Appl Microbiol Biotechnol (2002) 59:284–288 DOI 10.1007/s00253-002-0988-6
O R I G I N A L PA P E R
R. Duran · C. Deschler · S. Precigou · P. Goulas
Degradation of chlorophenols by Phanerochaete chrysosporium: effect of 3,4-dichlorophenol on extracellular peroxidase activities
Received: 19 November 2001 / Revised: 28 February 2002 / Accepted: 7 March 2002 / Published online: 1 June 2002 © Springer-Verlag 2002
Abstract Extracellular peroxidases play an important role in the degradation of chlorophenols by Phanerochaete chrysosporium. Depending on the moment of 3,4-dichlorophenol addition, the production of lignin peroxidase and manganese peroxidase in C-limited agitated cultures was affected in opposite ways. In cultures that received 3,4-dichlorophenol at the time of inoculation, fungal growth was reduced and peroxidases were not produced, whereas peroxidase activities were stabilized after 3,4-dichlorophenol addition to pregrown cultures. Further investigation revealed that mRNA encoding lignin peroxidase was not produced in cultures started with 3,4-dichlorophenol, suggesting that the onset of secondary metabolism was affected. In addition, the stabilization of lignin peroxidase activity was not the result of an activation of lignin peroxidase gene transcription, as shown by Northern blot experiments, but likely due to the inhibition of peroxidase degradation by extracellular proteases.
Introduction Phanerochaete chrysosporium, a lignin-degrading white rot fungus, is known to degrade a variety of environmentally persistent pollutants, including several chlorinated phenols (Hammel 1989; Barr and Aust 1994; Paszczynski and Crawford 1995). Under ligninolytic conditions, degradation of chlorophenols is initiated by oxidative dechlorination of the substrate to its corresponding p-quinone, catalyzed by two extracellular peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP; Hammel and Tardone 1988; Valli and Gold 1991; Joshi and Gold 1993; Reddy et al. 1998; Reddy and Gold 2000). Owing to its capacity to degrade chlorophenols, R. Duran · C. Deschler · S. Precigou · P. Goulas (✉) Laboratoire d’Ecologie Moléculaire, IBEAS, Université de Pau et des Pays de l’Adour, UFR Sciences, BP 1155, 64000 Pau Cedex, France e-mail:
[email protected] Tel.: +33-5-59923145, Fax: +33-5-59808311
some practical uses of P. chrysosporium have been explored, e.g., to decolorize and detoxify pulp and paper mill effluents, or to treat soils contaminated by wastes of the wood preservatives industry (Springer 1993; Paszczynski and Crawford 1995). However, microbial organochloride toxicity and the complexity of the metabolic activities of P. chrysosporium remain problems for the use of this white rot fungus as an agent of bioremediation. To better understand the metabolic pathways, we previously showed that P. chrysosporium was able to grow in a C-limited medium containing 3,4-dichlorophenol (Deschler et al. 1998). Although the complete disappearance of 3,4-dichlorophenol was achieved after about 2 weeks, only a weak amount of chloride was recovered, corresponding to approximately 15% of the starting material. Furthermore, in comparison with cultures without 3,4-dichlorophenol, we observed the lack of LiP and MnP production. Since the efficiency of dechlorination is strongly dependent on peroxidase activities, the aim of this work was to investigate the effect of 3,4-dichlorophenol on LiP activity under C-limited culture conditions.
Materials and methods Fungal strain and culture conditions P. chrysosporium ATCC 24725 was grown at 32 °C under an air atmosphere, in C-limited agitated liquid medium, using glycerol as the C-source and containing MnSO4·H2O (30 mg/l), as described by Deschler et al. (1998). The following culture volume was employed: 1,000 ml/6,000 ml (medium/Erlenmeyer flasks). The agitation rate was 140 rpm. A suspension of conidia in 0.1% Tween 80 served as the inoculum to obtain a final concentration of 105 conidia/ml. 3,4-Dichlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, and pentachlorophenol in methanol were added to the culture medium at a final concentration of 250, 250, 100, and 15 µM, respectively. All chemicals were purchased from Sigma-Aldrich. Enzymatic assays LiP and MnP activities were measured with veratryl alcohol and vanillylacetone as substrates, respectively (Bono et al. 1990). Pro-
285 tease activity was measured with azocoll, as described by Dosoretz et al. (1990a). Analytical methods The peroxidases were purified from the extracellular medium by fast protein liquid chromatography (FPLC) with a mono Q anionexchange column as described by Bono et al. (1990). Heme protein nomenclature (H1–H10) was based on elution properties and activity tests (Dass and Reddy 1990). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) in a 15% (w/v) polyacrylamide slab gel. Proteins were detected by staining with Coomassie brilliant blue G-250. RNA preparation P. chrysosporium mycelia were recovered by filtration through Whatman filter paper, washed with TE buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA), and stored at–20 °C prior to use. Frozen mycelia (3 g) were ground to a powder with a mortar and pestle under liquid N2 and suspended in 3 ml of lysis buffer [200 mM sodium acetate, pH 5.0, 10 mM EDTA, 1% SDS(w/v)]. One volume of prewarmed phenol (65 °C) and 0.5 vol of chloroform were added. Then the mixture was allowed to stand at room temperature for 10 min. The aqueous phase was recovered by centrifugation and then twice extracted with an equal volume of chloroform. RNA was precipitated with 2 M LiCl, resuspended in water, and stored at –20 °C. Following purification, the integrity of RNA samples was verified by electrophoresis through formaldehyde agarose gels stained with ethidium bromide. RNA concentrations were normalized with ribosomal RNA fluorescence. Aliquots (100 µg) of total RNA were prepared for Northern analysis. Northern analysis Total RNA was electrophoresed in 1.2% (w/v) agarose gels containing 2 M formaldehyde and then transferred to a Nylon membrane (Hybond-N, Amersham) by capillary blotting, as described by Sambrook et al. (1989). RNA was fixed onto the membrane by baking at 80 °C for 1 h. Filters were hybridized with a 32P-labeled LiP probe at 65 °C overnight. The oligonucleotide (5′-AGCTGCGTCTCGACGGAAGAACTGGGAGTCGAA-3′) which hybridizes to the coding region of all the LiP genes so far studied was used as a LiP probe. The probe was 5′ end-labeled with [γ-32P] ATP at 5,000 Ci/mmol (Amersham), using T4 polynucleotide kinase (Sambrook et al. 1989).
Results LiP and MnP activities This study was carried out with C-limited agitated cultures of P. chrysosporium, using glycerol as the carbon source under an air atmosphere (Deschler et al. 1998). Under these culture conditions, the formation of regular pellets was observed and high levels of LiP (1,000 units/l) and MnP (800 units/l) were obtained. Typically, LiP and MnP activities appeared on day 5 in the culture medium, reached a maximum on day 7 and disappeared on day 10 (Fig. 1). Four isoenzymes with LiP activity (H1, H2, Ha, H6) and two isoenzymes with MnP activity (H3, H4) were recovered after separation with a mono Q anion-exchange column (data not shown).
Fig. 1A, B Extracellular peroxidase activities of C-limited agitated cultures of Phanerochaete chrysosporium. A Lignin peroxidase (LiP) activity: circles without 3,4-dichlorophenol, triangles with 3,4-dichlorophenol added on day 7, squares with 3,4-dichlorophenol added at time of inoculation. B Manganese peroxidase (MnP) activity: circles without 3,4-dichlorophenol, triangles with 3,4dichlorophenol added on day 7, squares with 3,4-dichlorophenol added at time of inoculation. U Units of enzyme activity
The addition of 3,4-dichlorophenol changed appreciably the time-course of LiP and MnP activities. Indeed, contrary to control cultures, peroxidase activity stabilization was observed in the extracellular fluid of cultures with 3,4-dichlorophenol added on day 7 (Fig. 1). The peroxidase activity remained practically constant during the entire course of the experiments (15 days). Conversely, neither LiP nor MnP activities were observed when 3,4-dichlorophenol (250 µM) was added at the time of inoculation. However, under these conditions, smaller pellets were observed and a 30% biomass reduction was measured with regard to the control cultures (2 g/l, 2.85 g/l, respectively). To investigate the possible link between the lack of peroxidase activity and the deactivation of enzymes by inhibitors, the presence of extracellular peroxidases was analyzed in the supernatant of 7-day-old cultures. Culture fluids were recovered, concentrated by ultrafiltration, and analyzed by FPLC or precipitated with 10% trichloroacetic acid and subjected to SDS-PAGE. The FPLC profile monitored at 409 nm showed that no heme protein was retained on the mono Q anion-exchange column. The absence of peroxidases was confirmed by SDS-PAGE analysis. Extracellular proteins could not be detected with Coomassie brilliant blue staining (Fig. 2), suggesting that 3,4-dichlorophenol inhibited more or less directly the secretion or the biosynthesis of LiP and MnP. This phenomenon was not observed in cultures of P. chrysosporium established by inoculation with fungal spores in the presence of pentachlorophenol (15 µM), 2,4,5-trichlorophenol (100 µM), or 2,4-dichlorophenol (250 µM). Although fungal growth was also significantly reduced with regard to that obtained in control cultures
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Fig. 2 SDS-PAGE electrophoresis of P. chrysosporium extracellular proteins precipitated from 7-day-old cultures. Lane 1 Purified LiP (isoenzyme H2), lane 2 culture with 3,4-dichlorophenol added at time of inoculation, lane 3 culture without 3,4-dichlorophenol
Fig. 3A–C Northern blot analysis of LiP transcript accumulation after various days of growth. Total RNA extracted from P. chrysosporium was probed with the LiP-specific probe. Lanes 1–5 contained total RNA (100 µg) corresponding to days 3–7 of growth in cultures. A Without 3,4-dichlorophenol, B with 3,4-dichlorophenol added at time of inoculation, C with 3,4-dichlorophenol added on day 5
without chlorophenols, LiP activities were observed in these three cases, in contrast to the cultures with 3,4-dichlorophenol. Northern blot analysis Northern blot analysis was performed to investigate the role played by the xenobiotic 3,4-dichlorophenol on LiP gene expression. P. chrysosporium was grown in C-limited medium under three conditions: without 3,4-dichlorophenol, with 3,4-dichlorophenol added to pregrown cultures (day 5), and with 3,4-dichlorophenol added at the time of inoculation. Total RNA was extracted on days 3–7 of growth, subjected to agarose gel electrophoresis, and hybridized with a LiP-specific oligonucleotide probe (Fig. 3). LiP transcripts were observed on days 4–7 in P. chrysosporium cultures without 3,4dichlorophenol and on days 4 and 5 in cultures with 3,4-dichlorophenol added on day 5. In contrast, no transcript was observed in P. chrysosporium cultures with 3,4-dichlorophenol added at the time of inoculation, explaining the absence of peroxidase activity in the fluid of these cultures.
Fig. 4 Extracellular protease activity of C-limited agitated cultures of P. chrysosporium. Circles Without 3,4-dichlorophenol, triangles with 3,4-dichlorophenol added on day 6
followed the extracellular proteolytic activity of P. chrysosporium ligninolytic cultures. As expected, an extracellular protease activity was observed under C-limited agitated culture conditions. The addition of 3,4-dichlorophenol on day 6 led to an important decline in protease activity (Fig. 4) that may correspond to the maintenance of LiP activity.
Discussion Effect of 3,4-dichlorophenol on the protease activity The decline in LiP activity in ligninolytic cultures of P. chrysosporium has been correlated with the appearance of iodophasic extracellular protease activity (Dosoretz et al. 1990b). Aiming to understand the mechanism of stabilization of the LiP activity when 3,4-dichlorophenol was added to pregrown cultures, we
The white-rot fungus P. chrysosporium has been shown to degrade a variety of persistent pollutants. The enzymes responsible for pollutant degradation are normally involved in the degradation of wood (Cameron et al. 2000). From studies on the P. chrysosporium degradation of 2,4-dichlorophenol (Valli and Gold 1991), 3,4-dichlorophenol (Deschler et al. 1998), 2,4,5-trichlorophenol (Joshi and Gold 1993), 2,4,6-trichlorophenol (Armenante
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et al. 1994; Reddy et al. 1998), and pentachlorophenol (Mileski et al. 1988; Reddy and Gold 2000), it appears that the first step of the pathway can be either an extracellular oxidative dechlorination of the substrate catalyzed by LiP or MnP to its corresponding p-quinone (followed by the enzymatic or non enzymatic quinone reduction to p-dihydroxybenzene), or an intracellular o-hydroxylation of the polychlorophenol (Deschler et al. 1998). The (poly)chlorodihydroxybenzenes produced by these different pathways can undergo further successive intracellular reductive dechlorinations and hydroxylation to 1,2,4-trihydroxybenzene (Reddy et al. 1998; Reddy and Gold 2000). In contrast to the oxidative dechlorination step, the hydroxylations and reductive dechlorinations occur during both primary and secondary metabolic growth. Beside these degradative reactions, the o-methylation of polychlorophenols by P. chrysosporium has often been observed. For example, significant amounts of pentachloroanisole, which probably retains significant toxicity, have been found in soils contaminated by pentachlorophenol and treated with P. chrysosporium (Lamar and Dietrich 1990). In any case, it appears that the efficiency of chlorophenol degradation is strongly dependent on peroxidase activities. Previously, we showed that the first step in 3,4-dichlorophenol transformation by P. chrysosporium may occur by oxidation, methylation, and hydroxylation reactions to produce 2-chloro-1,4-benzoquinone, 3,4-dichloroanisole, and 4,5-dichlorocatechol, respectively (Deschler et al. 1998). Nevertheless, the maximum dechlorination yield was obtained in the presence of extracellular peroxidases. Our present results show that the quantity of LiP and MnP in the culture supernatants was affected by 3,4-dichlorophenol in contrasting ways, depending on t he time when it was added to the cultures. Peroxidases were not produced in cultures that received 3,4-dichlorophenol at the time of inoculation. LiP transcripts could not be detected in cells produced under such culture conditions, indicating that 3,4-dichlorophenol interferes with LiP biosynthesis at the transcriptional level. Furthermore, the lack of extracellular proteins strongly suggests that 3,4-dichlorophenol not only inhibits fungal growth, as do the other chlorophenols used, but also affects the onset of secondary metabolism. This may explain the weak dechlorination of the substrate under these conditions (Deschler et al. 1998). In contrast, 3,4-dichlorophenol stabilized LiP activity when added to pregrown cultures. This stabilization was apparently not the result of an activation of LiP gene transcription, as shown in the Northern blot experiments. Dosoretz et al. (1990a, b) showed that the decrease in LiP activity in ligninolytic cultures of P. chrysosporium was partly due to extracellular protease activity during the secondary metabolic phase. Two different proteases, one inhibited by phenylmethylsulfonyl fluoride and the other by pepstatin A, have been characterized by electrophoresis (Dass et al. 1995). A similar secondary protease activity was observed in our cultures. The addition of 3,4-dichlorophenol to pregrown cultures resulted in
a noticeable loss of protease activity, suggesting that 3,4-dichlorophenol inhibited the degradation of peroxidases by proteases, by a mechanism that remains to be characterized. Given its capacity to degrade chlorophenols, some potential applications of P. chrysosporium have been explored, e.g., to decolorize and detoxify pulp and paper mill effluents (Springer 1993), or to treat soils contaminated by wastes of the wood preservative industry (Paszczynski and Crawford 1995). Unfortunately, until now, P. chrysosporium has not fulfilled its promise as a bioremediation agent for industrial use, because its application appears too complex and unpredictable. The results presented here point out the complex regulation of fungal metabolism, illustrating the difficulties of using P. chrysosporium in industrial processes.
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