S O N
1,lo-phenanthroline suggesting contributions from both serine and metalloproteinases (Table 2). Proteinase C3 was not affected by any of the inhibitors tested, including pepstatin. Proteinase C2 was completely inhibited by PMSF suggesting that it was a serine proteinase, although one that was not affected by leupeptin. The effects of inhibitors on the other proteinases were more difficult to discern. C4 appeared similar to C2 in being sensitive to PMSF, while proteinases C5 and C6 were most affected by 1,lo-phenanthroline, although E D T A was much less inhibitory. A comparison with the data for C. versicolor azocaseinase activity suggests that the latter could be accounted for largely by the proteinases revealed by gelatin-SDS-PAGE. The intracellular C1 band was affected by the same agents as those which inhibited the intracellular azocaseinase activity, namely PMSF, EDTA and 1,lo-phenanthroline. Metalloproteinases were the major contributors to the extracellular azocaseinase activity, and so proteinases C5 and C6 were most likely involved.
2
51 s2 53 -
2
- 51 (extra) DISCUSSION
Fig. 6. Proteinase patterns o f S. lacrymans during starvation. Samples were obtained from the cultures harvested at (a) 2 weeks and (b) 4weeks after transfer o n t o 'starvation' medium. Lanes: 1, Intracellular; 2, extracellular. Gels were incubated a t pH 6.2. The pattern for the culture a t the time of transfer is shown in Fig. 2(a). Samples loaded: (a) lane 1, 8 p g protein (equivalent to 160 pl); lane 2, equivalent to 530 PI; (b) lane 1, 6.2 pg protein (equivalent t o 320 PI);lane 2, equivalent t o 400 pI. Fig. 2(a): lane 1, 12 pg protein (equivalent t o 80 pl); lane 2, equivalent t o 160 pI.
Gelatin-SDSPAGE of C. versicolor proteinases Gelatin-SDS-PAGE analysis of C. versicolor revealed a more complex proteolytic system than that of S. lacrymans (Fig. 3). In intracellular samples derived from 10 to 15-dold cultures there was a single proteinase band, C1, corresponding to an apparent M,of 62-64000. Its activity was detected over a wide range of pH but was highest at pH 6.2. The band was always diffuse even when different volumes and concentrations of sample were loaded, and it seems likely that more than one proteinase was responsible. Five bands in the M , range 50-100000 were detectable in extracellular samples. Of these, proteinase C3 represented the most active enzyme. Its activity was highest at pH 4.0, declined with increasing pH and was difficult to detect at pH 7.0. Three of the other proteinases (C2, C4 and C5) were most active around neutral pH but gave less defined bands. Proteinase C6 could be detected over the whole pH range tested (pH 4-0-7.0). A proteinase with similar mobility to C6 was sometimes apparent in intracellular samples. None of the extracellular proteinases exactly corresponded to intracellular proteinase C1. The activity of the C1 band was reduced by PMSF, EDTA and 1582
At present very little is known about the proteinases of wood-decay fungi. This analysis has shown that the two species studied here each produce a number of different proteinases typical of fungi (North, 1982), although the two species do not have similar enzymes. Four proteinases, S1, S2, S3 and S4, were the main proteolytic enzymes of 5.lacrymans. In many ways these were typical of fungal proteinases, being aspartic (S4) and metalloproteinases (Sl, S2 and S3). Proteinase S1, a metalloproteinase, is intracellular and appeared to be a 'leaking out' enzyme of the type described by Schanel e t al. (1971), where an intracellular enzyme is secreted into culture medium without any change to its molecular properties. S2 and S3 are apparently strictly intracellular enzymes and S4 is an extracellular aspartic proteinase. The pepstatinsensitive intracellular activity has not yet been matched to an enzyme detectable by gelatin-SDS-PAGE, and there is a probability that a second intracellular aspartic proteinase is present. The proteinase levels of S. lacrJmans changed with the physiological state of the culture. The intracellular proteinase S1, present in the samples of all ages of the culture, increased under starved conditions and gradually accumulated in the medium. Transfer of mycelium to starvation conditions resulted in the immediate release of a small amount of proteinase activity, mainly S4, but release was not sustained, and is more likely to have been due to mechanical breakage of cells than to specific induction. The data suggest possible physiological functions for the enzymes. S4 could be a digestive enzyme, solubilizing wood protein under acid conditions during growth. The hyphae of S.lacrymans grow actively on medium of high carbon:nitrogen ratio where pH as low as 2.8 can develop in culture, when the fall in pH during growth is proportional to the carbon: nitrogen ratio (Watkinson,
Proteolytic activities of wood-decaying fungi 1775), being usually attributed to the secretion of oxalic acid (Goksoyr, 1957). Wood itself has a high carbon: nitrogen ratio, typically approaching 500 : 1. The metalloproteinase S1 could be active in protein turnover, as it is intracellular and remained active during starvation. The proteinases seen in C. versicolor were significantly different, suggesting that the organization of proteolysis in vivo is different in these two fungi. In C. versicolor only one intracellular proteinase band (Cl) was found, which appeared to be a mixture of serine and metalloproteinases. This band was sensitive to PMSF, and the intracellular azocaseinase activity from C. versicolor had a broad pH optimum [features also found in the intracellular proteinase from Agariczls bisporzls stipes (Burton e t al., 1973)]. A complex of extracellular enzymes was found including serine proteinase, metalloproteinase, and a pepstatininsensitive acid proteinase. In the analysis of the proteinase of C. versicolor, Staszczak & Novak (1784) found seven distinct intracellular proteinase bands compared with the single one detected here. However, direct comparisons cannot be made because the culture conditions were different and the proteinases were analysed using different substrate and electrophoresis conditions. The extracellular bands found by Staszczak & Novak (1784) did resemble those described here, and were possibly equivalent. The location of extracellular proteinase released from colonies on solid agar medium was previously found to differ in S. lacrymans and C. versicolor (Venables & Watkinson, 1787b). In C. versicolor, extracellular proteolysis was found mainly in the region of growing hyphal tips, with much less in agar medium under older parts of the colony. With S. lacymans more activity was found beneath the central older part of the colony. In nature the two fungi differ both in physiology and morphology. S. lacr_ymans causes brown rot, degrading cellulose but not completely degrading lignin, while C. versicolor decomposes cellulose and lignin, causing white rot. S. lacrymans is able to grow long distances over non-nutrient surfaces, supported by nutrients transported through the hyphae and mycelial cords from a colonized food source. C. versicolor does not show this behaviour. The results of the analysis of the proteolytic enzymes support the view that intracellular protein mobilization plays a more important role in S. lacrymans than in C. versicolor.
REFERENCES Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-d ye binding. Anal Biochem 72, 248-254. Burton, K. S., Wood, D. A., Thurston, C. F. & Barker, P. J. (1993). Purification and characterisation of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus. J Gen Microbiol 139, 1379-1386. Dowson, C. G., Rayner, A. D. M. & Boddy, L. (1988). Foraging patterns of mycelial cord -forming basidiomycetes between discontinuous resource units in soil. F E M S Microbiol Ecol 53, 291-298.
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Biology of Higher Fungi, pp. 249-279. Edited by D. Moore, L. A. Casselton, D. A. Wood & J. C. Frankland. Cambridge: Cambridge University Press. Salvesen, G. & Nagase, H. (1989). Inhibition of proteolytic enzymes. In Proteohtic EnZymes: a Practical Approach, pp. 83-104. Edited by R. J. Beynon & J. S. Bond. Oxford: IRL. Santamaria, F. & Reyes, F. (1988). Proteases produced during autolysis of filamentous fungi. Trans Br Mycol Soc 91, 217-220. Schanel, L., Blaich, R. & Esser, K. (1971). Function of enzymes in wood decaying fungi. Arch Microbiol 77, 140-1 50. Staszczak, M. & Nowak, G. (1984). Proteinase pattern in Trametes versicolor in response to carbon and nitrogen starvation. Acta Biochim Pol 31, 431436. Venables, C. E. (1987). The nitrogen economy of wood decaying basidiomycetes. DPhil thesis. University of Oxford. Venables, C. E. & Watkinson, 5. C. (1989a). Medium induced changes in patterns of free and combined amino acids in mycelium of Serpula lacrymans. Mycol Res 92, 273-277. Venables, C. E. & Watkinson, 5. C. (1989b). Production and localization of proteinases in colonies of timber-decaying basidiomycete fungi. J Gen Microbioll35, 1369-1374. Watkinson, S. C. (1975). The relation between nitrogen nutrition and formation of mycelial strands in Serpula lacymans. Trans Br Mycol SOC64, 195-200. Watkinson, S. C. (1984). Morphogenesis of the Serpula lacymans colony in relation to its functions in nature. In The Ecology and Ph_ysiology of the Fungal Mycelium, pp. 165-184. Edited by D. H. Jenning & A. D. M. Rayner. Cambridge: Cambridge University Press. Watkinson, 5. C., Davison, E. M. & Bramah, 1. (1981). The effect of nitrogen availability on growth and cellulolysis by Serpula lacymans. New Ph_ytol89, 295-305. Received 24 November 1994; revised 14 February 1995; accepted 15 March 1995.
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