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The effects of tolaasin, the toxin produced by Pseudomonas tolaasii on tyrosinase activities and the induction of browning in Agaricus bisporus fruiting bodies
Physiological and Molecular Plant Pathology (1999) 55, 21–28 Article No. pmpp.1999.0200, available online at http :\\www.idealibrary.com on
The effects of tolaasin, the toxin produced by Pseudomonas tolaasii on tyrosinase activities and the induction of browning in Agaricus bisporus fruiting bodies C . S O L E R -R I V A S " , * , N . A R P I N # , J . - M . O L I V I E R $ a n d H . J . W I C H E R S " " Agrotechnological Research Institute (ATO-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands, # Laboratoire de Mycochimie, UniteT de Formation et de Recherche de Chimie et Biochimie, UniersiteT Claude Bernard, 69622 Villeurbanne Cedex, France, $ Institut National de la Recherche Agronomique, Station de Recherches sur les Champignons, Centre de Recherches de Bordeaux, 33883 Villenae d’Ornon Cedex, France (Accepted for publication February 1999) Infection of Agaricus bisporus with Pseudomonas tolaasii or treatment with a tolaasin containing preparation resulted in the activation of tyrosinase and the development of a brown discolouration of the fruitbody. In order to investigate whether tolaasin is responsible for these reactions, mushrooms were treated with different concentrations of HPLC-purified tolaasin. Tolaasin was found to induce both browning and the activation of tyrosinase, with the level of activation being related to the concentration of toxin applied. In water-treated controls, active tyrosinase isoforms with pIs of 4n5 and 4n4 were the main isoforms found but tolaasin treatment resulted in the activation of a tyrosinase isoform with a pI of 5n7. The different active isoforms could be distinguished as the tolaasin activated isoforms had a higher affinity for catechol as a substrate (Km 0n82 m) than the active isoforms in the control (Km 2n86 m). In mushrooms inoculated with P. tolaasii or treated with tolaasin containing extracts no significant differences were found in the mechanism of oxidation of tyrosine, γ-glutaminyl-4-hydroxybenzene or γ-glutaminyl-3,4-dihydroxybenzene to form melanin. In tolaasin treated samples, a constant total amount of melanin and sum of the three phenols was found. # 1999 Academic Press Keywords : tyrosinase ; Agaricus bisporus ; Pseudomonas tolaasii ; tolaasin ; phenols ; tyrosine ; GHB ; GDHB ; melanin ; brown blotch disease ; browning ; discolouration.
INTRODUCTION Pseudomonas spp. are common pathogens of many Basidiomycetes [2, 7, 22, 27, 29 ]. Pseudomonas tolaasii causes brown blotch disease in Agaricus bisporus which is characterised by brown spots on the surface of the sporophores and severe reductions in yield [5, 18 ]. Pseudomonas tolaasii culture filtrates cause blotch symptoms identical to those caused by the organism [16 ]. The causal factor, tolaasin, was purified by Nutkins et al. [17 ] and treatment of A. bisporus caps with it disrupts the plasma and vacuolar membranes and causes the collapse of the hyphae [20 ]. Tolaasin is a lipodepsipeptide with surface active properties [9 ] which is able to open ion channels in membranes [4 ]. * All correspondence should be addressed to : C. Soler-Rivas, Agrotechnological Research Institute (AT-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands. Abbreviations used in text : CF, cell-free culture filtrate ; GDHB, γ-glutaminyl-3,4-dihydroxybenzene ; GHB, γ-glutaminyl-4-hydroxybenzene ; IEF, isoelectric focusing ; L-DOPA, L-dihydroxyphenylananine ; PT, partially purified toxin ; T1, 0n61 mg tolaasin ml−" ; T2, 0n17 mg tolaasin ml−" ; T3, 0n06 mg tolaasin ml−".
0885-5765\98\070021j08 $30.00\0
The tolaasin isolated by Nutkins et al. [17 ] consisted of two principal components, Tol I, the major component, with a molecular mass of 1985 and Tol II, a minor component with a molecular mass of 1941. The composition of Tol II was found to be identical to that of Tol I apart from a single amino acid substitution. However, other tolaasins have been reported with different amino acid compositions. Eight isoforms were purified from a P. tolaasii strain isolated from Pleurotus ostreaus using a different purification method [24 ] than that used by Nutkins et al. [17 ]. Of these, Tox 4, the main toxin, possessed the same structure as Tol I while Tox 6 was identical to Tol II. Such diversity in structure is not uncommon for compounds synthesised by non-ribosomal mechanisms, and tolaasin appears to be synthesised by a peptidesynthetase complex of at least three enzymes (TL1, TL2 and TL3) encoded by a TL-gene cluster of 65 kbp located at one end of a 640 kbp PacI chromosomal fragment [21 ]. Other extra-genomic factors, required to mediate tolaasin production and activity, have been described [14 ]. In an earlier paper, it was reported that a protease free preparation of tolaasin induced blotch symptoms by # 1999 Academic Press
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activating tyrosinases, which are involved in the browning mechanism [25 ]. Tyrosinases are normally present in cells in a latent form but when activated, oxidise phenolic compounds such as tyrosine, γ-glutaminyl-4-hydroxybenzene (GHB) and γ-glutaminyl-3,4-dihydroxybenzene (GDHB) to form brown melanin-like compounds [3, 12, 26 ]. Although there is the possibility that a specific protease was present as an undetected contaminant of the tolaasin preparation [1 ], it seems more likely that tolaasin itself is involved in the activation of tyrosinase. This paper reports an investigation of the effects of an HPLC-purified tolaasin preparation on the activation of tyrosinase isoforms and the induction of tissue browning in A. bisporus. MATERIALS AND METHODS Fungal and bacterial material The mushroom strain used in this investigation, Agaricus bisporus U1 (Somycel) was grown in boxes at the Mushroom Experimental Station, Horst, The Netherlands. The boxes were transferred to ATO-DLO, Wageningen, a few days before the onset of fruitbody production. The boxes were maintained at 18mC and 80–85 % r.h. throughout the period of fruitbody production. Two bacterial strains of Pseudomonas tolaasii, SPC 8907 and SPC 8911 were used. Strain SPC 8907, a subculture of P. tolaasii 112 S (Preece) (NCPPB no. 3148), was pathogenic, white line test positive [31 ] and produced smooth colonies on Pseudomonas agar F medium. Strain SPC 8911 was non-pathogenic, white line test negative and produced rough colonies on Pseudomonas agar F medium. Strain SPC 8911 was isolated from mushrooms in 1985 at the Mushroom Experimental Station, Horst. The two strains were cultured as described by SolerRivas et al. [25 ] and mushrooms were inoculated with c. 9n5i10( cells ml−" of the required strain according to Soler-Rivas et al. [25 ]. Preparation of toxin-containing extracts and purification of tolaasin Toxin preparations were obtained from the pathogenic strain, SPC 8907 as described by Nutkins et al. [17 ]. After each stage of purification, the preparations were tested for their effects on mushrooms. These preparations were, 0n83 mg ml−" of the crude extract (CF ; cell free culture filtrate), 2n85 mg ml−" of the fraction obtained prior to HPLC separation (PT ; partially purified toxin) and a range of concentrations of a fraction, equivalent to Tol I, obtained after HPLC purification. The HPLC chromatograms obtained were similar to those of Nutkins et al. [17 ], and contained only two forms of tolaasin Tol I and Tol II. The Tol I fraction was lyophilized, dissolved in
DMSO and analysed by NMR. The fraction was about 82 % pure, calculated on proton number and this fraction was used at concentrations of 0n61 (T1), 0n17 (T2) and 0n06 mg ml−" (T3). A range of concentrations of the Tol I fraction were analysed by HPLC to obtain a standard curve for the relationship between peak area and tolaasin concentration. Tol I peak area in PT extract chromatograms was expressed as tolaasin concentration. The percentage of tolaasin in the PT extract was thus, calculated to be 65 %. Inoculation procedure and treatment with tolaasin preparations Mushrooms from the first flush with caps of between 30–40 mm diameter, in stages 2 or 3 of development according to the classification of Hammond and Nichols [8 ], were harvested and prepared as described by Moquet et al. [15 ]. They were inoculated with P. tolaasii strains or treated with the tolaasin preparations as described in Soler-Rivas et al. [25 ]. Two negative controls were included in each experiment, inoculation with the non-pathogenic strain SPC 8911 [5 ] and a water treatment. Three positive controls were also included, inoculation with the pathogenic strain SPC 8907, and treatments with CF, and PT [25 ]. The experimental treatments involved mushrooms treated with the tolaasin preparations obtained after HPLC (T1, T2, and T3). All measurements and assays were carried out 2 days after inoculation or treatment. Browning measurements and tyrosinase actiity Changes in colour of the mushroom tissue were measured with a Chroma Meter (Minolta CR-200, 1 cm slot) using the procedure described by Soler-Rivas et al. [25 ]. This procedure involves measuring several parameters ; L is a measure of brightness (from 0, white to 100, black), a and b are measures of chromaticity. The parameter a is a measure of change from green to red (positive values indicate red colour and negative green), and b a measure of change from blue to yellow (blue is negative and yellow positive). Browning was quantified as the change in colour (∆E l N[∆L#j∆a#j∆b#]) resulting from the treatment in relation to the colour of the tissue immediately before inoculation or treatment. After measurement of browning, samples were extracted as described previously [25 ] for tyrosinase analysis. The tyrosinase activity was measured using L-DOPA (Sigma) as substrate as described by Wichers et al. [30 ]. Kinetic measurements The kinetics of L-DOPA and catechol (Sigma) oxidation by the tyrosinases after treatment with either 0n17 mg ml−"
Tolaasin treatment of Agaricus bisporus 50 Tyrosinase activity (µkat g–1)
50
Browning level (DE)
40 30 20 10 0
T1
T2
T3
W
NP
P
CF
23
(a)
40
30
20
10
PT 0
F. 1. Browning levels (∆E) in Agaricus bisporus U1 caps treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W), crude culture filtrate (CF) and partially purified tolaasin preparation (PT), or inoculated with non-pathogenic (NP) and pathogenic (P) strains of Pseudomonas tolaasii (7, tolaasin treatments ; , negative controls ; , positive controls).
Isoelectric focusing Isoelectric focusing was carried out using IEF 3-6 gels (Servalyt2 Precotes2 150 µm, 125i125 mm, Serva). Samples of 10 µl adjusted to contain 15n5 µkat g−" dry wt. with 100 m sodium phosphate buffer (pH 6n5) were applied to each well and the gels were run for 3 h following the manufacturer’s instructions. IEF standards (Pharmacia) were used to calculate the pI of each band after the bands had been visualized by staining with Coomassie blue G250 (Merck). Gels were stained for tyrosinase activity by two methods. In the first method, 16n7 m L-DOPA in 100 m sodium phosphate buffer (pH 6n5), was used to stain the active tyrosinase isoforms only. In the second method, 16n7 m L-DOPA
7
T2
T3
W
NP
P
CF
PT
T2
T3
W
NP
P
CF
PT
(b)
6 Tyrosinase activity (%)
of the HPLC-purified fraction T2, or water were determined. Tyrosinase activity on L-DOPA was measured first [30 ] and then samples were diluted to give equal activity, approx. 0n3 µkat active tyrosine g−". SDS was not included in the reaction mixture, in order to assay the active forms of the enzymes only [32 ]. L-DOPA and catechol oxidation activities of the enzymes were then compared using a range of substrate concentrations (for L-DOPA from 0n8–50 m and for catechol from 0n2–45 m) but using the same amount of active tyrosinase. Catechol and L-DOPA were dissolved in 100 m sodium phosphate buffer (pH 6n5). Oxidation of L-DOPA was quantified by measuring the increase in absorbance at 478 nm and catechol oxidation was quantified by measuring the increase in absorbance at 410 nm [10 ]. The extinction coefficient of catechol was determined using the method described in Waite [28 ] and was found to be 1190 M−" cm−".
T1
5 4 3 2 1 0 T1
F. 2. Tyrosinase activity in A. bisporus U1 caps. (a) total tyrosinase activity, and (b) percentage of active tyrosinase in mushrooms treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W), crude culture filtrate (CF) and partially purified tolaasin preparation (PT), or inoculated with non-pathogenic (NP) and pathogenic (P) strains of Pseudomonas tolaasii (7, tolaasin treatments ; , negative controls ; , positive controls).
containing 0n11 % (w\v) SDS in sodium phosphate was used to stain both latent and active isoforms ; SDS activated the latent isoforms [32 ]. In each assay, 100 ml of solution were poured on the gels for 5 min, after which the gels were dried with a stream of cold air using a hairdryer.
Quantification of phenols and melanins Phenolic compounds were extracted according to Jolivet et al. [12 ], identified and quantified using HPLC. A reversed phase HPLC column (Hypersil BDS C18 5 µm 250i4n6 mm, Alltech) was used and eluted with a gradient of : 1n8 % tri-fluoroacetic acid (TFA) (A) and 10 % acetonitrile in 1n8 % TFA (B) ; 5 % to 46 % B over a time
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interval of 20 min (flow rate 1n0 ml min−"). Tyrosine, GHB and GDHB were detected by monitoring absorbance at 224 and 245 nm with a UV detector (Pharmacia). Peaks were identified from their retention times by comparison with standards (6n8, 10n0 and 13n1 min for GDHB, GHB and tyrosine (Serva) respectively). GDHB and GHB were purified according to Jolivet et al. [12 ]. Melanin was extracted from 10 mg of lyophilised mushroom powder according to the method of Lotan and Lotan [13 ] and analysed spectrophotometrically by measuring absorbance at 400 nm. Melanin concentrations were calculated from a standard curve derived using synthetic melanin (Sigma).
0n61 mg ml−" produced almost the same discolouration as did 9n5i10( cells ml−" of the pathogenic strain. The CF and PT extracts produced more intense browning than the more purified tolaasin extracts T1, T2 and T3 (0n61, 0n17 and 0n06 mg ml−" respectively). Intense pitting was observed [31 ], in response to treatment with CF, PT, and T1 preparations. Changes in tyrosinase actiity Analyses of tyrosinase activity were repeated twice. In each analysis, three mushrooms were used per treatment or inoculation and activities were measured in duplicate. The results, given in Fig. 2, are the averages of both analyses. Total tyrosinase activity in the mushrooms infected with the pathogenic strain, or treated with CF and PT extracts was lower than that of the water-treated control [Fig. 2(a)]. Mushrooms treated with the HPLC-purified toxin preparations T2 and T3 (0n17 and 0n06 mg ml−" respectively) showed no reduction in total tyrosinase activity. Only samples treated with T1 (0n61 mg ml−") showed a decrease in activity, but to a lesser extent than the samples inoculated with the pathogenic strain or treated with CF or PT. Mushrooms that turned brown also showed higher levels of active tyrosinase than the negative controls, and so, the percentage of active tyrosinases calculated relative to total tyrosinase activity (latent and active) [Fig. 2(b)],
RESULTS Increase of browning Six mushrooms were used per treatment or inoculation. All the preparations produced brown discolouration on the treated surface except for the negative controls (water treatment and inoculation with the non-pathogenic strain) (Fig. 1). All tolaasin fractions (T1, T2 and T3) induced symptoms as did the positive controls (inoculation with the pathogenic strain, and CF and PT treatments) and the level of browning was proportional to the amount of tolaasin applied. The increase in browning (∆E) was more closely correlated to ∆L (r# l 0n99), than to ∆a(r# l 0n96) or ∆b (r# l 0n71). Tolaasin at a concentration of
4.0 2 r = 0.997
3.5 3.0 2.5
1 µkatal–1
2.0 1.5 r2 = 0.991
1.0 0.5 0.0 –0.5 –1.0 –3
–2
–1
0
1
2
3
4
5
6
7
8
1/[catechol]–1 (mM)
F. 3. Lineweaver-Burk graph for active tyrosinases present in mushrooms treated with 0n17 mg tolaasin ml−" (4) and water (=) using catechol as substrate.
Tolaasin treatment of Agaricus bisporus W
T1
T2
25
higher affinities for catechol than the water-treated samples. In contrast, using L-DOPA as substrate Vmax was found to be 6n37 µkat and Km to be 0n75 m for both T2 treated and control samples.
T3
5.85
5.7
Latent and actie tyrosinase isoforms 5.5 5.4 5.3 5.20
4.55
4.5 4.4
In the gel stained to reveal latent and active isoforms (Fig. 4), two very intense bands of pIs 5n5 and 5n4 and two very weak bands of pIs 4n5 and 4n4 were found in all the samples. Bands of pI 5n3 were less intense in mushrooms treated with T1, T2 and T3 tolaasin preparations. In these samples, another intense band of pI 5n7 was present. When this gel was stained to reveal the active isoforms only (i.e. in the absence of SDS), only the low pI bands of 4n5 and 4n4 and the very intense band of pI 5n7 of mushroom extracts treated with T1, T2 or T3 were found indicating that the other isoforms were latent (gel not shown).
Substrates and products of mushroom tyrosinases 4.15
F. 4. IEF gel of A. bisporus U1 samples treated with water (W), 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3). The same total tyrosinase activity was applied to each well (15n5 nkat). The gel was stained for activity with L-DOPA, in the presence of SDS. The positions of the IEF standards are shown on the left and the pIs of each band are indicated on the right.
was higher than in the water-treated control. Only mushrooms treated with T2 or T3 (0n17 or 0n06 mg tolaasin ml−") provoked browning but the percentage of active enzyme did not differ significantly from the watertreated control. Mushrooms inoculated with the nonpathogenic strain showed the same values as the watertreated control. The samples treated with T1 (0n61 mg tolaasin ml−") gave the same level of browning as samples inoculated with the pathogenic strain and also contained almost the same percentage of active tyrosinase. Kinetic characteristics of actie tyrosinases The L-DOPA and catechol oxidation activities of the samples treated with T2 (0n17 mg tolaasin ml−") and with water (control) were measured (two mushrooms were used per treatment and activity was measured in duplicate). Using catechol as substrate (Fig. 3), Vmax was found to be similar (6n26 µkat) for both T2 and watertreated mushrooms but the Km’s were different (0n82 m for the T2 treated sample and 2n86 m for the watertreated control). This indicates that samples treated with the T2 preparation contained tyrosinase isoforms with
The amount of tyrosine, GHB and GDHB was measured in mushrooms treated with purified tolaasin preparations T1, T2 and T3 (eight replicates per datum point). Tyrosine was the phenol which was least oxidized. Its level decreased with increasing tolaasin concentrations but in no case was more than 35 % oxidized as determined by comparison with the level in the water-treated control. In PT treated mushrooms, or mushrooms infected with the pathogenic strain a higher proportion of the tyrosine was oxidized [Fig. 5(a)]. GHB levels [Fig. 5(b)] were more strongly affected by toxin treatments than the other two phenols. In samples treated with T3 (0n06 mg tolaasin ml−"), about 21 % was oxidized but in samples treated with 0n61 mg tolaasin ml−" more than 70 % was oxidized. Samples treated with PT or infected with the pathogenic strain also showed higher levels of GHB oxidation than tyrosine. GDHB [Fig. 5(C)] oxidation in tolaasin-treated samples was also very high. With the T3 preparation (0n06 mg ml−"), more than 35 % was oxidized compared to the water-treated control. With higher toxin preparations T2 or T1 (0n17 or 0n61 mg ml−" respectively), greater amounts of GDHB were oxidised leaving around 44 % unoxidized slightly less than that of GHB. PTtreated samples or samples infected with the pathogenic strain of P. tolaasii showed high levels of oxidation of GDHB and in the PT treated samples very little unoxidized GDHB or GHB was left. Melanin contents (measured in duplicate) and total phenol concentrations (the sum of tyrosine, GHB and GDHB levels) were compared [Fig. 5(d)]. A constant
C. Soler-Rias et al. (a)
GHB conc. (µmol g–1)
4 3 2 1 0
4
T1 T2 T3
W NP
P
(c)
3 2 1 0
T1 T2 T3
W NP
P
PT
4
(b)
3 2 1 0
PT Concentration (mg g–1)
GDHB conc. (µmol g–1)
Tyrosine conc. (µmol g–1)
26
T1 T2 T3
W NP
P
PT
W NP
P
PT
12 (d) 10 8 6 4 2 0
T1 T2 T3
F. 5. Phenolic compounds and melanin in A. bisporus caps treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W) and partially purified tolaasin preparation (PT) or inoculated with non-pathogenic (NP) and pathogenic (P) strains of P. tolaasii. (a) Tyrosine, (b) GHB and (c) GDHB (7, mushroom treated with tolaasin ; , negative controls ; , positive controls). (d) Melanins ( ) and sum of the three phenols ( ) (total height of columns are thus the sum of substrates and products).
value (approx. 5 mg g−") for the sum of substrates and products was shown in all treatments, except for the PT treated samples. DISCUSSION In earlier reports, browning and pitting symptoms produced in mushroom fruitbodies after the application of tolaasin were scored visually on a scale from 0 to 5. Threshold values of 0n01 mg tolaasin ml−" required to cause pitting [9 ] and 0n03 mg tolaasin ml−" for browning [4 ] were reported. The levels of browning found in our studies increased with the concentration of tolaasin applied. The lowest tolaasin concentration used (0n06 mg ml−"), was double the threshold value for browning reported by Brodey et al. [4 ] and this level produced a light brown discolouration of the cap as well as pitting. These observations are in line with those of Hutchison and Johnstone [9 ], who reported that 0n5 mg ml−" of toxin produced intense pitting, browning and tissue splitting, 0n1 mg ml−" produced pitting and significant browning while 0n05 mg ml−" produced pitting but only marginal browning. The more detailed study of browning we have carried out revealed a high correlation between the browning level (∆E) and brightness (∆L, r# l 0n99). This indicates that the most important change of colour in the browning process resulting from tolaasin treatment was a decrease in brightness. Healthy mushrooms were reported to turn reddish during senescence (increase in a) [23 ], and so the discolouration caused by the toxin treatment appears to
differ from natural senescence, which might indicate a different mechanism of melanogenesis. The partly purified preparation we used was calculated to contain approx. 1n8 mg Tol I ml−" and 0n1 mg Tol II ml−", since the ratio Tol II\Tol I was 5n6 % based on HPLC peak areas. Thus, the expected level of browning, if it was correlated with toxin concentration, would be expected to be more than three times that in mushrooms treated with the T1 preparation of tolaasin (0n61 mg ml−"). However, a lower level of browning was produced indicating that a more than saturating concentration of tolaasin was present in those samples. Total tyrosinase activity in mushrooms infected with the pathogenic strain of P. tolaasii, or treated with CF and PT extracts was lower than that in the water-treated control. The production of specific proteases by the invading bacteria [1 ] or released from the mushroom tissues due to membrane disruption [19 ], may have resulted in tyrosinase degradation. The threshold concentration of tolaasin required to induce enzyme activity appeared to lie between 0n61 and 0n17 mg ml−". At the highest tolaasin concentration (T1), only 3 % of the total tyrosinase content was active, and no significant activation occurred at lower concentrations (T2 and T3), although the latter samples showed higher levels of browning than the control. Perhaps, in these samples the levels of tolaasin were too low for tyrosine activation but were high enough to cause some membrane disruption [19 ], thus allowing already active forms of mushroom tyrosinase access to phenolic substrates. The higher affinity of active tyrosinase for catechol as a
Tolaasin treatment of Agaricus bisporus substrate, as observed in the samples that were treated with the low tolaasin concentration (T2), may be due to the fact that different isoforms are active in the treated tissues than in the untreated control mushrooms. This possibility was supported by isoelectric focusing, where a 5n7 pI active band was more intense in tolaasin treated samples than in water-treated control extracts. Active isoforms at pIs 4n4 and 4n5 were found in all treatments and similar bands have been described by Flurkey [6 ], who reported two active bands at pI 4n7 and 4n8 in other Agaricus bisporus strains. Tolaasin may cause various effects. Through its detergent properties it could disrupt membranes allowing contact between tyrosinases and their substrates. In addition, it could also activate specific tyrosinase isoforms because detergents are known to have such action [11, 32 ]. Tolaasin has been reported to display a broad spectrum of activities, including the ability to lyse horse erythrocytes, to form ion channels in planar lipid bilayers, to act as a biosurfactant, and to cause colloid osmotic lysis of protoplasts [4, 9, 20 ]. In this study it is shown that it can induce mushroom tyrosinase activation. Under our experimental conditions, 0n06 mg ml−" appeared to be sufficient for the induction of an active isoform of pI 5n7. In mushrooms treated with purified preparations of tolaasin a general oxidation of mushroom phenols occurred. Tyrosine was the phenol which was the least oxidised while GHB and GDHB were more strongly oxidised. The melanin content was found to be highly correlated to the level of browning ∆E (r# l 0n98) indicating that discolouration was mainly due to the amount of melanin formed. Since the sum of substrates and products of tyrosinase found in the different samples was always constant the three phenolic substrates appear to be the main factors in the melanogenesis induced by tolaasin. However mushrooms treated with PT contained higher levels of melanin and phenols than the other treatments indicating that other compounds may be involved in melanin formation, as suggested by Moquet et al. [15 ]. This work was supported by a grant (AIR 1 CT 94-5959) from the European Commission under the framework of the Agriculture, Agro-industry and Fisheries (AIR) programme. REFERENCES 1. Baral A, Fox PF, O’Connor TP. 1995. Isolation and characterisation of an extracellular proteinase from Pseudomonas tolaasii. Phytochemistry 39 : 757–762. 2. Bessette AE. 1984. Distribution of brown blotch bacteria in wild and cultivated species of Basidiomycetes. Applied and Enironmental Microbiology 48 : 878–880. 3. Boekelheide K, Graham DG, Mize PD, Anderson CW, Jeffs PW. 1979. Synthesis of γ-L-glutaminyl-[3,5-3H]4-
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hydroxybenzene and the study of reactions catalysed by the tyrosinase of Agaricus bisporus. The Journal of Biological Chemistry 254 : 12185–12191. 4. Brodey CL, Rainey PB, Tester M, Johnstone K. 1991. Bacterial blotch disease of the cultivated mushroom is caused by an ion channel forming lipodepsipeptide toxin. Molecular Plant-Microbe Interactions 4 : 407–411. 5. Cutri SS, Macauley BJ, Roberts WP. 1984. Characteristics of pathogenic non-fluorescent (smooth) and nonpathogenic fluorescent (rough) forms of Pseudomonas tolaasii and Pseudomonas gingeri. Journal of Applied Bacteriology 57 : 291–298. 6. Flurkey WH. 1991. Identification of tyrosinase in mushrooms by isoelectric focusing. Journal of Food Science 56 : 93–95. 7. Geels FP, Hesen LPW, Griensven LJLD van. 1994. Brown discolouration of mushrooms caused by Pseudomonas agarici. Journal of Phytopathology 140 : 249–259. 8. Hammond JBW, Nichols R. 1975. Changes in respiration and soluble carbohydrates during the post-harvest storage of mushrooms (Agaricus bisporus). Journal of the Science of Food and Agriculture 26 : 835–842. 9. Hutchison ML, Johnstone K. 1993. Evidence for the involvement of the surface active properties of the extracellular toxin tolaasin in the manifestation of brown blotch disease symptoms by Pseudomonas tolaasii on Agaricus bisporus. Physiological and Molecular Plant Pathology 42 : 373–384. 10. Ingebrigtsen J, Kang B, Flurkey WH. 1989. Tyrosinase activity and isoenzymes in developing mushrooms. Journal of Food Science 54 : 128–131. 11. Jime! nez Cervantes C, Garci! a Borro! n JC, Lozano JA, Solano F. 1995. Effect of detergents and endogenous lipids on the activity and properties of tyrosinase and its related proteins. Biochimica et Biophysica acta 1243 : 421–430. 12. Jolivet S, Voiland A, Pellon G, Arpin N. 1995. Main factors involved in the browning of Agaricus bisporus. Mushroom Science 14 : 695–702. 13. Lotan R, Lotan D. 1980. Stimulation of melanogenesis in a human melanoma cell line by retinoids. Cancer Research 40 : 3345–3350. 14. Mamoun M, Moquet F, Laffitte J, Olivier JM. 1997. Pseudomonas tolaasii : extra genomic factor mediates toxin production and efficiency. FEMS Microbiology Letters 153 : 215–219. 15. Moquet F, Mamoun M, Olivier JM. 1996. Pseudomonas tolaasii and tolaasin : Comparison of symptom induction on a wide range of Agaricus bisporus strains. FEMS Microbiology Letters 142 : 99–103. 16. Nair NG, Fahy PC. 1973. Toxin production by Pseudomonas tolaasii Paine. Australian Journal of Biological Sciences 26 : 509–512. 17. Nutkins JC, Mortishire Smith RJ, Packman LC, Brodey CL, Rainey PB, Johnstone K, Williams DH. 1991. Structure determination of tolaasin, an extracellular lipodepsipeptide produced by the mushroom pathogen. Pseudomonas tolaasii Paine. Journal of the American Chemical Society 113 : 2621–2627. 18. Olivier JM, Guillaumes J, Martin D. 1978. Study of a bacterial disease of mushroom caps. Proceedings of the 4th International Conference in Plant Pathology and Bacteriology, Angers, 903–916. 19. Preece TF, Wong WC. 1982. Quantitative and scanning electron microscope observations on the attachment of Pseudomonas tolaasii and other bacteria to the surface of Agaricus bisporus. Physiological Plant Pathology 21 : 251–257.
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20. Rainey PB, Brodey CL, Johnstone K. 1991. Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii. Physiological and Molecular Plant Pathology 39 : 57–70. 21. Rainey PB, Brodey CL, Johnstone K. 1993. Identification of a gene cluster encoding three high-molecular-weight proteins, which is required for synthesis of tolaasin by the mushroom pathogen Pseudomonas tolaasii. Molecular Microbiology 8 : 643–652. 22. Richardson PN. 1993. Stipe necrosis of cultivated mushrooms (Agaricus bisporus) associated with a fluorescent pseudomonad. Plant Pathology 42 : 927–929. 23. Sapers GM, Miller RL, Miller FC, Cooke PH, Choi SW. 1994. Enzymatic browning control in minimally processed mushrooms. Journal of Food Science 59 : 1042–1047. 24. Shirata A, Sugaya K, Takasugi M, Monde K. 1995. Isolation and biological activity of toxins produced by a Japanese strain of Pseudomonas tolaasii, the pathogen of bacterial rot of cultivated oyster mushroom. Annals of the Phytopathological Society of Japan 61 : 493–502. 25. Soler-Rivas C, Arpin N, Olivier JM, Wichers HJ. 1997. Activation of tyrosinase in Agaricus bisporus strains following infection by Pseudomonas tolaasii or treatment with a tolaasin-containing preparation. Mycological Research 101 : 375–382.
26. Soulier LFV, Arpin N. 1993. Occurrence of agaritine and γ-glutaminyl-4-hydroxybenzene (GHB) in the fructifying mycelium of Agaricus bisporus. Mycological Research 97 : 529–532. 27. Tsuneda A, Suyama K, Murakami S, Ohira I. 1995. Occurrence of Pseudomonas tolaasii on fruiting bodies of Lentinula edodes formed on Quercus logs. Mycoscience 36 : 283–288. 28. Waite JH. 1976. Calculating extinction coefficients for enzymatically produced o-quinones. Analytical Biochemistry 75 : 211–218. 29. Wells JM, Sapers GM, Fett WF, Butterfield JE, Jones JB, Bouzar H, Miller FC. 1996. Postharvest discoloration of the cultivated mushroom Agaricus bisporus caused by Pseudomonas tolaasii, P. ‘‘ reactans ’’, and P. ‘‘ gingeri ’’. Phytopathology 86 : 1098–1104. 30. Wichers HJ, Peetsma GJ, Malingre! TM, Huizing HJ. 1984. Purification and properties of a phenol oxidase derived from suspension cultures of Mucuna pruriens. Planta 162 : 334–341. 31. Wong WC, Preece TF. 1979. Identification of Pseudomonas tolaasii : the white line in agar and mushroom tissue block rapid pitting tests. Journal of Applied Bacteriology 47 : 401–407. 32. Yamaguchi M, Hwang PM, Campbell JD. 1970. Latent o-diphenol oxidase in mushrooms (Agaricus bisporus). Canadian Journal of Biochemistry 48 : 198–202.
The effects of tolaasin, the toxin produced by Pseudomonas tolaasii on tyrosinase activities and the induction of browning in Agaricus bisporus fruiting bodies C . S O L E R -R I V A S " , * , N . A R P I N # , J . - M . O L I V I E R $ a n d H . J . W I C H E R S " " Agrotechnological Research Institute (ATO-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands, # Laboratoire de Mycochimie, UniteT de Formation et de Recherche de Chimie et Biochimie, UniersiteT Claude Bernard, 69622 Villeurbanne Cedex, France, $ Institut National de la Recherche Agronomique, Station de Recherches sur les Champignons, Centre de Recherches de Bordeaux, 33883 Villenae d’Ornon Cedex, France (Accepted for publication February 1999) Infection of Agaricus bisporus with Pseudomonas tolaasii or treatment with a tolaasin containing preparation resulted in the activation of tyrosinase and the development of a brown discolouration of the fruitbody. In order to investigate whether tolaasin is responsible for these reactions, mushrooms were treated with different concentrations of HPLC-purified tolaasin. Tolaasin was found to induce both browning and the activation of tyrosinase, with the level of activation being related to the concentration of toxin applied. In water-treated controls, active tyrosinase isoforms with pIs of 4n5 and 4n4 were the main isoforms found but tolaasin treatment resulted in the activation of a tyrosinase isoform with a pI of 5n7. The different active isoforms could be distinguished as the tolaasin activated isoforms had a higher affinity for catechol as a substrate (Km 0n82 m) than the active isoforms in the control (Km 2n86 m). In mushrooms inoculated with P. tolaasii or treated with tolaasin containing extracts no significant differences were found in the mechanism of oxidation of tyrosine, γ-glutaminyl-4-hydroxybenzene or γ-glutaminyl-3,4-dihydroxybenzene to form melanin. In tolaasin treated samples, a constant total amount of melanin and sum of the three phenols was found. # 1999 Academic Press Keywords : tyrosinase ; Agaricus bisporus ; Pseudomonas tolaasii ; tolaasin ; phenols ; tyrosine ; GHB ; GDHB ; melanin ; brown blotch disease ; browning ; discolouration.
INTRODUCTION Pseudomonas spp. are common pathogens of many Basidiomycetes [2, 7, 22, 27, 29 ]. Pseudomonas tolaasii causes brown blotch disease in Agaricus bisporus which is characterised by brown spots on the surface of the sporophores and severe reductions in yield [5, 18 ]. Pseudomonas tolaasii culture filtrates cause blotch symptoms identical to those caused by the organism [16 ]. The causal factor, tolaasin, was purified by Nutkins et al. [17 ] and treatment of A. bisporus caps with it disrupts the plasma and vacuolar membranes and causes the collapse of the hyphae [20 ]. Tolaasin is a lipodepsipeptide with surface active properties [9 ] which is able to open ion channels in membranes [4 ]. * All correspondence should be addressed to : C. Soler-Rivas, Agrotechnological Research Institute (AT-DLO), Bornsesteeg 59, 6708 PD Wageningen, The Netherlands. Abbreviations used in text : CF, cell-free culture filtrate ; GDHB, γ-glutaminyl-3,4-dihydroxybenzene ; GHB, γ-glutaminyl-4-hydroxybenzene ; IEF, isoelectric focusing ; L-DOPA, L-dihydroxyphenylananine ; PT, partially purified toxin ; T1, 0n61 mg tolaasin ml−" ; T2, 0n17 mg tolaasin ml−" ; T3, 0n06 mg tolaasin ml−".
0885-5765\98\070021j08 $30.00\0
The tolaasin isolated by Nutkins et al. [17 ] consisted of two principal components, Tol I, the major component, with a molecular mass of 1985 and Tol II, a minor component with a molecular mass of 1941. The composition of Tol II was found to be identical to that of Tol I apart from a single amino acid substitution. However, other tolaasins have been reported with different amino acid compositions. Eight isoforms were purified from a P. tolaasii strain isolated from Pleurotus ostreaus using a different purification method [24 ] than that used by Nutkins et al. [17 ]. Of these, Tox 4, the main toxin, possessed the same structure as Tol I while Tox 6 was identical to Tol II. Such diversity in structure is not uncommon for compounds synthesised by non-ribosomal mechanisms, and tolaasin appears to be synthesised by a peptidesynthetase complex of at least three enzymes (TL1, TL2 and TL3) encoded by a TL-gene cluster of 65 kbp located at one end of a 640 kbp PacI chromosomal fragment [21 ]. Other extra-genomic factors, required to mediate tolaasin production and activity, have been described [14 ]. In an earlier paper, it was reported that a protease free preparation of tolaasin induced blotch symptoms by # 1999 Academic Press
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activating tyrosinases, which are involved in the browning mechanism [25 ]. Tyrosinases are normally present in cells in a latent form but when activated, oxidise phenolic compounds such as tyrosine, γ-glutaminyl-4-hydroxybenzene (GHB) and γ-glutaminyl-3,4-dihydroxybenzene (GDHB) to form brown melanin-like compounds [3, 12, 26 ]. Although there is the possibility that a specific protease was present as an undetected contaminant of the tolaasin preparation [1 ], it seems more likely that tolaasin itself is involved in the activation of tyrosinase. This paper reports an investigation of the effects of an HPLC-purified tolaasin preparation on the activation of tyrosinase isoforms and the induction of tissue browning in A. bisporus. MATERIALS AND METHODS Fungal and bacterial material The mushroom strain used in this investigation, Agaricus bisporus U1 (Somycel) was grown in boxes at the Mushroom Experimental Station, Horst, The Netherlands. The boxes were transferred to ATO-DLO, Wageningen, a few days before the onset of fruitbody production. The boxes were maintained at 18mC and 80–85 % r.h. throughout the period of fruitbody production. Two bacterial strains of Pseudomonas tolaasii, SPC 8907 and SPC 8911 were used. Strain SPC 8907, a subculture of P. tolaasii 112 S (Preece) (NCPPB no. 3148), was pathogenic, white line test positive [31 ] and produced smooth colonies on Pseudomonas agar F medium. Strain SPC 8911 was non-pathogenic, white line test negative and produced rough colonies on Pseudomonas agar F medium. Strain SPC 8911 was isolated from mushrooms in 1985 at the Mushroom Experimental Station, Horst. The two strains were cultured as described by SolerRivas et al. [25 ] and mushrooms were inoculated with c. 9n5i10( cells ml−" of the required strain according to Soler-Rivas et al. [25 ]. Preparation of toxin-containing extracts and purification of tolaasin Toxin preparations were obtained from the pathogenic strain, SPC 8907 as described by Nutkins et al. [17 ]. After each stage of purification, the preparations were tested for their effects on mushrooms. These preparations were, 0n83 mg ml−" of the crude extract (CF ; cell free culture filtrate), 2n85 mg ml−" of the fraction obtained prior to HPLC separation (PT ; partially purified toxin) and a range of concentrations of a fraction, equivalent to Tol I, obtained after HPLC purification. The HPLC chromatograms obtained were similar to those of Nutkins et al. [17 ], and contained only two forms of tolaasin Tol I and Tol II. The Tol I fraction was lyophilized, dissolved in
DMSO and analysed by NMR. The fraction was about 82 % pure, calculated on proton number and this fraction was used at concentrations of 0n61 (T1), 0n17 (T2) and 0n06 mg ml−" (T3). A range of concentrations of the Tol I fraction were analysed by HPLC to obtain a standard curve for the relationship between peak area and tolaasin concentration. Tol I peak area in PT extract chromatograms was expressed as tolaasin concentration. The percentage of tolaasin in the PT extract was thus, calculated to be 65 %. Inoculation procedure and treatment with tolaasin preparations Mushrooms from the first flush with caps of between 30–40 mm diameter, in stages 2 or 3 of development according to the classification of Hammond and Nichols [8 ], were harvested and prepared as described by Moquet et al. [15 ]. They were inoculated with P. tolaasii strains or treated with the tolaasin preparations as described in Soler-Rivas et al. [25 ]. Two negative controls were included in each experiment, inoculation with the non-pathogenic strain SPC 8911 [5 ] and a water treatment. Three positive controls were also included, inoculation with the pathogenic strain SPC 8907, and treatments with CF, and PT [25 ]. The experimental treatments involved mushrooms treated with the tolaasin preparations obtained after HPLC (T1, T2, and T3). All measurements and assays were carried out 2 days after inoculation or treatment. Browning measurements and tyrosinase actiity Changes in colour of the mushroom tissue were measured with a Chroma Meter (Minolta CR-200, 1 cm slot) using the procedure described by Soler-Rivas et al. [25 ]. This procedure involves measuring several parameters ; L is a measure of brightness (from 0, white to 100, black), a and b are measures of chromaticity. The parameter a is a measure of change from green to red (positive values indicate red colour and negative green), and b a measure of change from blue to yellow (blue is negative and yellow positive). Browning was quantified as the change in colour (∆E l N[∆L#j∆a#j∆b#]) resulting from the treatment in relation to the colour of the tissue immediately before inoculation or treatment. After measurement of browning, samples were extracted as described previously [25 ] for tyrosinase analysis. The tyrosinase activity was measured using L-DOPA (Sigma) as substrate as described by Wichers et al. [30 ]. Kinetic measurements The kinetics of L-DOPA and catechol (Sigma) oxidation by the tyrosinases after treatment with either 0n17 mg ml−"
Tolaasin treatment of Agaricus bisporus 50 Tyrosinase activity (µkat g–1)
50
Browning level (DE)
40 30 20 10 0
T1
T2
T3
W
NP
P
CF
23
(a)
40
30
20
10
PT 0
F. 1. Browning levels (∆E) in Agaricus bisporus U1 caps treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W), crude culture filtrate (CF) and partially purified tolaasin preparation (PT), or inoculated with non-pathogenic (NP) and pathogenic (P) strains of Pseudomonas tolaasii (7, tolaasin treatments ; , negative controls ; , positive controls).
Isoelectric focusing Isoelectric focusing was carried out using IEF 3-6 gels (Servalyt2 Precotes2 150 µm, 125i125 mm, Serva). Samples of 10 µl adjusted to contain 15n5 µkat g−" dry wt. with 100 m sodium phosphate buffer (pH 6n5) were applied to each well and the gels were run for 3 h following the manufacturer’s instructions. IEF standards (Pharmacia) were used to calculate the pI of each band after the bands had been visualized by staining with Coomassie blue G250 (Merck). Gels were stained for tyrosinase activity by two methods. In the first method, 16n7 m L-DOPA in 100 m sodium phosphate buffer (pH 6n5), was used to stain the active tyrosinase isoforms only. In the second method, 16n7 m L-DOPA
7
T2
T3
W
NP
P
CF
PT
T2
T3
W
NP
P
CF
PT
(b)
6 Tyrosinase activity (%)
of the HPLC-purified fraction T2, or water were determined. Tyrosinase activity on L-DOPA was measured first [30 ] and then samples were diluted to give equal activity, approx. 0n3 µkat active tyrosine g−". SDS was not included in the reaction mixture, in order to assay the active forms of the enzymes only [32 ]. L-DOPA and catechol oxidation activities of the enzymes were then compared using a range of substrate concentrations (for L-DOPA from 0n8–50 m and for catechol from 0n2–45 m) but using the same amount of active tyrosinase. Catechol and L-DOPA were dissolved in 100 m sodium phosphate buffer (pH 6n5). Oxidation of L-DOPA was quantified by measuring the increase in absorbance at 478 nm and catechol oxidation was quantified by measuring the increase in absorbance at 410 nm [10 ]. The extinction coefficient of catechol was determined using the method described in Waite [28 ] and was found to be 1190 M−" cm−".
T1
5 4 3 2 1 0 T1
F. 2. Tyrosinase activity in A. bisporus U1 caps. (a) total tyrosinase activity, and (b) percentage of active tyrosinase in mushrooms treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W), crude culture filtrate (CF) and partially purified tolaasin preparation (PT), or inoculated with non-pathogenic (NP) and pathogenic (P) strains of Pseudomonas tolaasii (7, tolaasin treatments ; , negative controls ; , positive controls).
containing 0n11 % (w\v) SDS in sodium phosphate was used to stain both latent and active isoforms ; SDS activated the latent isoforms [32 ]. In each assay, 100 ml of solution were poured on the gels for 5 min, after which the gels were dried with a stream of cold air using a hairdryer.
Quantification of phenols and melanins Phenolic compounds were extracted according to Jolivet et al. [12 ], identified and quantified using HPLC. A reversed phase HPLC column (Hypersil BDS C18 5 µm 250i4n6 mm, Alltech) was used and eluted with a gradient of : 1n8 % tri-fluoroacetic acid (TFA) (A) and 10 % acetonitrile in 1n8 % TFA (B) ; 5 % to 46 % B over a time
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interval of 20 min (flow rate 1n0 ml min−"). Tyrosine, GHB and GDHB were detected by monitoring absorbance at 224 and 245 nm with a UV detector (Pharmacia). Peaks were identified from their retention times by comparison with standards (6n8, 10n0 and 13n1 min for GDHB, GHB and tyrosine (Serva) respectively). GDHB and GHB were purified according to Jolivet et al. [12 ]. Melanin was extracted from 10 mg of lyophilised mushroom powder according to the method of Lotan and Lotan [13 ] and analysed spectrophotometrically by measuring absorbance at 400 nm. Melanin concentrations were calculated from a standard curve derived using synthetic melanin (Sigma).
0n61 mg ml−" produced almost the same discolouration as did 9n5i10( cells ml−" of the pathogenic strain. The CF and PT extracts produced more intense browning than the more purified tolaasin extracts T1, T2 and T3 (0n61, 0n17 and 0n06 mg ml−" respectively). Intense pitting was observed [31 ], in response to treatment with CF, PT, and T1 preparations. Changes in tyrosinase actiity Analyses of tyrosinase activity were repeated twice. In each analysis, three mushrooms were used per treatment or inoculation and activities were measured in duplicate. The results, given in Fig. 2, are the averages of both analyses. Total tyrosinase activity in the mushrooms infected with the pathogenic strain, or treated with CF and PT extracts was lower than that of the water-treated control [Fig. 2(a)]. Mushrooms treated with the HPLC-purified toxin preparations T2 and T3 (0n17 and 0n06 mg ml−" respectively) showed no reduction in total tyrosinase activity. Only samples treated with T1 (0n61 mg ml−") showed a decrease in activity, but to a lesser extent than the samples inoculated with the pathogenic strain or treated with CF or PT. Mushrooms that turned brown also showed higher levels of active tyrosinase than the negative controls, and so, the percentage of active tyrosinases calculated relative to total tyrosinase activity (latent and active) [Fig. 2(b)],
RESULTS Increase of browning Six mushrooms were used per treatment or inoculation. All the preparations produced brown discolouration on the treated surface except for the negative controls (water treatment and inoculation with the non-pathogenic strain) (Fig. 1). All tolaasin fractions (T1, T2 and T3) induced symptoms as did the positive controls (inoculation with the pathogenic strain, and CF and PT treatments) and the level of browning was proportional to the amount of tolaasin applied. The increase in browning (∆E) was more closely correlated to ∆L (r# l 0n99), than to ∆a(r# l 0n96) or ∆b (r# l 0n71). Tolaasin at a concentration of
4.0 2 r = 0.997
3.5 3.0 2.5
1 µkatal–1
2.0 1.5 r2 = 0.991
1.0 0.5 0.0 –0.5 –1.0 –3
–2
–1
0
1
2
3
4
5
6
7
8
1/[catechol]–1 (mM)
F. 3. Lineweaver-Burk graph for active tyrosinases present in mushrooms treated with 0n17 mg tolaasin ml−" (4) and water (=) using catechol as substrate.
Tolaasin treatment of Agaricus bisporus W
T1
T2
25
higher affinities for catechol than the water-treated samples. In contrast, using L-DOPA as substrate Vmax was found to be 6n37 µkat and Km to be 0n75 m for both T2 treated and control samples.
T3
5.85
5.7
Latent and actie tyrosinase isoforms 5.5 5.4 5.3 5.20
4.55
4.5 4.4
In the gel stained to reveal latent and active isoforms (Fig. 4), two very intense bands of pIs 5n5 and 5n4 and two very weak bands of pIs 4n5 and 4n4 were found in all the samples. Bands of pI 5n3 were less intense in mushrooms treated with T1, T2 and T3 tolaasin preparations. In these samples, another intense band of pI 5n7 was present. When this gel was stained to reveal the active isoforms only (i.e. in the absence of SDS), only the low pI bands of 4n5 and 4n4 and the very intense band of pI 5n7 of mushroom extracts treated with T1, T2 or T3 were found indicating that the other isoforms were latent (gel not shown).
Substrates and products of mushroom tyrosinases 4.15
F. 4. IEF gel of A. bisporus U1 samples treated with water (W), 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3). The same total tyrosinase activity was applied to each well (15n5 nkat). The gel was stained for activity with L-DOPA, in the presence of SDS. The positions of the IEF standards are shown on the left and the pIs of each band are indicated on the right.
was higher than in the water-treated control. Only mushrooms treated with T2 or T3 (0n17 or 0n06 mg tolaasin ml−") provoked browning but the percentage of active enzyme did not differ significantly from the watertreated control. Mushrooms inoculated with the nonpathogenic strain showed the same values as the watertreated control. The samples treated with T1 (0n61 mg tolaasin ml−") gave the same level of browning as samples inoculated with the pathogenic strain and also contained almost the same percentage of active tyrosinase. Kinetic characteristics of actie tyrosinases The L-DOPA and catechol oxidation activities of the samples treated with T2 (0n17 mg tolaasin ml−") and with water (control) were measured (two mushrooms were used per treatment and activity was measured in duplicate). Using catechol as substrate (Fig. 3), Vmax was found to be similar (6n26 µkat) for both T2 and watertreated mushrooms but the Km’s were different (0n82 m for the T2 treated sample and 2n86 m for the watertreated control). This indicates that samples treated with the T2 preparation contained tyrosinase isoforms with
The amount of tyrosine, GHB and GDHB was measured in mushrooms treated with purified tolaasin preparations T1, T2 and T3 (eight replicates per datum point). Tyrosine was the phenol which was least oxidized. Its level decreased with increasing tolaasin concentrations but in no case was more than 35 % oxidized as determined by comparison with the level in the water-treated control. In PT treated mushrooms, or mushrooms infected with the pathogenic strain a higher proportion of the tyrosine was oxidized [Fig. 5(a)]. GHB levels [Fig. 5(b)] were more strongly affected by toxin treatments than the other two phenols. In samples treated with T3 (0n06 mg tolaasin ml−"), about 21 % was oxidized but in samples treated with 0n61 mg tolaasin ml−" more than 70 % was oxidized. Samples treated with PT or infected with the pathogenic strain also showed higher levels of GHB oxidation than tyrosine. GDHB [Fig. 5(C)] oxidation in tolaasin-treated samples was also very high. With the T3 preparation (0n06 mg ml−"), more than 35 % was oxidized compared to the water-treated control. With higher toxin preparations T2 or T1 (0n17 or 0n61 mg ml−" respectively), greater amounts of GDHB were oxidised leaving around 44 % unoxidized slightly less than that of GHB. PTtreated samples or samples infected with the pathogenic strain of P. tolaasii showed high levels of oxidation of GDHB and in the PT treated samples very little unoxidized GDHB or GHB was left. Melanin contents (measured in duplicate) and total phenol concentrations (the sum of tyrosine, GHB and GDHB levels) were compared [Fig. 5(d)]. A constant
C. Soler-Rias et al. (a)
GHB conc. (µmol g–1)
4 3 2 1 0
4
T1 T2 T3
W NP
P
(c)
3 2 1 0
T1 T2 T3
W NP
P
PT
4
(b)
3 2 1 0
PT Concentration (mg g–1)
GDHB conc. (µmol g–1)
Tyrosine conc. (µmol g–1)
26
T1 T2 T3
W NP
P
PT
W NP
P
PT
12 (d) 10 8 6 4 2 0
T1 T2 T3
F. 5. Phenolic compounds and melanin in A. bisporus caps treated with 0n61 mg tolaasin ml−" (T1), 0n17 mg tolaasin ml−" (T2) and 0n06 mg tolaasin ml−" (T3), water (W) and partially purified tolaasin preparation (PT) or inoculated with non-pathogenic (NP) and pathogenic (P) strains of P. tolaasii. (a) Tyrosine, (b) GHB and (c) GDHB (7, mushroom treated with tolaasin ; , negative controls ; , positive controls). (d) Melanins ( ) and sum of the three phenols ( ) (total height of columns are thus the sum of substrates and products).
value (approx. 5 mg g−") for the sum of substrates and products was shown in all treatments, except for the PT treated samples. DISCUSSION In earlier reports, browning and pitting symptoms produced in mushroom fruitbodies after the application of tolaasin were scored visually on a scale from 0 to 5. Threshold values of 0n01 mg tolaasin ml−" required to cause pitting [9 ] and 0n03 mg tolaasin ml−" for browning [4 ] were reported. The levels of browning found in our studies increased with the concentration of tolaasin applied. The lowest tolaasin concentration used (0n06 mg ml−"), was double the threshold value for browning reported by Brodey et al. [4 ] and this level produced a light brown discolouration of the cap as well as pitting. These observations are in line with those of Hutchison and Johnstone [9 ], who reported that 0n5 mg ml−" of toxin produced intense pitting, browning and tissue splitting, 0n1 mg ml−" produced pitting and significant browning while 0n05 mg ml−" produced pitting but only marginal browning. The more detailed study of browning we have carried out revealed a high correlation between the browning level (∆E) and brightness (∆L, r# l 0n99). This indicates that the most important change of colour in the browning process resulting from tolaasin treatment was a decrease in brightness. Healthy mushrooms were reported to turn reddish during senescence (increase in a) [23 ], and so the discolouration caused by the toxin treatment appears to
differ from natural senescence, which might indicate a different mechanism of melanogenesis. The partly purified preparation we used was calculated to contain approx. 1n8 mg Tol I ml−" and 0n1 mg Tol II ml−", since the ratio Tol II\Tol I was 5n6 % based on HPLC peak areas. Thus, the expected level of browning, if it was correlated with toxin concentration, would be expected to be more than three times that in mushrooms treated with the T1 preparation of tolaasin (0n61 mg ml−"). However, a lower level of browning was produced indicating that a more than saturating concentration of tolaasin was present in those samples. Total tyrosinase activity in mushrooms infected with the pathogenic strain of P. tolaasii, or treated with CF and PT extracts was lower than that in the water-treated control. The production of specific proteases by the invading bacteria [1 ] or released from the mushroom tissues due to membrane disruption [19 ], may have resulted in tyrosinase degradation. The threshold concentration of tolaasin required to induce enzyme activity appeared to lie between 0n61 and 0n17 mg ml−". At the highest tolaasin concentration (T1), only 3 % of the total tyrosinase content was active, and no significant activation occurred at lower concentrations (T2 and T3), although the latter samples showed higher levels of browning than the control. Perhaps, in these samples the levels of tolaasin were too low for tyrosine activation but were high enough to cause some membrane disruption [19 ], thus allowing already active forms of mushroom tyrosinase access to phenolic substrates. The higher affinity of active tyrosinase for catechol as a
Tolaasin treatment of Agaricus bisporus substrate, as observed in the samples that were treated with the low tolaasin concentration (T2), may be due to the fact that different isoforms are active in the treated tissues than in the untreated control mushrooms. This possibility was supported by isoelectric focusing, where a 5n7 pI active band was more intense in tolaasin treated samples than in water-treated control extracts. Active isoforms at pIs 4n4 and 4n5 were found in all treatments and similar bands have been described by Flurkey [6 ], who reported two active bands at pI 4n7 and 4n8 in other Agaricus bisporus strains. Tolaasin may cause various effects. Through its detergent properties it could disrupt membranes allowing contact between tyrosinases and their substrates. In addition, it could also activate specific tyrosinase isoforms because detergents are known to have such action [11, 32 ]. Tolaasin has been reported to display a broad spectrum of activities, including the ability to lyse horse erythrocytes, to form ion channels in planar lipid bilayers, to act as a biosurfactant, and to cause colloid osmotic lysis of protoplasts [4, 9, 20 ]. In this study it is shown that it can induce mushroom tyrosinase activation. Under our experimental conditions, 0n06 mg ml−" appeared to be sufficient for the induction of an active isoform of pI 5n7. In mushrooms treated with purified preparations of tolaasin a general oxidation of mushroom phenols occurred. Tyrosine was the phenol which was the least oxidised while GHB and GDHB were more strongly oxidised. The melanin content was found to be highly correlated to the level of browning ∆E (r# l 0n98) indicating that discolouration was mainly due to the amount of melanin formed. Since the sum of substrates and products of tyrosinase found in the different samples was always constant the three phenolic substrates appear to be the main factors in the melanogenesis induced by tolaasin. However mushrooms treated with PT contained higher levels of melanin and phenols than the other treatments indicating that other compounds may be involved in melanin formation, as suggested by Moquet et al. [15 ]. This work was supported by a grant (AIR 1 CT 94-5959) from the European Commission under the framework of the Agriculture, Agro-industry and Fisheries (AIR) programme. REFERENCES 1. Baral A, Fox PF, O’Connor TP. 1995. Isolation and characterisation of an extracellular proteinase from Pseudomonas tolaasii. Phytochemistry 39 : 757–762. 2. Bessette AE. 1984. Distribution of brown blotch bacteria in wild and cultivated species of Basidiomycetes. Applied and Enironmental Microbiology 48 : 878–880. 3. Boekelheide K, Graham DG, Mize PD, Anderson CW, Jeffs PW. 1979. Synthesis of γ-L-glutaminyl-[3,5-3H]4-
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