(Received for publication, October 2, 1996, and in revised form, December 20, 1996)
From the Department of Biochemistry, Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, São Paulo 05508-900, SP, Brazil
The induction of cellulases by cellulose, an insoluble polymer, in the filamentous fungus Trichoderma reesei is puzzling. We previously proposed a mechanism that is based on the presence of low levels of cellulase in the uninduced fungus; this basal cellulase activity would digest cellulose-releasing oligosaccharides that could enter the cell and trigger expression of cellulases. We now present experiments that lend further support to this model. We show here that transcripts of two members of the cellulase system, cbh1 and egl1, are present in uninduced T. reesei cells. These transcripts are induced at least 1100-fold in the presence of cellulose. We also show that a construct containing the hygromycin B resistance-encoding gene driven by the cbh1 promoter confers hygromycin B resistance to T. reesei cells grown in the absence of cellulose. Moreover, cellulose-induced production of the cbh1 transcript was suppressed when antisense RNA against three members of the cellulase system was expressed in vivo. Experiments are presented indicating that extracellular cellulase activity is the rate-limiting event in induction of synthesis of the cellulase transcripts by cellulose. The results reveal a critical requirement for basal expression of the cellulase system for induction of synthesis of its own transcripts by cellulose.
Cellulose, a -(1,4)-linked glucose polymer, is a product of the
utilization of solar energy and carbon dioxide by plants, through
photosynthesis, a process which is estimated to produce 7.2 × 1010 tons of cellulosic biomass annually (1). The
degradation and oxidation of cellulose to carbon dioxide, a process
carried out mainly by microorganisms, is a key transformation step in
the biological carbon cycle in nature (2). The filamentous fungus Trichoderma reesei is considered to be the most efficient
producer of cellulases (3, 4). Its cellulases are classified into two
broad classes: cellobiohydrolases (CBH),1
whose major activity involves the cleavage of cellobiose residues consecutively from the ends of the cellulose chains (5), and endoglucanases (EG), whose major activity involves the cleavage of
-glycosidic bonds in the cellulose chain (6). The members of this
system act synergistically and are necessary for the efficient hydrolysis of cellulose to soluble oligosaccharides (7, 8). The genes
encoding two cellobiohydrolases, cbh1 and cbh2,
and two endoglucanases, egl1 and egl2, have been
isolated and characterized (9-15).
In T. reesei, cellulase transcripts are induced when the
natural inducer cellulose is the only carbon source available (16). The
natural cellulose is insoluble; hence, we developed an interest in
studying how this insoluble polymer, which cannot traverse the cell
membrane, would induce cellulase production. Previously published
observations suggested that basal levels of cellulolytic enzymes may be
necessary to generate soluble breakdown products of cellulose such as
sophorose (2-O--glucopyranosyl-D-glucose), a
soluble disaccharide inducer of cellulase synthesis (17), which could
pass through the cell wall and initiate the inductive process.
Experimental evidence in vitro was reported supporting this
mechanism. The potential soluble disaccharide sophorose was detected,
as well as other glucose disaccharides, during growth of T. reesei on cellobiose (18), and after hydrolysis of cellulose with
the T. reesei cellulase system (19). A membrane-bound
-glucosidase is believed to catalyze the formation of sophorose
through its transglycosylation activity (20). Indirect evidence was
also reported showing that the presence of antibodies to the major cellulase inhibited production of the cbh1 transcript with
cellulose present. However, the antibodies did not affect transcription of the cbh1 transcript when sophorose was present (21).
In this study, we present evidence for the requirement of basal levels of cellulases by showing: 1) the presence of the transcripts of two major members of the cellulase system, cbh1 and egl1, in uninduced T. reesei cells; 2) the activity of a reporter gene driven by the cbh1 promoter in the absence of cellulose; and 3) the inhibition of cbh1 expression under celulose but not under sophorose by expressing antisense RNA against three members of the cellulase system. We also show that increasing the concentration of added cellulase decreases the time necessary for the induction of synthesis of the cbh1 and egl1 transcripts. Moreover, extracellularly added purified cellulases also have a positive effect on cbh1 and egl1 expression, consistent with a model in which induction is dependent on the extracellular activity of cellulases.
[-32P]ATP,
[
-32P]dATP, and [
-32P]dCTP (specific
radioactivity: 3000 Ci/mmol) were purchased from Amersham Corp.,
GeneAmp DNA Amplification kit was from Perkin-Elmer Co. Avicel (PH101,
microcrystalline cellulose) was generously provided by Forlab-Kelrio
S/A, Brazil.
The plasmids
pCBH1-Hph-2.2 and pCBH1R-Hph-2.2 containing the the 5-flanking region
of the cbh1 gene fused in the correct and the opposite
orientation to the hygromycin B resistance gene (hph) were
constructed as follows. A 2.2-kb EcoRI-BglI DNA
fragment containing the 5
-flanking region of the cbh1 gene
was treated with T4 DNA polymerase and ligated to a BamHI
linker. The coding region for the hph gene was amplified
using the plasmid pLG90 (22) in conjunction with PCR in which a
BamHI site was included at the 5
ends of the primers. The
5
-flanking region of the cbh1 gene and the amplified
hph coding sequence were digested with BamHI,
ligated and then inserted into the BamHI site of a
pBluescript containing a polyadenylation signal. A construct containing
the 5
-flanking region, the hph gene and the polyadenylation
signal placed in the correct orientation was isolated and designated pCBH1-Hph-2.2. The pCBH1R-Hph-2.2 was constructed by placing the 5
-flanking region in opposite orientation using the SacII
sites located 10 bp 5
of the ATG.
The antisense plasmid, pJO51, was constructed as follows. Specific
oligonucleotides, in which suitable restriction enzymes sites were
placed at the 5 end, were used in conjunction with PCR to amplify the
required cbh2 and egl2 sequences. The amplified cbh2 DNA fragment contained 399 bp (from base 1477 to 1876)
from the fourth exon of the gene (14). The amplified egl2
DNA fragment contained 398 bp (from base 982 to 1380) from the second
exon of the gene (11). A 587-bp KpnI-EcoRI DNA
fragment (from base 86 to 673) from the first exon of the
egl1 gene (10) was isolated and used in the construction of
the plasmid pJO51. After digestion with the suitable restriction
enzymes, the fragments were ligated and inserted in the reverse
orientation between the TrpC promoter and the 3
terminator
from Aspergillus nidulans (23, 24).
Total RNA was isolated as
described (25). RNA (10 µg) was separated by electrophoresis on a
1.2% agarose gel, after denaturation with glyoxal and dimethyl
sulfoxide (26), and transferred to a Zeta-Probe membrane. Membranes
were hybridized with a random primer [-32P]dCTP
radiolabeled EcoRI fragment (720 bp) or
KpnI-PstI fragment (1050 bp) containing part of
the coding region of the cbh1 or egl1
respectively (10, 12).
Each of the forward
primers was a 21-mer oligonucleotide complementary to the mRNA;
cb1 and eg1 primers correspond to DNA sequences
740-760 and 885-905 of the second exon of cbh1 (12) and
egl1 (10) genes, respectively. Each of the reverse primers was a 21-mer oligonucleotide of the same polarity as the mRNA; cb2 and eg2 primers correspond to DNA sequence
612-632 and 711-731 of the first exon of cbh1 (12) and
egl1 (10) genes, respectively. cDNA was synthesized by
mixing 1 µg of total RNA, 1 µM of forward primers
(cb1 for cellobiohydrolase I and eg1 for
endoglucanase I), 2.5 units/µl of murine leukemia virus reverse
transcriptase (Perkin-Elmer) in polymerase buffer (10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM
MgCl2, 0.01% (w/v) gelatin) and 1.25 mM each
dNTP. Samples were incubated 60 min at 42 °C, and then second strand synthesis and 25 cycles of amplification were performed by addition of
1 µM reverse primers (cb2 for
cellobiohydrolase I and eg2 for endoglucanase I), 2.5 units
of Taq DNA polymerase, and 20 µCi of
[-32P]dATP (3000 Ci/mmol). Each cycle of PCR consisted
of 1.5 min of denaturation at 94 °C, 1.5 min of annealing at
55 °C, and 3 min of polymerization at 72 °C. Genomic
amplification was performed as described above, except that 10 ng of
genomic DNA clone were used in place of RNA, and the first strand
synthesis was omitted.
T. reesei was induced for 6 days with 2% Avicel. The culture filtrate was concentrated and chromatographed on a Sephacryl S-200 column, and the fraction containing the excluded cellulolytic activity was used as crude cellulase. The major members of the cellulolytic system composed of CBHI, CBHII, EGI, and EGII were purified and assayed as described previously (21).
Inoculum, Culture Conditions, and TransformationMaintenance of T. reesei (QM9414) cultures, inocula preparation, culture medium, and washing of Avicel were carried out as described previously (21). Mycelia from germinated spores were centrifuged, washed twice with 100 mM potassium phosphate buffer (pH 6.0), and incubated on a rotary shaker for 2 h. Mycelia (2 mg, dry weight) were suspended in 5 ml of culture medium and cellulose, sophorose, or cellulase was added to the reaction mixture as indicated. All cultures were incubated on a rotary shaker (200 rpm) at 28 °C for the indicated time.
Preparation and transformation of protoplasts were carried out as described (27, 28). After transformation, the protoplasts were plated on minimal medium in agarose overlay. The minimal medium used was as described by Pentillä et al. (28) except that glucose was substituted with 0.4% glycerol.
The
transcripts of two members of the cellulase system, cbh1 and
egl1, could not be detected by Northern analysis in total RNA isolated from uninduced T. reesei cells (Fig.
1). Moreover, the physical heterogeneity of substrates
and the complexity of the cellulase system's multiple enzymes,
synergism, and glucanases with overlapping specificity (6), present
obstacles to assaying the expression of a single member of the
cellulase system, specially at low activity levels. To detect low
levels of transcription of cellulases in uninduced T. reesei
cells, we assayed for the presence of cbh1 and
egl1 transcripts using reverse transcription-PCR. The
strategy of the amplification process is presented in Fig. 2A. The position of the primer and the
expected amplified product using RNA or DNA as a template is also
shown. cDNAs were synthesized from total RNA extracted from
Avicel-induced and glycerol-grown (uninduced) T. reesei
cells and then amplified by PCR. This procedure produced 82- and 125-bp
segments of the cbh1 and egl1 transcripts, respectively (Fig. 2B; refer to details in Fig.
2A). However, if T. reesei genomic DNA was used
as a template instead of mRNA, the presence of introns in
cbh1 and egl1 genes, resulted in fragments of 149 and 196 bp, respectively (Fig. 2C). In addition, digestion of amplified products by appropriate restriction enzymes produced fragments of the expected sizes (Fig. 2C; see also Fig.
2A). This excluded any possible artifactual amplification of
contaminating DNA and confirmed that the amplified DNA fragments
corresponded to spliced cbh1 and egl1
transcripts. Quantitation of both transcripts from uninduced relative
to induced T. reesei cells was performed using 5
32P-labeled reverse primers (cb2 for
cbh1 and eg2 for egl1) and comparing
the extent of amplification achieved after different numbers of PCR
cycles as described by Chelly et al. (29). The levels of
cbh1 and egl1 transcripts were, respectively,
1156- and 1148-fold lower in uninduced cells relative to induced cells. It is worth mentioning that similar results were obtained when total
RNA was isolated from uninduced T. reesei cells grown in a
culture medium in which peptone was omitted.
The Promoter of the cbh1 Gene Drives Basal Expression of a Heterologous Gene Placed under Its Control
We used a heterologous
gene fusion to demonstrate that the promoter of the major member of the
cellulase system, cbh1, is functionally active in the
absence of cellulose, and that this low basal promoter activity is
sufficient to endow a new phenotype on transformed T. reesei
cells. Series of plasmids were constructed in which the
Escherichia coli gene encoding hygromycin B
phosphotransferase (22) was placed downstream from the 5-flanking DNA
sequence of the cbh1 gene. The enzyme, hygromycin B
phosphotransferase, catalyzes the phosphorylation and subsequent
inactivation of the antibiotic hygromycin B, a potent inhibitor of
protein synthesis in both pro- and eukaryotes (30). The resistance to
hygromycin B of transformed T. reesei cells is shown in Fig.
3. The results demonstrate that the cbh1
promoter fused to the hygromycin B phosphotransferase gene in the
correct orientation (plasmid pCBH1-Hph-2.2) conferred resistance to the
antibiotic in noninduced cells. No transformants were obtained in
control experiments in which the cbh1 promoter was fused in
the opposite orientation (plasmid pCBH1R-Hph-2.2) or with promoterless
construct (pHph). In six independent transformations, an average of 500 transformants/µg of the plasmid pCBH1-Hph-2.2 was usually obtained
(Fig. 3). It is important to note that in T. reesei
transformants harboring the plasmid pCBH1-Hph-2.2, the transcript of
hygromycin B phosphotransferase was found to be induced with cellulose
or sophorose in a manner resembling that of the cbh1 gene
(data not shown). These results indicate that the promoter of the
cbh1 gene has basal transcription activity in the absence of
an inducer and that low levels of mRNA do indeed give rise to
hygromycin B phosphotransferase activity and subsequent resistance of
transformed T. reesei cells to the antibiotic.
Expression of Antisense RNA against cbh2, egl1, and egl2 mRNA Leads to Suppression of Cellulose-induced Expression of the cbh1 Transcript
We designed an antisense strategy to establish
convincingly that the inductive mechanism of the cellulase mRNA by
cellulose in T. reesei requires basal expression of the
cellulase transcripts. The cellulase system of T. reesei is
made up of hydrolases catalyzing the cleavage of -(1,4)-glycosidic
bonds in the cellulase chain. The members of this system include at
least two cellobiohydrolases, CBHI and CBHII, and two major
endoglucanases, EGI and EGII, that act synergistically (7, 8) in the
hydrolysis of cellulose to oligosaccharides. Therefore, we decided to
examine the effect that the expression of an antisense RNA against
CBHII, EGI, and EGII mRNAs would have on the induction of
cbh1. We reasoned that if the basal activity of
cbh2, egl1, and egl2 was necessary for induction by cellulose, the expression of antisense transcripts against
those three cellulases should inhibit the induction of cbh1
by cellulose but not by sophorose. A 399-bp DNA sequence from the
fourth exon of the cbh2 gene, a 587-bp DNA sequence from the
first exon of the egl1 gene and a 398-bp DNA sequence from the second exon of the egl2 gene were ligated together and
then inserted in reverse orientation between the TrpC
promoter and the 3
terminator (23, 24) from A. nidulans
(Fig. 4, A and B). The
TrpC promoter was found to be functional in T. reesei (16). This antisense construct, pJO51, was transformed into QM9414 T. reesei cells, and five stable transformants were
isolated. Analyses of DNA from those transformants showed that the
plasmid pJO51 was integrated in the fungal genome (data not shown). The effect of cellulose and sophorose on the expression of the
cbh1 transcript were analyzed and the results of one of
those transformants, QMJO51, and the original QM9414 cells are
presented in Fig. 4, C and D. The cbh1
transcript was examined by Northern blot analysis of total RNA isolated
from QMJO51 and QM9414 cells exposed to cellulose for 17, 20, and
24 h, or sophorose for 4 and 6 h. Using cellulose as an
inducer, the results revealed reduction of the expression of the
cbh1 transcript by at least 85% in cells carrying the
antisense construct QMJO51, compared with the original QM9414 (Fig.
4C). However, expression of the antisense RNA did not
repress the synthesis of the cbh1 transcript if the soluble
disaccharide sophorose was used as an inducer (Fig. 4D).
Effect of the Addition of Exogenous Cellulase on the Induction of cbh1 and egl1 Transcripts
Studies on the nature of the
physiological inducer of the cellulases by growing T. reesei
on cellobiose have indicated that sophorose might be one of the
naturally occurring inducers of the cellulase system (18). Therefore we
examined the kinetics of induction of the cbh1 and
egl1 transcript by the soluble disaccharide sophorose and
compared it with that of cellulose. The results show that the
transcripts could not be detected up to 14 h after induction with
cellulose, while 4 h were needed for induction with sophorose
(Fig. 5A). Similar results were obtained with
the egl1 transcript (data not shown). This result is
expected if the formation of sophorose is dependent on the initial
hydrolysis of cellulose by the basal cellulase activity and suggest
that the concentration of the extracellular cellulase system could be
the rate-limiting step in the induction of the cellulase transcript with cellulose. Therefore, we examined whether the induction of the
cbh1 and egl1 transcripts by cellulose could be
detected earlier in the presence of added cellulase. To this end, a
crude cellulase fraction was prepared as described under
"Experimental Procedures." The induction of cbh1 and
egl1 transcripts by cellulose was detected earlier in the
presence of the crude cellulase fraction, and the response was also
dependent on the amount of added cellulase (Fig. 5B, compare
also lanes 6 and 7). The enzymes of the
cellulolytic system, composed of CBHI, CBHII, EGI, and EGII were
purified to homogeneity from the crude cellulase fraction as described
under "Experimental Procedures." All of them individually
accelerated the induction of cbh1 and egl1
transcripts (Fig. 5C). In the above-mentioned experiments,
actin transcript presented no alteration (data not shown). A summary of
these kinetic studies is presented in Fig. 5D. The time
required for induction of the cbh1 and egl1
transcripts using cellulose, cellulose + cellulase, and sophorose is
14, 10, and 4 h, respectively. These results implicate
extracellular cellulase activity in the generation of soluble(s)
inducer(s), such as sophorose, from cellulose and are consistent with
the proposed mechanism for cellulase induction by cellulose.
In T. reesei, cellulase is an inducible enzyme system that has drawn considerable interest regarding the mechanism by which the insoluble inducer cellulose triggers synthesis of the cellulase transcripts. The proposed mechanism of cellulase induction is that the fungus produces basal levels of cellulase and that the activity of these extracellular enzymes on cellulose produces a soluble inducer, which can enter the cell and effect induction (18, 31). In support of this mechanism, we have previously shown that antibodies against CBHI, CBHII, EGI, and EGII blocked the expression of cbh1 gene in the presence of cellulose but not the soluble inducer sophorose (21). However, these constitutive levels of cellulases and their direct role in cellulase induction have not been conclusively demonstrated.
The results presented here show that the mRNAs of the major members
of the cellulase system, cbh1 and egl1, are
transcribed under uninduced conditions, and that induction with
cellulose results in at least 1100-fold increase of both transcripts.
To examine further the basal activity of the promoter of the
cbh1 gene, we constructed a chimeric vector in which the
gene encoding hygromycin B phosphotransferase (22) was placed under the
control of the 5-flanking DNA sequence of the cbh1 gene.
Indeed, under uninduced conditions, resistance to the antibiotic
hygromycin B was observed with T. reesei cells transformed
with this construct and grown on medium lacking cellulose. These sets
of experiments indicate that the promoters of cbh1 and
egl1 are transcriptionally active under uninduced
conditions, and that the basal activity of the cbh1 promoter
is sufficient to drive expression of a heterologous gene such as the
hygromycin B resistance-encoding gene.
We also used an antisense RNA strategy to present direct and in vivo evidence for the requirement of the basal expression of the cellulase in induction of the cellulase transcripts by cellulose. The major cellulases from T. reesei, CBHI, CBHII, EGI, and EGII, have glucanase activities with overlapping specificity (6). Therefore, we decided to express an antisense RNA composed of parts of the coding sequences against cbh2, egl1, and egl2 mRNAs in T. reesei and examine the expression of the cbh1 transcript. The results show that the expression of this antisense RNA produced marked effects on the induction of the cbh1 transcript using cellulose but not sophorose as an inducer. The reduction of the expression of the cbh1 transcript by cellulose was between 80 and 90% in experiments with three independent transformants.
In this study we also present evidence indicating that the initial
hydrolysis of cellulose is the rate-limiting step in the inductive
process. This conclusion is based on the fact that the addition of the
cellulase system or its purified enzyme members to a culture of
T. reesei, in the presence of cellulose, resulted in earlier
detection of the cbh1 and egl1 transcripts. That
the inducer is produced from the cell wall of T. reesei
cells, and not from cellulose, or that cellulase itself acts as inducer
are ruled out, since the addition of cellulase in the absence of
cellulose does not affect expression of cellulase transcripts (data not shown). The time required for induction of cbh1 and
egl1 transcripts using cellulose, cellulose + cellulase, or
sophorose is 14, 10, and 4 h, respectively. This result supports
our hypothesis that oligosaccharide(s) is(are) formed in
vivo from cellulose by the activity of a low, constitutive, and
extracellular cellulase activity. The relatively slow induction by
sophorose could be explained by the fact that the inductive process is
protein synthesis-dependent (data not shown). In addition,
it has recently been shown that a sophorose-inducible -diglucoside
permease is involved in the induction of the cellulase system in
T. reesei (32).
In summary, our results demonstrate that in T. reesei (i) the transcripts of two members of the cellulase system, cbh1 and egl1, are expressed at a low level in the absence of added inducer and are induced at least 1100-fold by cellulose; (ii) the promoter of the major member of the cellulase system, cbh1, is able to drive basal expression of a reporter gene placed under its control; (iii) expression of antisense RNA against the cbh2, egl1, and egl2 mRNAs resulted in inhibition of induction of the cbh1 transcript by cellulose but not sophorose; and (iv) the kinetic of expression of the cbh1 and egl1 transcripts by cellulose is influenced by the addition of cellulase to the culture medium.
Taken together, these results support a model that requires the presence of basal activity of cellulases for the induction of their own transcripts by cellulose. The identification of genes involved in this process and their mode of action will be critical to completely understand how the induction occurs at the molecular level.