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J. Biol. Chem., Vol. 276, Issue 26, 24309-24314, June 29, 2001

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ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei^*

Nina Aro, Anu Saloheimo, Marja Ilmén, and Merja Penttilä

From VTT Biotechnology, Tietotie 2, FIN-02044 VTT, Espoo, Finland

Received for publication, April 28, 2000, and in revised form, March 1, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2 encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bind in vitro to the 5'-GGCTAATAA site present in the cbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects of ace2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30-70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by sophorose was not affected by ace2 deletion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The most abundant plant materials produced by photosynthesis are cellulose and hemicelluloses. They can be degraded and used as an energy source by numerous microorganisms that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars, such as to glucose in the case of cellulose. Yet filamentous fungi play a special role because many yeasts, such as Saccharomyces cerevisiae, lack the ability to hydrolyze cellulose and hemicellulose. The cellulolytic system of the soft rot fungus Trichoderma reesei is one of the best characterized among microorganisms. Mutant strains have been reported to produce over 35 g/liter extracellular protein (1), and nearly all of the secreted protein consists of cellulases and hemicellulases. Of these cellobiohydrolase I encoded by a single gene forms the major part (2). The synergistic activities of cellobiohydrolases (CBHs),¹ endoglucanases (EGs), and -glucosidases are necessary for the efficient hydrolysis of cellulose. The hemicellulolytic system of Trichoderma consists of a more complex set of enzymes among which are the two endo--xylanases, the -mannanase, and the side chain cleaving enzymes.

The production of the main cellulases in Trichoderma is regulated at the transcriptional level depending on the carbon source available (3), the genes being repressed tightly by glucose and induced up to several thousand fold by cellulose or the disaccharide sophorose (4-6). Carbon catabolite repression of cellulase genes has been extensively studied, and the repressor gene cre1 of Trichoderma has been shown to mediate glucose repression of cellulase expression (7). In the various conditions studied expression of the main cellulase genes, cbh1, cbh2, egl1, and egl2, has been shown to be coordinate, and expression of the cbh1 gene encoding cellobiohydrolase I has been shown to be always the strongest (4). Analysis of relative expression levels of various hemicellulase genes on different carbon sources and inducing compounds indicate that several regulatory mechanisms operate, some of which may be shared by genes encoding cellulases and hemicellulases (8). However, little information is available on the molecular mechanism involved in the strong activation of the cellulase genes or various hemicellulase genes.

To isolate cellulase regulators we have developed a novel yeast-based method to isolate transcriptional activators by selecting simultaneously for their promoter binding and activation properties. This led to the discovery of the transcriptional regulator, ace1 (9). In this article we describe the cloning and functional studies of another cellulase regulator, ACEII, obtained in a similar screening. We show that this novel transcription factor binds to the cbh1 promoter and provide evidence that ace2 has a role in the induction of the major cellulase and xylanase genes of Trichoderma.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Strains and Culture Media-- Escherichia coli strain DH5 was used for plasmid construction, and strain BL21(DE3)LysS was used as a host for production of GST fusion proteins. S. cerevisiae strain DBY746 (ATCC44773, , his3-1, leu2-3, leu2-112, ura3-52, trp1-289, cyh^r, cir⁺) (D. Bothstein, Massachusetts Institute of Technology, Cambridge, MA) was used as a host for the screening of activators. The deletion of ace2 was made in a low protease mutant strain ALKO2221² originating from the hypercellulolytic strain VTT-D-79125 (10).

Synthetic selection media lacking the appropriate nutrients were used for the plasmid-carrying yeast strains (11). The Trichoderma strains were grown in minimal medium described by Ilmén et al. (4) supplemented with either 2% glycerol or 1% Solka floc cellulose (James River Corp.) as the carbon source. In the 6-day-long Solka floc cellulose cultivation, 0.2% proteose peptone was also added. 1 mM -sophorose (Serva) was added to the glycerol medium to induce cellulase expression.

Isolation of the ace2 cDNA and the Chromosomal Gene-- ace2 was isolated from a T. reesei cDNA library expressed in S. cerevisiae as described by Saloheimo et al. (9). To isolate the chromosomal ace2 gene the genomic  library of T. reesei QM9414 (12) was screened with the ace2 cDNA from pAS26. The chromosomal ace2 gene was subcloned as a 6.5-kb HindIII-EcoRI fragment into pZErO-1 vector (Invitrogen). This plasmid, pAS33, contained 0.6 and 4.9 kb, respectively, of the ace2 5'- and 3'-flanking sequences.

Production and Purification of Glutathione S-Transferase-ACEII Fusion Protein-- To construct the GST-ACEII fusion expression vector, a DNA fragment encompassing the ACEII DNA-binding domain (amino acids 1-58) was amplified by polymerase chain reaction and cloned in frame with the GST gene in the pGEX-2T plasmid (Amersham Pharmacia Biotech) to obtain pARO17. Production of GST-ACEII_1-58 in E. coli BL21(DE3) cells transformed with pARO17 and purification by glutathione Sepharose 4B (Amersham Pharmacia Biotech) were performed according to the supplier's manual.

DNA Mobility Shift Assays-- Short double-stranded DNA for binding assays was made by annealing complementary oligonucleotides designed to produce a 3' recessed end that was then filled in with [-³²P]dCTP using the Klenow fragment of DNA polymerase I. Fragments longer than 100 bp were amplified by polymerase chain reaction using sequence specific primers, gel-purified, and labeled with [-³²P]ATP by using T4 polynucleotide kinase (New England Biolabs).

Binding assays were performed at room temperature in a 20-µl reaction mixture containing 50 mM Tris (pH 8.0), 50 mM KCl, 10% glycerol, 4 mM spermidine, 2 µg of poly(dI·dC), 2.5 µM ZnCl₂ with 0.5-1 µg of the GST-ACEII_1-58 fusion protein and 1 ng (about 20,000 cpm) of the labeled double-stranded DNA. In competition experiments unlabeled DNA was added to the reaction in 20-200-fold excess over the labeled DNA fragment. Electrophoresis was performed as described by Saloheimo et al. (9).

Deletion of ace2 from the Trichoderma Genome-- The plasmid containing the ace2 deletion cassette was constructed as follows. The hygromycin resistance cassette was cloned from pRLM_EX30 (13) as a XhoI-HindIII fragment into the XhoI-HindIII cut pBluescript KS+ (Stratagene) to create pARO21. An Asp718-SalI fragment from pAS33 containing 2.2 kb of the 3'-flanking sequence of ace2 was cloned into the Asp718-XhoI sites of pARO21. Then the NruI fragment containing 1.6 kb of the 5'-flanking sequence of ace2 gene was cloned directly from a  clone containing chromosomal ace2 into the SmaI site of pARO21, which already contained the 3'-flanking sequences of ace2, to obtain pAS40. The deletion cassette was released from pAS40 by an Asp718-BamHI digestion, and 5 µg was used for transformation of the ALKO2221 strain according to Penttilä et al. (14) except that the transformants were selected on plates containing 100 µg/ml hygromycin. Fungal DNA was isolated from transformants by the Easy-DNA kit according to the manufacturer's protocol number 3 (Invitrogen) and subjected to Southern analysis to verify the replacement of the ace2 gene in the genome and the presence of no other copies of the deletion cassette.

Cultivation of the Host and the ace2 Deletants-- For the 6-day cultivation on Solka floc cellulose 50 ml of growth medium in two parallel shake flasks of ace2 deletants and three of the host strain ALKO2221 were inoculated with 10⁷ spores. The cultures were carried out in 250-ml flasks with shaking at 200 rpm at 28 °C. Culture medium samples were collected for enzyme activity measurements from each flask. For RNA isolation mycelia were combined from the two parallel shake flasks of the ace2 deletants. Mycelia from the host cultures were treated separately.

In the experiment designed to study induction kinetics of cellulases, ace2 deletants and ALKO2221 were each cultivated in three parallel 2-liter shake flasks on 400 ml of glycerol medium for 72 h, after which mycelia of each strain were combined, and an equal amount was transferred into two shake flasks containing Solka floc cellulose medium and one shake flask containing glycerol medium (control). Samples of mycelium were collected for RNA isolation 9, 12, 15, 18, and 32 h after transfer. For sophorose induction studies strains were grown on glycerol medium for 72 h after which 1 mM sophorose was added, and mycelial samples were collected 1, 2, 3, and 6 h after the sophorose addition.

Northern Analysis of Cellulase Expression of ace2 Deletants-- Total RNA was isolated with the Trizol reagent kit (Life Technologies, Inc.). The probes for Northern analyses were the entire cDNAs of cbh1, cbh2 (15), egl1 (16), and egl2 (previously called egl3) (17) released from vector sequences. The probes for the -xylanases xyn1 and xyn2 (18, 19) were 350-bp fragments prepared by polymerase chain reaction (8). The membranes were hybridized with the actin fragment and a glyceraldehyde-3-phosphate dehydrogenase encoding (gpd1) cDNA fragment as internal RNA loading controls. The probes were labeled using the random primed DNA labeling kit (Roche Molecular Biochemicals) and [-³²P]dCTP (Amersham Pharmacia Biotech). The amounts of the hybridized mRNAs were quantified by densitometric scanning using ImageQuant software of Phosphoimager SI (Molecular Dynamics) and normalized for the total amount of mRNA by using the amount of gpd1 and actin mRNA as a loading control for each filter.

Enzyme Activity Assays and Total Protein Measurement-- CBHI and EGI activity in the culture supernatants were measured using 4-methylumbelliferyl--D-lactoside (MUL) (Sigma) as a substrate according to van Tilbeurgh et al. (20) using 0.17 mM MUL and 10 min of incubation time at pH 5.0 and 50 °C. The endoglucanase activity in the culture supernatant was measured as liberation of reducing sugars using 1% hydroxyethyl cellulose (HEC) (Fluka) as a substrate in 10 min of incubation time at 50 °C and pH 4.8. Total protein amount was measured according to Lowry et al. (21) from proteins precipitated from the culture medium with 20% trichloroacetic acid.

Sequence Comparisons-- The amino acid similarities of ACEII with other proteins were calculated, and the multiple alignment of Fig. 2 was generated with the Clustal W program (22) with a gap opening penalty of 10.0 and a gap extension penalty of 0.2. The similarity searches of data bases were done using the BLAST program with an existence gap penalty of 11 and an extension gap penalty of 1.

Other Methods-- All procedures not described above were carried out using standard methods.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of ace2 by Expression in Yeast-- To isolate the transcriptional activators of cellulase genes, the 1.15-kb-long promoter of the T. reesei cbh1 gene was fused to the S. cerevisiae HIS3 gene to provide a reporter construct, pAS3, in which the HIS3 gene is not expressed without activation from the cbh1 promoter (9). A Trichoderma cDNA expression library present on a multicopy vector was transformed to a his3 mutant yeast containing the reporter construct pAS3, and the transformants were selected on plates containing no histidine. One of the growing colonies contained a cDNA library plasmid named pAS26. pAS26 supported growth only when the reporter construct was also present in the cell, indicating activation of HIS3 expression through binding to the cbh1 promoter (Fig. 1).

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Activation of HIS3 transcription by ACEII through the T. reesei cbh1 promoter in S. cerevisiae DBY746. Growth of yeast colonies each carrying two of the following plasmids is shown: the cbh1 promoter-containing reporter plasmid pAS3, the corresponding plasmid, pMS95 containing nonrelevant DNA instead of the cbh1 promoter (9), the ace2 cDNA clone pAS26, or the corresponding empty vector pAJ401. The colonies were streaked on test plates lacking histidine (SCLeuUraHis) and on plates containing histidine (SCLeuUra). Only the presence of both plasmids pAS3 and pAS26 supported growth of the yeast in the absence of histidine.

The cDNA in plasmid pAS26 contains an open reading frame coding for a protein of 341 amino acids with a calculated molecular mass of 38 kDa. This new gene was named ace2 for activator of cellulase expression (GenBank^TM accession number AF220671).

The ace2 Gene Encodes a Zinc Binuclear Cluster Protein-- The N-terminal part of the deduced ACEII protein has a typical zinc binuclear cluster DNA-binding domain of the fungal type (Zn(II)2Cys₆), first characterized in S. cerevisiae (23). Fig. 2 shows an alignment of the ACEII zinc binuclear cluster domain with the corresponding domains of 12 other proteins showing the highest similarity with the ACEII DNA-binding domain and that of XlnR, a factor regulating xylanase expression in Aspergillus nidulans. The DNA-binding domain of ACEII is most similar (70%) to the DNA-binding domains found in ACU-15 (SWISS-PROT entry P87000), a positive regulator of acetate induction of Neurospora crassa, and in the A. nidulans FacB (70%), the major regulatory protein involved in acetamide and acetate utilization (24).

View larger version (66K): [in this window] [in a new window]   Fig. 2.   Alignment of the zinc binuclear cluster DNA-binding domain of ACEII with the corresponding domains of 12 fungal transcription factors most similar in amino acid sequence to ACEII. The accession numbers from top to bottom are AF220671, P87000, AAB63564, M15210, K01486, CAA62160, CAA55139, S50246, AAD24767, AAC98670, P52958, AAB67707, and BAA78564, respectively. The DNA-binding domain of the transcriptional regulator XlnR of A. niger (28) (accession number CAA05082) is shown at the bottom. Amino acids identical with those in ACEII are indicated with a gray background.

Aside from the zinc binuclear cluster domain, no significant similarities with known proteins or sequences contained in the expressed sequence tag data bases were found by the BLAST program. The overall similarity of ACU-15 and FacB with ACEII is low (25 and 23%, respectively). However, the DNA-binding domain of ACEII is followed by a histidine-rich area (amino acids 53-66) in which a His-Xaa sequence is repeated seven times. A similar histidine repeat of unknown function is found in the Zn(II)2Cys₆-type aflatoxin regulatory protein AflR of two Aspergillus species (25, 26). Furthermore, a glutamine-rich region (⁸²QQQEQQQGQPQHPPPPVQ⁹⁹) resembling the glutamine-rich activation domains of other transcription factors, e.g. the human transcription factor Sp1 (27), is present in ACEII.

Characterization of the ACEII-binding Site-- Because the cbh1 promoter used in the initial screening was about 1.15 kb, further experiments were needed to characterize the ACEII-binding site more precisely. Therefore, the ACEII segment coding for the zinc binuclear cluster domain (amino acids 1-58) was fused to GST, and the ability of the resulting of GST-ACEII_1-58 hybrid to bind to DNA was studied by electrophoretic mobility shift assays with four polymerase chain reaction amplified fragments: fragment 1 (from 133 to 392 upstream from ATG), fragment 2 (621 to 941), fragment 3 (886 to 1177), and fragment 4 (1116 to 1421). DNA-protein complexes were detected with fragments 2 and 3 (data not shown). Because binding to fragment 2 produced the strongest specific shift, it was further characterized by amplifying shorter DNA fragments and designing specific oligonucleotides to further delimit the ACEII-binding region. Of the four partially overlapping oligonucleotides named 2B (843 to 809), 2C (818 to 780), 2D (789 to 764), and 2G (772 to 736), GST-ACEII_1-58 binding was observed only to oligonucleotide 2D. No competition was observed by the addition of nonspecific DNA to the binding reaction, whereas addition of excess of oligonucleotide 2D clearly competed out the binding (data not shown). Interestingly, 2D (Fig. 3) contains a sequence 5'-GGCTAA that is similar to the one (5'-GGCTAAA) recently shown to bind the xylanase activator XlnR of Aspergillus niger (28). To study whether this sequence in 2D was the recognition site for GST-ACEII_1-58, four mutations of nucleotide triplets were made to the 2D oligonucleotide. Fig. 3 shows that the mutation of the three nucleotides upstream from the 5'-GGCTAA sequence in the cbh1 promoter did not affect the binding of GST-ACEII_1-58; instead the following three nucleotides (GGC) were essential for binding. The mutations of the following two TAA triplets in the promoter each reduced ACEII binding. The cbh1 promoter also contains regions identical to the proposed XlnR-binding site (5'-GGCTAAA). One is found at position 825, present in the oligo 2B, but GST-ACEII_1-58 did not bind to it (data not shown).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Gel mobility shift analysis of the binding of GST-ACEII1-58 to the double-stranded oligonucleotide 2D corresponding to the native cbh1 promoter sequences from 789 to 764 and to its mutated forms mut2D1-mut2D4. The oligonucleotide 2D and the four variants (mut2D1, mut2D2, mut2D3, and mut2D4), each containing a different 3-bp (underlined) mutation (bold type) are shown on top. +, addition of GST-ACEII1-58 to the binding reaction; , no protein.

Effect of ace2 Deletion on Cellulase Expression-- To investigate the role the of ace2 gene in carbon utilization, the protein coding region of the ace2 chromosomal fragment was replaced with the hygromycin selectable marker hph in the T. reesei strain ALKO2221. The ace2 deletants VTT-D-99729, VTT-D-99757, and VTT-D-99758 were selected for further experiments.

Because the induction and high level expression of cellulase genes occurs in the presence of cellulose as the sole carbon source, the possible negative effect of ace2 deletion on cellulase expression in cellulose-based cultivations was analyzed. In the first approach several parallel cultures of the three deletants and the host strain were cultivated on medium containing Solka floc cellulose as the sole carbon source. Cellobiohydrolase and endoglucanase activities were measured from culture supernatants with MUL and HEC as substrates, respectively (Table I). The values for MUL mainly reflect the amount of CBHI and EGI produced by the fungus, and the values for HEC reflect the amount of endoglucanases (e.g. EGI and EGII). The ace2 deletants clearly produced less cellobiohydrolase and endoglucanase activities than the host strain. At day 3 the ace2 deletants only had on the average 20% of the cellobiohydrolase activity when compared with the host strain ALKO2221, and after 6 days the cellobiohydrolase and endoglucanase activities produced by the ace2 deletants were on the average 55% of the activities of ALKO2221. Biomass determination cannot be made from the cultures containing cellulose particles, but the pH level of the medium indicated that ace2 deletant may have grown slower than the host. This would be expected if the lower cellulase levels would lead to reduced amount of sugar available for the fungus. No difference in biomass accumulation was observed on glucose- or glycerol-based cultures.

To verify that the effect of ace2 deletion is seen also at transcriptional levels at later time points of the culture and not only at the enzyme level, the expression of three of the main cellulase genes, cbh1, cbh2, and egl2, was analyzed by Northern analysis from the mycelia collected after 6 days of cultivation. Transcript levels of all of the cellulases analyzed were lower in the three ace2 deletants than in their host strain ALKO2221; the mean values of the transformants were at this time point 70, 70, and 80% for cbh1, cbh2, and egl2 mRNA, respectively, of the values of the host (data not shown).

The finding that ace2 deletion resulted in reduced expression levels of cellulases after 3 and 6 days of cultivation on Solka floc cellulose prompted us to analyze the role of ace2 in induction kinetics of cellulase genes at earlier time points in cellulose-based cultures. For this study we cultivated two parallel shake flasks of both the ace2 deletant VTT-D-99729 and ALKO2221. The strains were first pregrown to accumulate biomass on glycerol, a neutral carbon source with respect to cellulase expression (4), whereafter the mycelia were transferred to Solka floc cellulose to induce expression and to glycerol medium as a control. After 6, 9, 12, 15, 18, and 32 h of the transfer, mycelium was harvested, and Northern blot analyses were performed. The differences between the ace2 deletant and the host were seen in the level of induction of the cbh1, cbh2, and egl1 (Fig. 4) mRNAs studied. Quantification of the signals showed that on the average the signals of the three cellulase mRNAs of the ace2 deletant were 30-75% of the amount seen in the ALKO2221 strain at time points 9-18 h after transfer but were closer to the control levels at later time points (Fig. 4B). No cellulase signals were detected from the mycelia transferred to the glycerol medium (data not shown). Taken together the results obtained on Solka floc cellulose medium show that cellulase transcript levels of the ace2 mutant strain are on the average lower throughout the cultivation until day 6. The difference is greater at the early stages of cultivation, indicating that induction has become slower in the mutant.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Time course of cbh1, cbh2, egl1, xyn1, and xyn2 mRNA accumulation in two parallel shake flasks each of the ace2 deletant VTT-D-99729 and the host ALKO2221 at the indicated hours after transfer from glycerol into medium containing Solka floc cellulose. A, Northern blot analysis of cbh1, xyn1, xyn2, and actin (as control). The probes are indicated on the left side. B, densitometric scanning of the cellulase and xylanase mRNA signals normalized with actin mRNA. X axis represents the different time points (hours) after the mycelia were transferred to the cellulose medium. The gray bars represent the values of the host strain ALKO2221, and the white bars represent the values of the ace2 deletant VTT-D-99729. The values are shown relative to the highest value of the ALKO2221 mRNA signal, which was set as 100.

It was of interest whether ace2 functions also in the induction caused by a known strong and rapid cellulase inducer, the disaccharide sophorose. Each of the strains VTT-D-99729, VTT-D-99757, and ALKO2221 were grown in two parallel shake flasks on glycerol medium for 4 days, after which sophorose was added into the culture. mRNA analyses were performed 1, 2, 3, and 6 h after sophorose addition. The induction of cbh1 (Fig. 5) and of cbh2 and egl1 (data not shown) was followed by Northern analysis. The levels of cellulase mRNA were very similar between the two independent ace2 deletants and the control strain ALKO2221. This result suggests that ace2 deletion does not affect sophorose induction of cbh1 and the other cellulase genes.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Accumulation of the cbh1 mRNA at 1, 2, 3, and 6 h after addition of 1 mM sophorose to glycerol cultivations. A, Northern blot of cbh1 and gpd1 (control) mRNA isolated from two parallel shake flask cultivations of the ace2 deletants VTT-D-99729 (lanes 1, 2, 7, 8, 13, 14, 19, and 20) and VTT-D-99757 (lanes 3, 4, 9, 10, 15, 16, 21, and 22) and the host strain ALKO2221 (lanes 5, 6, 11, 12, 17, 18, 23, and 24) at time points indicated at the top. The 6-h time points shown here are from another filter that contained also samples from the 3-h time points allowing the comparison of the mRNA amounts between different time points. B, quantification of the cbh1 mRNA. The columns represent cbh1 mRNA normalized with gpd1 mRNA. The highest value obtained from densitometry scanning was set as 100.

Effect of ace2 Deletion on Xylanase Expression-- We wanted to study whether ace2 deletion would also affect the transcription of the two main endo--xylanase genes, xyn1 and xyn2, encoding enzymes that hydrolyze the main chain of xylan. The same filter as probed for cellulase expression was probed with the xyn1- and xyn2-specific probes. After transfer of mycelia from glycerol medium to Solka floc cellulose medium the xyn1 gene was induced weakly at 18 h and more strongly at 32 h after transfer to cellulose medium (Fig. 4A). The expression of xyn2 was induced more strongly already at 18 h. Fig. 4 shows that the expression of xyn2 is much more weakly induced in the ace2 deletant than in the host strain ALKO2221, and the quantification reveals that the xyn2 signal in VTT-D-99729 is only 30-45% of that of the host strain ALKO2221 at 15, 18, and 32 h. No significant difference in the xyn1 expression level between the host and the ace2 deletant strain can be seen in this experiment (Fig. 4B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Despite the importance of plant material breakdown in basic biology and biotechnology, very little is yet known about the molecular mechanisms that regulate expression of the genes encoding the tens or even hundreds of hydrolytic enzymes needed for the efficient breakdown of the polymeric substrates, cellulose and hemicellulose. In addition to the glucose repressor protein CREI/CREA (7, 29) the only other regulatory proteins described are the XlnR activator protein of A. niger (28); the T. reesei protein ACEI, having a Cys₂-His₂ DNA-binding domain (9); and ACEII described here. The development of a novel yeast-based cloning method, which selects simultaneously for the capability of the protein to bind the cbh1 promoter and to activate transcription (9), allowed us to identify the two new factors ACEI and ACEII.

GST-ACEII_1-58 bound to the 5'-GGCTAATAA sequence in the cbh1 promoter at 779 relative to ATG. Mutation of the triplet GGC completely abolished ACEII binding, and changing the next two TAA triplets reduced it. The binding site is not a repeated sequence as has been shown for most other transcription factors with zinc binuclear cluster DNA-binding domains reviewed by Todd and Andrianopoulos (30). In this respect ACEII appears to be similar to the A. niger XlnR.

The promoter of the other cellobiohydrolase gene, cbh2, also contains a 5'-GGCTAATAA sequence at position 326. Although our data suggest that ACEII prefers a long AT-rich region 3' of GGC, it remains possible that ACEII binds also for instance 5'-GGCTAA(A/T) in certain sequence contexts. The promoters of the two most strongly expressed T. reesei cellulase genes, cbh1 (Fig. 6) and cbh2, contain six and three sites, respectively, having the core 5'-GGCTAA. The 1-kb-long egl1 promoter contains a 5'-GGCTAAA sequence and that of the minor endoglucanase, EGV (egl5), two 5'-GGCTAAT sequences. The 525-bp sequence available for the egl2 promoter and the 330-bp xyn2 promoter do not contain these types of site, but it remains possible that binding sequences are located further upstream. The 0.5-kb-long xyn1 promoter contains two 5'-GGCTAA sequences.

View larger version (6K): [in this window] [in a new window]   Fig. 6.   Schematic representation of the localization of putative transcription factor-binding sites within in the cbhl promoter. The in vitro binding site for ACEII is indicated by a closed triangle, and additional GGCTAA sites are indicated by open triangles. The four sites shown to bind CREI in vitro (N. Belshaw, M. Ilmén, M. Penttilä, and D. Archer, unpublished data) are indicated with filled squares, and the additional sequences fitting to the binding consensus (5'-SYGGRG) for CREA are indicated with open squares. Seven sites containing the core sequence 5'-AGGCA shown to bind ACEI in vitro (9) are indicated by crosses. The CCAAT sequences are indicated by filled circles, and the major transcription start point at 90 is indicated by an arrow (P. Lehtovaara, unpublished data). The shortest promoter inducible by sophorose is shown by a hatched box. The promoter region proposed to mediate cellulose induction is shown by a vertically striped box. Mutation of CREI-binding sites in the region indicated by an open box causes glucose derepression in vivo (34).

Now that data concerning the two new cellulase regulators, ACEI and ACEII, are available including their putative promoter-binding sites, it becomes meaningful to outline the cbh1 promoter structure (Fig. 6). The putative ACEII-binding site at 779 determined in this work is located close to the three CREI-binding sites around 700 shown to mediate glucose repression of cbh1 in vivo (7). This region is surrounded by four CCAAT sequences, and it contains also one in vitro binding site for ACEI at 803 (9). CCAAT sequences are required for high level expression of A. nidulans amdS (acetamidase) (31) and could serve as binding sites for a complex similar to AnCF of A. nidulans, which influences the chromatin structure 5' of the amdS gene (32). The cbh1 promoter region from 700 to 750 has been shown to be hypersensitive to micrococcal nuclease³ and thus possibly represents an internucleosomal region easily accessible to transcriptional regulators. It is noteworthy that there are several putative ACEI-binding sites scattered along the cbh1 promoter that have been shown to bind ACEI in vitro (9) and that there are many 5'-GGCTAA sites that may or may not bind ACEII or the putative equivalent of XlnR in vivo (Fig. 6). Assuming that the level of transcriptional activity is determined to a large extent by the number of transcription activator-binding sites, this could explain the strong expression level of the cbh1 gene.

Deletion of ace2 clearly reduced expression levels and induction kinetics of the main cellulase genes on Solka floc cellulose but interestingly did not affect their induction by sophorose. Sophorose, consisting of two -1,2-linked glucose units, has been generally considered to be formed during cellulose hydrolysis through the transglycosylation activity of some of the cellulases (33) and to be at least partly responsible for the cellulase expression obtained on cellulose-containing media (3). The present study suggests that the induction of cellulase genes in T. reesei by sophorose and cellulose, respectively, use at least partially different molecular mechanisms or that these mechanisms can at least be distinguished in certain experimental conditions. This statement is in agreement with our data, which demonstrated that short forms of the cbh1 promoter (161-210 bp; Fig. 6) were still inducible by sophorose, albeit at a reduced level compared with the full-length promoter (34), but were not induced when the fungus was grown in the presence of Solka floc cellulose or the microcrystalline cellulose Avicel.⁴ The results of Henrique-Silva et al. (35) support this by indicating that the region from 373 to 204 in the cbh1 promoter mediates induction by cellulose (Fig. 6), whereas the sequences downstream do not. There is one putative ACEII-binding site (5'GGCTAA) at 209 that could mediate the induction.

Zeilinger et al. (36) reported that certain mutations in the sequence 5'-ATTGGGTAATA in the T. reesei cbh2 promoter abolished induction by sophorose completely in a replacement assay. They proposed that this element consists of a CCAAT box in one orientation and an overlapping GGGTAA sequence binding ACEII in another orientation. The results presented here showed that ace2 deletion had no effect on sophorose induction, suggesting that the mutations in the cbh2 promoter affected binding of some factors other than ACEII.

We demonstrate here that the cellulase regulator ACEII also affects xylanase expression in T. reesei, seen as reduced amounts of xyn2 transcript. A common regulatory factor for these two classes of enzymes is foreseeable because some polymeric substrates and oligosaccharides induce both cellulase and hemicellulase expression, although at different relative levels (8). More extensive studies are necessary to clarify the contribution of ACEII in the complex pattern of cellulase and xylanase expression in the various inducing conditions known. These studies are further complicated by the fact that the two xylanase genes, xyn1 and xyn2, appear to be differentially expressed, at least in respect to pH,⁵ carbon catabolite repression, and the carbon source (8, 36).

The A. niger XlnR was first identified as a xylanase regulator (28) but was later shown to regulate other hemicellulases (37) and also cellulases (38). The suggested XlnR-binding site 5'-GGCTAAA (28) was later narrowed to 5'-GGCTAA, because the longer sequence was not found in all the promoters affected by an xlnR loss of function mutation (37). The suggested putative binding sites for XlnR and ACEII contain an identical core. Further experiments would, however, be required to determine the functional in vivo consensus binding sites for these two factors. ACEII and XlnR are different in size and show no amino acid sequence similarity except in the DNA-binding domain. Even here the identity is only 20%, much less than when ACEII is compared with other proteins with zinc binuclear cluster domains (Fig. 2). It seems highly unlikely that ACEII and XlnR are homologous factors. It remains possible that equivalents for both ACEII and XlnR (and ACEI) are present in both fungal genera and that they contribute to varying extents to the relative expression levels of cellulase and hemicellulase genes in different inducing conditions. The effect of the XlnR loss of function mutation in A. nidulans (38) appears to be more severe than the effect of the ace2 deletion on the expression levels of the genes studied here. This XlnR mutation, however, does not reside in the DNA-binding domain. It remains to be studied whether binding of the mutant XlnR prevents binding of other factors, such as an ACEII type factor and in this way causes severe reduction of (hemi)cellulase expression. On the other hand, the deletion of ace2 in the present work with Trichoderma would not have prevented a possible XlnR type factor from activating gene expression.

Taken together, our current understanding of (hemi)cellulase expression in Trichoderma points to the existence of several factors involved in activation of cellulase gene expression. Neither ACEI (9) nor ACEII alone can be solely responsible for the expression on cellulose-containing medium, and their contribution to induction by sophorose still requires further studies. The presence of a number of putative binding sites for ACEI and ACEII (and a possible XlnR equivalent) in a promoter further complicates the analysis of the relative contributions in vivo of the regulatory factors known today.

	ACKNOWLEDGEMENT

We warmly thank Seija Nordberg for skilled technical assistance.

	FOOTNOTES

^* This work was supported by the Foundation for Biotechnical and Industrial Fermentation Research, Helsinki Graduate School in Biotechnology and Molecular Biology, Roal Oy, and the National Technology Agency (Tekes).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF220671.

To whom correspondence should be addressed: VTT Biotechnology, Tietotie 2, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland. Tel.: 358-9-4561; Fax: 358-9-4552103; E-mail: nina.aro@vtt.fi.

Published, JBC Papers in Press, April 13, 2001, DOI 10.1074/jbc.M003624200

² A. Mäntylä, unpublished data.

³ N. Belshaw, M. Ilmén, M. Penttilä, and D. Archer, manuscript in preparation.

⁴ M. Ilmén, unpublished data.

⁵ N. Aro, A. Saloheimo, M. Ilmén, and M. Penttilä, unpublished data.

	ABBREVIATIONS

The abbreviations used are: CBH, cellobiohydrolase; EG, endoglucanase; GST, glutathione S-transferase; kb, kilobase pair(s); bp, base pair(s); MUL, 4-methylumbelliferyl--D-lactoside; HEC, hydroxyethyl cellulose.

	REFERENCES

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