A novel endo-β-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum

JOURNAL OF BACTERIOLOGY, Dec. 1995, p. 6937–6945 Vol. 177, No. 23 0021-9193/95/$04.0010 Copyright q1995, American Society for Microbiology A Novel Endo-b-1,3-Glucanase, BGN13.1, Involved in the Mycoparasitism of Trichoderma harzianum JESU ´SDE LA CRUZ, 1 JOSE ´A. PINTOR-TORO, 2 TAHI ´A BENI ´TEZ, 3 ANTONIO LLOBELL, 1 * AND LUIS C. ROMERO 2 Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Consejo Superior de Investigaciones Cientificas y Universidad de Sevilla, 1 Instituto de Recursos Naturales y Agrobiologı´a, Consejo Superior de Investigaciones Cientificas, 2 and Departamento Gene´tica, Universidad de Sevilla, 3 Seville, Spain Received 20 June 1995/Accepted 25 September 1995 The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular b-1,3glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for b-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this b-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls. b-1,3-Glucanases are widely distributed among bacteria, fungi, and higher plants (49), and many of them have been purified and characterized (10, 26, 40, 63, 73). They are classified as exo-b-1,3-glucanases (b-1,3-glucan glucanohydrolase [EC 3.2.1.58]) and endo-b-1,3-glucanases (b-1,3-glucan glucanohydrolase [EC 3.2.1.6 or EC 3.2.1.39]). The corresponding b-1,3-glucanase genes from the aforementioned organisms have also been isolated and characterized, with the exception of genes from filamentous fungi (29, 56, 58, 61, 70, 73). The physiological functions of b-1,3-glucanases depend on their source. In plants, these enzymes are thought to be a type of defense system against fungal pathogens (25, 37), although roles in cell differentiation have also been suggested (6, 20, 42). In bacteria, which lack b-1,3-glucan, a nutritional role has been assigned (73). In fungi, b-1,3-glucanases seem to have different functions. First, a physiological role in morphogenetic-morpholytic processes during fungal development and differentiation has been indicated (47). Second, b-1,3-glucanases have been related to the mobilization of b-glucans under conditions of carbon and energy source exhaustion, functioning as autolytic enzymes (48, 62). b-1,3-Glucanases are also involved in fungal pathogen-plant interactions, degrading callose (b-D-1,3glucan) in the host’s vascular tissues during pathogen attack (56). Finally, a nutritional role in saprophytes and mycoparasites has been suggested (9, 35, 59). Soilborne fungi of the genus Trichoderma have been especially studied for both their cellulolytic activity (38) and their antagonistic properties against fungal plant pathogens (46). The degradation and further assimilation of phytopathogenic fungi, namely, mycoparasitism, has been proposed as the major mechanism accounting for their antagonism against plant pathogens (19). A number of Trichoderma isolates produce hydrolytic enzymes such as chitinases, b-1,3and b-1,6-glucanases, and proteases when grown on laminarin (b-1,3-glucan), chitin, or fungal cell walls as the carbon source (14, 19). This observation, together with the fact that chitin and b-1,3glucan are the main structural components of fungal cell walls (47), suggests that chitinases and b-1,3-glucanases produced by some Trichoderma spp. are the key enzymes in the lysis of cell walls during their mycoparasitic action against phytopathogenic fungi (12, 13, 19, 58). In this study, we report the purification, molecular cloning, and characterization of an endo-b-1,3-glucanase, namely, BGN13.1, from the mycoparasitic fungus Trichoderma harzianum, which has been described as an agent of biological control of phytopathogenic fungi (11). Expression of the gene and enzyme secretion occur when T. harzianum grows in fungal cell wall components, and these processes are specifically induced by growing the fungus in autoclaved mycelia from different fungi. These conditions have been described as simulating mycoparasitism (24, 34). Also, on the basis of sequence comparison, BGN13.1 may be classified as a new class of b-1,3glucanase enzyme. A possible role of this enzyme in mycoparasitism is discussed. MATERIALS AND METHODS Fungal material and growth conditions. T. harzianum CECT 2413 (Spanish Type Culture Collection, Valencia, Spain) was used in this study. Potato dextrose agar was used for the maintenance of the culture. Production of the enzyme was carried out under the two-step culture conditions previously described (14). Autoclaved cells from Saccharomyces cerevisiae (baker’s yeast; La Cinta Roja, Seville, Spain) or mycelia from Botrytis cinerea CECT 2100, Giberella fujikuroi IMI 58289 (Imperial Mycological Institute, Kew, Surrey, United Kingdom), Phytophthora syringae CECT 2351, Rhizoctonia solani CECT 2815, or T. harzianum CECT 2413 were used as carbon sources. Mycelia from the different filamentous fungi were obtained as described elsewhere (33). Mycelia were lyophilized and added to T. harzianum second-step cultures. b-1,3-Glucanase assay and protein determination. b-1,3-Glucanase activity was determined by measuring the amount of reducing sugar released from * Corresponding author. Mailing address: Instituto de Bioquı´mica Vegetal y Fotosintesis, CSIC y Universidad de Sevilla, Apartado 1113, E-41080 Seville, Spain. Phone: (34) (5) 4557080. Fax: (34) (5) 4620154. 6937 on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from laminarin (Sigma Chemical Co., St. Louis, Mo.). The standard assay mixture (1 ml) contained enzyme solution properly diluted, 4 mg of laminarin, and 50 mM potassium acetate buffer, pH 5.5. The reactions were run for 30 min at 378C and then stopped by boiling for 5 min, and the reducing-sugar content was determined by the procedure of Somogyi (60) and Nelson (43). Enzyme and substrate blanks were also included. A unit of b-1,3-glucanase activity is defined as the amount of enzyme catalyzing the release of 1 mmol of glucose equivalent per min. Protein concentration was measured by the method of Bradford, with ovalbumin as the standard (5). For mycelium growth determination, total protein was measured as described elsewhere (14, 36). Enzyme purification. (i) Step 1. Unless otherwise indicated, all enzyme purification steps were carried out at 48C. Supernatant fluids (200 ml) from 48-h T. harzianum chitin cultures were obtained by filtering the media through Whatman no. 1 paper and centrifuging at 10,000 3gfor 10 min. The supernatant was then precipitated with (NH 4 ) 2 SO 4 (80% saturation) at 48C for 2 h. The precipitate was recovered by centrifugation (12,000 3gfor 20 min at 48C), dissolved in the minimal amount of distilled water, and dialyzed extensively against 2 liters of 50 mM potassium acetate buffer, pH 5.5. (ii) Step 2. Dialyzed aliquots were then adsorbed to pustulan (b-1,6-glucan; Calbiochem, La Jolla, Calif.) as described elsewhere (13). Adsorbed fractions were washed three times with 1 M NaCl–70 mM phosphate buffer, pH 6.0; resuspended in 50 mM potassium acetate buffer, pH 5.5, containing 1 mM sodium azide and 1 mM phenylmethylsulfonyl fluoride; and incubated at 378C overnight. Clarified solutions, after pustulan digestion, were dialyzed extensively against 2 liters of 25 mM imidazole-HCl buffer, pH 7.4. (iii) Step 3. The dialyzed solution (named pustulan digestion) was subjected to chromatofocusing (pH 7.4 to 4) on a Polybuffer exchanger column, PBE 94 (1 by 20 cm; Pharmacia, Uppsala, Sweden), equilibrated in 25 mM imidazole-HCl buffer, pH 7.4. Protein elution and gradient conditions were exactly as described elsewhere (13). Most active fractions were pooled and concentrated to approximately 0.5 ml on Centricon 10 concentrators (Amicon, Beverley, Mass.). (iv) Step 4. The concentrated pool was filtered through a Sephacryl S200 HR column (1.6 by 40 cm; Pharmacia) equilibrated with 1 M NaCl–100 mM potassium acetate buffer, pH 5.5. Elution was performed in the same buffer at a flow rate of 7 ml/h, collecting fractions of 0.65 ml. Most active fractions were pooled, washed, and concentrated in 50 mM potassium acetate buffer, pH 5.5, on Centricon 10 concentrators and finally stored at 2208C. Basic chromatofocusing. Basic chromatofocusing was performed by using a column (1 by 20 cm) of PBE 94 resin equilibrated in 25 mM ethanolamineNaOH, pH 9.4. The column was eluted at a flow rate of 9 ml/h with Polybuffer 96, which had been diluted eightfold with water and adjusted to pH 9.4 with NaOH. The chromatofocusing was monitored by measuring the pH of the eluted fractions. Gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) was performed by the method of Laemmli (31) with 4% acrylamide in the stacking gel and 12% acrylamide in the separating gel. Gels were stained with Coomassie R-250 brilliant blue. Low-molecular-mass standard proteins (Bio-Rad, Hercules, Calif.) were used for molecular mass determination. Glycoprotein stain assays were done in the gels by using either the periodic acid-Schiff reagent procedure (Sigma) or the silver nitrate procedure (18). Extracellular yeast invertase (Sigma) was used as a glycoprotein positive control. Also, for estimating possible mobility changes, deglycosylation reactions prior to SDS-PAGE were performed with endo-b-N-acetylglucosaminidase) H (endo-H) (Boehringer, Mannheim, Germany) (70) or with a-mannosidase (74). The presence of carbohydrates in the protein was also estimated by the binding of purified b-1,3-glucanase to various amounts of concanavalin A-Sepharose 4B (Pharmacia) (54). Isoelectrofocusing was performed at a pI range of 3.5 to 10 as described elsewhere (51). Proteins were visualized by Coomassie staining. b-1,3-Glucanase activity was detected after isoelectrofocusing by using Ampholine PAG plates, pH 3.5 to 9.5 (Pharmacia), in a Multiphor system (Pharmacia) according to the manufacturer’s instructions. The activity was assayed by using the laminarin– 2,3,5-triphenyltetrazolium chloride method previously described (45). The reagents were purchased from Sigma. Western blots (immunoblots) were incubated with antiserum against T. harzianum b-1,6-glucanase II (13) or antiserum for tobacco b-1,3-glucanase antiPR2 or tobacco basic b-1,3-glucanase (26). Immunological detection of the antigen-antibody complexes was carried out as described previously (13). Antibodies against tobacco b-1,3-glucanases were a generous gift from B. Fritig, Institut de Biologie Mole´culaire des Plantes, Strasbourg, France. N-terminal and internal amino acid peptide sequencing. N-terminal and internal amino acid peptide sequencing was performed by using an Applied Biosystem 470 A Gas Phase Protein Sequencer, equipped with a 120 A on-line phenylthiohydantoin analyzer, as described elsewhere (4). Kinetic parameters, temperature optimum, and thermal stability. MichaelisMenten constants were determined from Lineweaver-Burk representations of data obtained by measuring the initial rate of laminarin hydrolysis under the assay conditions described above and using a range of 20 to 0.5 mg of laminarin per ml. The effect of d-gluconolactone (Sigma) on the activity of the purified enzyme was measured as described previously (44). The temperature optimum and thermal stability were determined as described elsewhere (13), by adding laminarin as the substrate. The inactivation temperature was defined as the one at which the specific activity was reduced by 50% under the conditions described above. Substrate specificity and analysis of b-1,3-glucanase reaction products. The spectrum of substrates hydrolyzed by the enzyme was tested with other aand b-glucans at a final concentration of 5 mg/ml. Reaction products were determined by the standard assay described above. b-1,3-Glucanase reaction products were also determined by high-performance liquid chromatography (HPLC) on an HPX 42-A column (Bio-Rad) at 608Cas described elsewhere (13). Hydrolysis of fungal cell walls. Lytic activity by the b-1,3-glucanase on fungal cell walls was monitored as described previously (13, 65). Extraction and blotting analysis of DNA and RNA. Total DNA from T. harzianum was prepared as described elsewhere (41) and analyzed by Southern blotting under conditions described previously (32). Total RNA was isolated according to the method of Wadsworth et al. (71). RNA was separated in 1% agarose–formaldehyde gels by standard protocols (53, 71). During Northern (RNA) analysis, the amount of RNA loaded onto the gel was monitored by methylene blue staining of the membranes. Equal amounts of RNA were loaded into the wells. PCR conditions and screening of the cDNA library. A cDNA library on lgt11-Sfi-Not was constructed from poly(A) 1 RNA isolated from T. harzianum grown for 33 h on a chitin medium as a second-step culture (32). Total DNA was isolated from this library and used as a template for PCR using as a sense primer a degenerated oligonucleotide [59-AATACICA(A/G)AATGGITATACIG GIGG-39] corresponding to an internal sequenced peptide of the protein (p12) and as an antisense primer a commercial lgt11 reverse primer. The amplification protocol used was as follows: 958C, 1 min; 578C, 1.5 min; 728C, 2.0 min. This standard cycle was repeated 35 times and was followed by incubation at 728C for 5 min. The single amplified band was purified from 0.8% agarose gels, subcloned into pGEM-T vector (Promega), and analyzed by sequencing. Approximately 4 310 4 PFU from the lgt11-Sfi-Not cDNA library was screened by plaque hybridization, with the 32 P-labeled PCR-amplified cDNA fragment described above used as a probe. Hybridizations were carried out by a standard protocol previously described (32). DNA sequencing. Nucleotide sequencing was performed by the dideoxynucleotide chain termination method (55) with a Pharmacia ALF automatic sequencing system. Computer analyses of sequencing data were performed by using the University of Wisconsin Genetics Computer Group programs (15). Nucleotide sequence accession number. The nucleotide sequence reported in this article has been submitted to the GenBank/EMBL Data Bank under accession number X84085. RESULTS Enzyme production. Enzyme production was attempted under two-step culture conditions. In the first step, minimal medium containing 10% glucose (repression conditions) was used to grow the mycelia; in the second step, minimal medium containing the selected carbon source (induction conditions) was used and the levels of extracellular b-1,3-glucanase activity were measured. Table 1 shows the b-1,3-glucanase-specific activity found after 48 h of induction. The b-1,3-glucanase activity was detected in abundance in chitin, pustulan, and autoclaved yeast cells and in filamentous fungal mycelia used as carbon sources. Activity, though less abundant, was also detected when 0.1 and 2% glucose, 1% laminarin, and chitin supplemented with 2% glucose were used; finally, at a concentration of 10% glucose there was no detection of the activity. Results, therefore, suggest repression at a high glucose concentration, basal activity at a lower glucose concentration, and induction in the presence of fungal cell wall polymer sources, yeast cells, or fungal mycelia. In order to test whether the induction detected in these experiments is due to the presence of different b-1,3-glucanase isoenzymes, enzymatic detection on isoelectrofocusing gels was carried out with ammonium sulfate-concentrated supernatants of the previous cultures after 48 h of induction. As Fig. 1 shows, no b-1,3-glucanase isoforms were detected in the presence of 2 and 10% glucose. Under starvation conditions (0.1% glucose), a slight basic band was occasionally detected; in the presence of different fungal cell wall polysaccharides, the basic isoenzyme and at least three additional acidic b-1,3-glucanases 6938 DE LA CRUZ ET AL. J. BACTERIOL. on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from were found. Finally, in the presence of either autoclaved yeast cells or different autoclaved filamentous fungi the basic isoform was preferentially induced. Thus, the measured b-1,3glucanase activity is due to at least four proteins, the most basic enzyme being specifically induced under conditions that could simulate mycoparasitism. Commercially available chitin was a good inducer of b-1,3glucanase activity (Table 1). Figure 2 shows mycelial growth and b-1,3-glucanase production during the 96 h of induction in chitin as the carbon source. The appearance of extracellular b-1,3-glucanase activity was time dependent but was not related to mycelial growth. In addition, three peaks of b-1,3glucanase activity (pI of .7.4, pI of 6.2, and pI of 5.3, respectively) were resolved when concentrated extracellular samples were fractionated by acidic chromatofocusing (Fig. 3). Results indicated that in this case the measured b-1,3-glucanase activity is due to at least three proteins, the most basic being also the most active one. For these reasons, further studies were focused on the basic b-1,3-glucanase isoform, namely, BGN13.1 (b-glucanase-1,3 first isoform). Purification of BGN13.1 and size determination. Purification of BGN13.1 was based on the affinity of b-glucanases on particulated b-glucans. The supernatants of chitin-supplemented cultures (200 ml) were concentrated by 80% saturation ammonium sulfate, and concentrated proteins were then subjected to pustulan adsorption and further digestion. Most of the recovered enzymatic activity in the pustulan digestion was b-1,6-glucanase, as expected with an affinity adsorption, but 7% of the total b-1,3-glucanase activity was also present. Acidic chromatofocusing separated the pustulan digestion into two fractions, a nonadsorbed fraction which retained 10 to 15% b-1,6-glucanase activity and the total b-1,3-glucanase activity and a second fraction as a peak at pH 5.8, which contains the rest of the b-1,6-glucanase activity (13). Active fractions of the nonadsorbed volume were pooled, concentrated by using Centricon 10 concentrators, and finally purified by gel filtration on a Sephacryl S-200 HR chromatography column. Figure 4 shows the elution pattern for b-1,3-glucanase and b-1,6-glucanase found by S-200 HR chromatography, and Table 2 summarizes the purification procedure. The final purified prepaFIG. 1. b-1,3-Glucanase isoenzymes on isoelectrofocusing gels. Carbon sources used were as follows: lane 1, glucose, 0.1%; lane 2, glucose, 2%; lane 3, glucose, 10%; lane 4, chitin, 1%; lane 5, laminarin, 1%; lane 6, pustulan, 1%; lane 7, autoclaved yeast cells, 1%; lane 8, autoclaved mycelia from B. cinerea, 1%; lane 9, autoclaved mycelia from G. fujikuroi, 1%; lane 10, autoclaved mycelia from T. harzianum, 1%. Activity was developed as indicated in Materials and Methods. FIG. 2. Analysis of extracellular production of b-1,3-glucanase activity from T. harzianum growing in chitin. Shown is a time course curve of b-1,3-glucanase activity produced by T. harzianum during growth in 1.5% chitin as the sole carbon source. F,b-1,3-glucanase activity; E, extracellular protein; Ç, growth expressed as total protein. FIG. 3. Chromatofocusing analysis of extracellular b-1,3-glucanase isoenzymes from T. harzianum growing with chitin. An 80% (NH 4 ) 2 SO 4 fraction from a 48-h chitin T. harzianum culture fluid was loaded onto the column. F,b-1,3glucanase activity; Ç, protein measured as A 280 ;E, pH. TABLE 1. Extracellular b-1,3-glucanase activity production by T. harzianum pregrown in 10% glucose and incubated for 48 h with different carbon sources Carbon source a (% [wt/vol]) b-1,3-Glucanase activity (mU/mg of protein) Glc (0.1).................................................................................. 10 Glc (2)..................................................................................... 75 Glc (10)................................................................................... BD b Chit (1) ................................................................................... 640 Chit (1) 1Glc (2)................................................................. 50 Lam (1)................................................................................... 78 Pust (1) ................................................................................... 340 Yeast (1)................................................................................. 1,000 Bot (1) .................................................................................... 800 Gib (1).................................................................................... 790 Rhizo (1) ................................................................................ 785 Phyto (1)................................................................................. 700 Tricho (1) ............................................................................... 670 a Glc, glucose; Chit, chitin; Lam, laminarin; Pust, pustulan; Yeast, autoclaved cells from S. cerevisiae; Bot, autoclaved mycelia from B. cinerea; Gib, autoclaved mycelia from G. fujikuroi; Rhizo, autoclaved mycelia from R. solani; Phyto, autoclaved mycelia from Phytophthora citrophthora; Tricho, autoclaved mycelia from T. harzianum. b BD, below limit of detection (,0.5 mU/mg of protein). VOL. 177, 1995 b-1,3-GLUCANASES FROM TRICHODERMA HARZIANUM 6939 on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from ration migrated as a single band on SDS-PAGE, indicating a homogeneous protein with an apparent molecular mass of 66 kDa (Fig. 5). When the molecular mass was calculated by gel filtration, after calibration of the Sephacryl S-200 HR column, the value obtained was 5 to 10 kDa. Biochemical analysis of the purified BGN13.1. The isoelectric points of the pure enzyme, determined by both electrofocusing and basic chromatofocusing (pH range, 9.4 to 6), were estimated to be pH 7.7 and pH 8.0, respectively. Data were confirmed after enzymatic determination in isoelectrofocusing gels, since the pI of the purified enzyme was a basic one and around pI 8.0 (data not shown). Purified BGN13.1, subjected to SDS-PAGE, was not stained by the silver nitrate carbohydrate procedure (18) or by the Schiff’s reagent (results not shown). endo-H or a-mannosidase treatment also failed to alter the apparent size on SDS gels (results not shown). Also, the enzyme was not retained by concanavalin A-Sepharose at concentrations from 0 to 1.4 mg/ml (results not shown). Positive controls with highly glycosylated extracellular yeast invertase were included for the experiments described in this section. The optimum temperature for the enzymatic activity was 408C. After a 30-min preincubation at various temperatures, the inactivation temperature was 558C. The K m value with laminarin as the substrate was 3.3 mg/ml. At the same time, a V max of 75 mmol of product per min zmg of protein was calculated. The purified b-1,3-glucanase was not detected by immunoblotting with either an antiserum against plant acidic extracellular b-1,3-glucanase or basic vacuolar b-1,3-glucanase (data not shown). Furthermore, there was no cross-reaction when anti-T. harzianum b-1,6-glucanase II antiserum was used. N-terminal and internal amino acid sequences. The NH 2 - terminal sequence, determined with a confidence level up to 11 residues, is shown in Table 3. Four internal peptide sequences are also shown. Substrate specificity and hydrolytic action. As shown in Table 4, the enzyme specifically hydrolyzed molecules containing the b-1,3-glucoside bond, including laminarin and periodate-oxidized laminarin, pachyman, and yeast glucan. A slight activity was also found with pustulan (a b-1,6-glucan), probably because of b-1,3 bonds in the pustulan branch points. Slight hydrolysis was observed with the assayed filamentous fungus or yeast purified cell walls. In addition, no clearing activity for the b-1,3-glucanase protein alone, measured as described in Materials and Methods, was observed in agar plates containing fungal cell walls (data not shown). However, when the enzyme was combined with T. harzianum b-1,6-glucanases I and II, activity was detected in agar plates containing yeast cell walls (13). The mode of action of BGN13.1 toward laminarin was examined by measuring the rate of glucose production versus that of reducing-sugar production at different times and also analyzing the corresponding hydrolysis products by HPLC (Fig. 6). Incubation of laminarin with the enzyme resulted in the production of larger amounts of reducing sugars than of glucose (data not shown). After HPLC analysis, a series of oligosaccharides was detected, laminaritetraose, laminaribiose, and glucose being the final products of hydrolysis. When the enzyme was incubated with laminaribiose, no further hydrolysis was obtained (results not shown). In addition, no inhibition was observed with d-gluconolactone (1 to 20 mM), which has been described as a potent inhibitor of exo-b-glucanases at low concentrations (44). Thus, all these results indicate an endotype mode of action for the purified enzyme. Molecular cloning of the bgn13.1 gene. The molecular cloning of the b-1,3-glucanase bgn13.1 gene was attempted by PCR techniques, on the basis of the amino acid sequence of an FIG. 4. Gel filtration chromatography through a Sephacryl S-200 HR column. F,b-1,3-glucanase activity; E,b-1,6-glucanase activity; Ç, protein measured as A 280 . FIG. 5. SDS-PAGE of the purified endo-b-1,3-glucanase. std., molecular mass standards (10 mg); 1, purified b-1,3-glucanase (10 mg). Proteins were stained with Coomassie blue. Numbers on the left refer to molecular masses of protein standards. TABLE 2. Purification of a b-1,3-glucanase from T. harzianum Step Vol (ml) Total protein (mg) Total activity (U) Sp act (U/mg) Yield (%) Purification (fold) 80% (NH 4 ) 2 SO 4 18.0 121 428 3.5 100 1 Pustulan digestion 9.0 4.5 29 6.4 7 2 Chromatofocusing eluate 0.4 0.6 10 16.6 2.3 5 Sephacryl S-200 HR eluate 0.4 0.05 2 40.0 0.5 12 6940 DE LA CRUZ ET AL. J. BACTERIOL. on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from internal peptide of the protein. For PCRs, DNA from a unidirectional cDNA library in lgt11-Sfi-Not vector was used as the template and a degenerate oligonucleotide, corresponding to the internal sequence NTQNGYTGG (p12 [Table 3]), and the lgt11 reverse primers were used. Under the conditions described in Materials and Methods, a single 250-bp fragment was repetitively amplified and was further subcloned and sequenced. The amplified DNA was the C-terminal region of the b-1,3-glucanase protein, corresponding mostly to the 39untranslated region of the gene. This PCR-amplified band was used as a probe for screening the aforementioned cDNA library. Upon screening around 40,000 plaques, several positive clones were selected and isolated and the one containing the longest insert was chosen for further characterization. The cDNA insert of this phage (named bgn13.1) was cloned in the EcoRI-NotI sites of the pBluescript SKII (1) vector (Promega) and then was sequenced. Sequence analysis and homologies with other b-glucanases. Nucleotide and amino acid sequences are shown in Fig. 7. The cloned cDNA has a size of 2,484 bp, of which 32 bp corresponds to the 59untranslated region, 164 bp corresponds to the 39untranslated region, and 2,288 bp corresponds to the unique open reading frame present in the cDNA. Translation of the open reading frame indicates that the bgn13.1 gene product (BGN13.1) has 762 amino acids with a predicted molecular mass of 81,246 Da. Comparison of the deduced sequence with the N-terminal amino acid sequence from the isolated b-1,3glucanase protein (Table 3) indicates that the mature protein starts at residue 34, immediately preceded by the basic residues Lys and Arg, which have been described as the recognition sequence of KEX2-like endoproteinases (22). Therefore, the mature b-1,3-glucanase contains 728 amino acid residues and has a predicted molecular mass of 77,972 Da and an estimated isoelectric point of 7.3. The predicted protein contains the five peptides sequenced from the purified protein, demonstrating that the bgn13.1 gene codes for the b-1,3-glucanase described above (Fig. 7 and Table 3). Comparison of the nucleotide and amino acid sequences of BGN13.1 with sequences present in the databases does not show any significant homology to any a-orb-glucanases or other related protein families. This result clearly indicates that BGN13.1 represents a new class of b-1,3-glucanase. In fact, alignment of BGN13.1 with known b-1,3-glucanase sequences from bacteria, yeast strains, and plants shows that BGN13.1 has none of the conserved domains present in the other proteins. A phylogenetic tree created with the algorithms DISTANCE and GROWTREE by comparison of 63 glucanase sequences indicates that BGN13.1 is taxonomically far from sequences of bacterial, yeast, or plant b-1,3-glucanase families; that it is not related to either the b-glucosidase (BGL1 [3]) or the b-1,6-glucanase II (BGN16.2 [33]) genes from Trichoderma spp., and that it may more likely be related to Trichoderma cellulases (data not shown). Analysis of the protein clearly distinguishes three different regions. The first 33 amino acids mark a hydrophobic sequence and correspond to the leader peptide responsible for secretion and possible KEX2-mediated activation of the protein. It had been demonstrated that several secreted enzymes are synthesized in an inactive form and are activated prior to secretion by a KEX2 endoproteinase (22). Although the presence of KEX2-like proteinase in T. harzianum has not been demonstrated, other secreted enzymes, like CHIT42 chitinase (23), BGN16.2 b-1,6-glucanase (33), and PRB1 protease (24), as well as XYN2 xylanase from T. reesei (67), contain a similar leader peptide ending in the pair Lys-Arg. The region between amino acids 33 and 520 has no apparent structural relevance, and so it could contain the catalytic site. Even though the protein contains several Glu residues, the flanked conserved FIG. 6. HPLC analysis of b-1,3-glucanase action on laminarin. Laminarin (5 mg/ml) was incubated as described in Materials and Methods with 2 mgofthe purified enzyme, for the indicated times. G1, G2, and G4 indicate glucose oligomers; numbers indicate the degree of polymerization. TABLE 3. NH 2 -terminal and peptide sequences from BGN3.1 Peptide Sequence a N terminal ........................ATSFYYFNMDHV p5.......................................NPTYGDVTSSNTRPSALAPG p12.....................................LGXGTFINTQNGYTGGXTPGGGDVXP p13.....................................FNTDTILMGDPTN p14.....................................XVGEA(M)ATITGNGNFFK a The most probable amino acids are in parentheses. X, unknown amino acid. TABLE 4. Substrate specificity of the purified b-1,3-glucanase from T. harzianum Substrate a Main linkage type (monomer) b-1,3-Glucanase activity b (%) Laminarin b-1,3 (Glc) 100 IO 4 -oxidized laminarin b-1,3 (Glc) 100 Glucan (S. cerevisiae)b-1,3 (Glc) 97 Pachyman b-1,3 (Glc) 46 Pustulan b-1,3:b-1,6 (Glc) 5 Carboxymethyl cellulose b-1,4 (Glc) 0 Colloidal chitin b-1,4 (GlcNAc) 0 Glycol chitosan b-1,4 (GlcN) 0 Nigeran a1,3:a-1,4 (Glc) 0 Soluble starch a-1,4:a-1,6 (Glc) 0 Dextran a-1,6 (Glc) 0 B. cinerea cell walls Glucan:chitin 1 G. fujikuroi cell walls Glucan:chitin 1 P. syringae cell walls Glucan:cellulose 4 S. cerevisiae cell walls Glucan 2 a All substrates were used at a concentration of 5 mg/ml. b 100% activity corresponds to 28 U/mg of protein. VOL. 177, 1995 b-1,3-GLUCANASES FROM TRICHODERMA HARZIANUM 6941 on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from residues around the Glu catalytic site described for the b-glucanase studied (8) are absent in BGN13.1. The third region is localized at the C-terminal end and extends from residue 520 to the end of the protein. This region contains eight of the nine cysteine residues present in the protein. Finally, according to DNA-Southern blot hybridizations carried out at high and low stringencies there appears to be only one copy of the bgn13.1 gene in the T. harzianum genome, and no other related sequences were detected (Fig. 8). Expression pattern of bgn13.1. Northern analysis was performed to establish whether the induction of both activity and protein could also be found at the RNA level. As Fig. 9 shows, after 48 h of induction the mRNA levels were below the detection limit when glucose and pustulan were used as carbon sources and the mRNA was detected when laminarin and chitin were used. Also, mRNA accumulation was observed when autoclaved yeast cells or mycelia were used. Thus, these data are in agreement with those obtained when enzymatic activity and protein were determined (Table 1 and Fig. 1) and clearly indicate that bgn13.1 is controlled at the transcriptional level. Therefore, bgn13.1 is subjected to both repression by glucose and induction by fungal cell wall polymers, yeast cells, or fungal mycelia. However, when pustulan and laminarin were used as carbon sources, a lack of correlation between the activity and the mRNA levels was observed, suggesting that regulation at the translational level may also occur. This phenomenon has also been observed with a b-1,6-glucanase (BGN16.2) from the same microorganism (33) and could be FIG. 7. Nucleotide and predicted amino acid sequences of the bgn13.1 cDNA clone. Available peptide sequences from the purified protein are underlined. Cysteine residues are marked by circles. Mature BGN13.1 starts at the sequence ATS after the boxed KEX2 recognition sequence. FIG. 8. Southern blot analysis of the bgn13.1 gene. The experiment was performed by using 15 mg of chromosomal DNA digested with EcoRI (ERI), BamHI (BHI), HindIII (HIII), or XbaI (XI). Highand low-stringency conditions show the same band pattern. FIG. 9. Northern blot analysis. Carbon sources used were as follows: glucose (Gluc), 0.1 or 2%; laminarin (Lam), 1%; pustulan (Pust), 1%; chitin (Chit), 1%; yeast autoclaved cells (Yeast), 1%; autoclaved mycelia from P. syringae (Phyt), 1%; autoclaved mycelia from B. cinerea (Bot), 1%; autoclaved mycelia from R. solani (Rhiz), 1%; autoclaved mycelia from T. harzianum (Tric), 1%. Equal amounts of RNA were loaded into the wells and tested by methylene blue staining. 6942 DE LA CRUZ ET AL. J. BACTERIOL. on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from explained by the high level of stability of these enzymes in the culture media. The enzymatic activity at 48 h in pustulan would be due to an earlier increase of mRNA levels, further repressed by glucose produced by action of the enzyme over its substrate. DISCUSSION T. harzianum CECT 2413 is a mycoparasitic fungus that secretes at least three enzymes with b-1,3-glucanase activity in a chitin-containing medium. The function for these fungal enzymes is unknown, but they might have a nutritional role with b-glucans as substrates (62), a direct role in mycoparasitism (9), or morphogenetic functions in growth and development (47). We report herein the following: first, the expression patterns of these enzymes in different carbon sources (Table 1 and Fig. 1), and second, the purification, molecular cloning, and characterization of the b-1,3-glucanase protein, named BGN13.1, corresponding to the major extracellular activity from supernatants of T. harzianum growing in chitin as the sole carbon source. By the enzymatic detection of b-1,3-glucanase on isoelectrofocusing gels (45), we have shown the different conditions of induction of b-1,3-glucanase isoenzymes from T. harzianum CECT 2413. Acidic and basic b-1,3-glucanases were induced in the presence of various polysaccharides, but the basic enzyme (BGN13.1) was specifically induced by autoclaved yeast cell walls or filamentous fungi (Fig. 1). Preliminary results obtained with the mycoparasitic fungus Stachybotrys elegans have also shown different induction conditions for some extracellular chitinase and b-1,3-glucanase proteins (68). Although transcription of the bgn13.1 gene correlated with the data obtained from enzymatic assays (Table 1, Fig. 1, and Fig. 8), the regulation appears to be at either the transcriptional or the translational level. In addition, b-1,3-glucanases from T. harzianum were produced coordinately with b-1,6-glucanases, proteases, and chitinases, even though the medium with chitin does not contain b-glucan or protein as a carbon source (14, 24). In Trichoderma longibrachiatum, the b-1,3-glucanase activity is constitutively produced and depends on the fungal growth (66), which suggests a morphogenetic role. In other fungi, such as B. cinerea (62), Penicillium italicum (55), Penicillium oxalicum (10), and Sclerotium glucanicum (48), a derepression of the b-1,3-glucanase activity in starvation is associated with the mobilization of b-glucans and involved in the autolysis of cell walls. However, this is not the case in T. harzianum (Table 1, Fig. 1, and Fig. 8). A coordinated induction of chitinases, b-1,3and b-1,6-glucanases, and proteases may occur in T. harzianum when mycoparasitic action is established, as it occurs in plants for chitinases and b-1,3-glucanases during defense responses against pathogens (72). As previously discussed (13, 14, 16), a coordinated induction of the enzymes by an isolated polysaccharide would be of biological relevance, since the substrates of these enzymes are present simultaneously in fungal cell walls (47). The purification method included specific adsorption to pustulan (b-1,6-glucan) and digestion, followed by chromatofocusing and gel filtration. This procedure is novel, quick, and easy, and even if the yields seem to be poor, it is good enough to purify protein for an extensive biochemical characterization. In addition, the process allows purification of two other proteins with b-1,6-glucanase activity. Data from SDS-PAGE and S-200 HR gel filtration indicated apparent molecular masses of 66 and 5 to 10 kDa, respectively. This result indicated an affinity for Sephacryl support that has previously been observed for a number of extracellular enzymes, including other fungal cell wall hydrolases (12, 13, 50, 66). In addition, the molecular mass predicted from the amino acid sequence is 77.9 kDa. Disparity between calculated and deduced molecular weights has also been observed for the endo-b-1,3-glucanase from S. cerevisiae (BGL2), which shows an apparent size of 29 kDa by SDSPAGE and a predicted one of 33.5 kDa from the amino acid sequence. In this last case, it has been demonstrated that the difference in the molecular size is due to anomalous migration of the protein in the gel rather than to posttranslational processing of the polypeptide chain (40). The comparison of the amino acid sequence of BGN13.1 with those of BGL2 and other b-glucanases did not show any significant homology. Comparison between sequences of yeast and plant b-glucanases showed that relevant regions of the deduced primary structure are highly conserved, especially around the glutamine residues involved in the catalysis. The boxes VXEXGWP and EXXXGXF located at the nucleophilic and acidic catalytic sites, respectively, are not present in BGN13.1 (8, 27). Likewise, the box EXDXE present in the bacterial counterpart is also absent in BGN13.1. Therefore, the primary structure of BGN13.1 showed two significant differences with regard to other b-glucanases: (i) BGN13.1 lacks the conserved regions present in the studied b-glucanases, and (ii) BGN13.1 contains a cysteine-rich motif absent in other known b-glucanases, except for the b-1,3-glucanase from the bacterium Oerskovia xanthineolytica. Deletion of the cysteine-rich carboxyl-terminal region in the bacterial protein does not affect the enzymatic activity but abolishes the capability to interact with the yeast cell wall and therefore exert its lytic activity (58). A similar Cys-rich motif has also been found in other protein families which are able to bind specific membrane components, like the saccharide-binding plant lectins (e.g., wheat germ agglutinin); the lipid transfer proteins of animals, fungi, and plants; and class I chitinases (7, 17). The Cys-rich domain of BGN13.1 may function by interacting with other components of fungal cell walls. In fact, BGN13.1 is the only b-1,3-glucanase from T. harzianum able to interact with polysaccharides which are not substrates for the enzyme (like pustulan). Since the enzymatic activity of BGN13.1 is specific only for b-1,3 linkages, this putative cell wall binding domain may have functional relevance in the mycoparasitism of T. harzianum by recognizing a cell wall component of the target fungi. This hypothesis is currently being studied in our laboratories. The unique structure of the basic b-glucanase from T. harzianum was confirmed by the fact that sera raised against b-1,3and b-1,6-glucanases from plants (26) and fungi (13), respectively, did not cross-react with any of the b-1,3-glucanase enzymes from T. harzianum. Furthermore, extracellular hydrolases of fungi are normally glycosylated (76), as are cellulases from Trichoderma reesei (30, 52). Our results, including those obtained with specific carbohydrate stains, endo-H or a-mannosidase treatments, and concanavalin A-binding assays, indicated that either the purified BGN13.1 is not glycosylated or the glycosylation level is so low as to be not detected under the conditions used. These results, however, are similar to those found with other extracellular b-glucanases and chitinases from T. harzianum (12, 13). The measured K m for laminarin (3.3 mg/ml) of the purified enzyme was substantially higher than those reported for other b-1,3-glucanases (21, 54, 66, 69), with the exception of one b-1,3-glucanase of the fungus S. glucanicum (48) and that of Rhizopus chinensis (75). The enzyme purified in this work was specific for b-1,3 linkages in polysaccharides (Table 4), hydrolyzing laminarin and yeast glucan with the same efficiency and pachyman less efficiently. The slight activity found with pustulan (linear b-1,6-glucan) could be related to the binding of this VOL. 177, 1995 b-1,3-GLUCANASES FROM TRICHODERMA HARZIANUM 6943 on July 25, 2016 by USE/BCTA.GEN UNIVERSITARIAhttp://jb.asm.org/Downloaded from enzyme to this substrate and the presence of some b-1,3 linkages in it. Unlike the exo-b-1,3-glucanases previously described for T. harzianum (28) and Trichoderma reesei (2), the enzyme purified in this work behaves as an endo-splitting hydrolase as judged from the liberation of more reducing sugars than of glucose from laminarin, the presence of activity on periodateoxidized laminarin (unable to be hydrolyzed for exo-b-1,3glucanases), the noninhibitory effect of d-gluconolactone on the enzymatic activity, the inability to hydrolyze substrates such as laminaribiose, and the pattern of reaction products detected by HPLC. In contrast with some fungal endo-b-1,3-glucanases that degrade b-1,3-glucans to generate oligosaccharides of high molecular mass and that are classified as larger-oligosaccharide-producing types of enzyme (1), the endo-b-1,3-glucanase characterized in this work hydrolyzes b-1,3-glucans in an endo manner with glucose, laminaribiose, and laminaritetraose as end products and therefore, as are other bacterial and fungal endo-b-1,3-glucanases, is classified as a smaller-oligosaccharide-producing type (39, 64). Finally, the enzyme alone releases reducing sugars but is unable to form hydrolytic haloes when incubated with purified cell walls of several filamentous fungi, including phytopathogenic ones. Furthermore, when it was combined with other fungal cell wall hydrolases from T. harzianum, such as b-1,6glucanases and chitinases (13), not only did the enzyme give clearing activity on fungal cell walls but also an inhibition of the growth of phytopathogenic fungi was observed. Similar results have been found with T. harzianum P1 and also with other organisms. A synergistic, inhibitory effect for chitinases, N-acetyl-b-glucosaminidases, and b-glucanolytic enzymes from strain P1 of T. harzianum has been described (34, 35). In plants, a combination of b-1,3-glucanases with chitinases is essential for observation of inhibitory effects against some phytopathogenic fungi (37). Also, in the lyticase from the bacterium O. xanthineolytica ab-1,3-glucanase and a protease activity act in combination in yeast cell lysis (57). In conclusion, because of the specific induction of this enzyme under simulated mycoparasitic conditions, as well as its antifungal action when combined with other fungal cell walldegrading enzymes from T. harzianum, a contribution of this b-1,3-glucanase during the first steps of mycoparasitism could be expected, although the results did not discard the possibility of the implication of this b-1,3-glucanase in other, unknown saprophytic roles. At present, our work is focused on the overexpression of this enzyme in transgenic T. harzianum, which could result in the improvement of this fungus as a biocontrol agent. ACKNOWLEDGMENTS We thank F. Dominguez for his helpful assistance during all biochemical experimental work. We also thank E. Martinez-Force for his help with HPLC experiments and R. Rhett for proofreading the manuscript. We thank R. Geremia and G. Bauw at the Laboratorium voor Genetika (Gent, Belgium) for the N-terminal and peptide amino acid determinations. J.C. is a recipient of a fellowship from the Ministerio de Educacio´n y Ciencia. This work was supported by grant BIO91-1078 from CICYT (Spain) and grant TS3-CT92-0140 from the European Community. REFERENCES 1. Bacon, J. C., A. H. Gordon, D. Jones, I. F. Taylor, and D. M. Webley. 1970. The separation of b-glucanases produced by Citophaga johnsonii and their role in the lysis of yeast cell walls. Biochem. J. 120:67–71. 2. Bamforth, C. W. 1980. 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