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Applied and Environmental Microbiology, October 1998, p. 3615-3619, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Transcriptional Activator XlnR Regulates Both
Xylanolytic and Endoglucanase Gene Expression in
Aspergillus niger
Noël N. M. E.
van Peij,
Marco M. C.
Gielkens,
Ronald P.
de
Vries,
Jaap
Visser, and
Leo H.
de Graaff*
Section Molecular Genetics of Industrial
Microorganisms, Wageningen Agricultural University, NL-6703 HA
Wageningen, The Netherlands
Received 11 March 1998/Accepted 5 July 1998
 |
ABSTRACT |
The expression of genes encoding enzymes involved in xylan
degradation and two endoglucanases involved in cellulose degradation was studied at the mRNA level in the filamentous fungus
Aspergillus niger. A strain with a loss-of-function
mutation in the xlnR gene encoding the transcriptional
activator XlnR and a strain with multiple copies of this gene were
investigated in order to define which genes are controlled by XlnR.
The data presented in this paper show that the
transcriptional activator XlnR regulates the transcription of the
xlnB, xlnC, and xlnD genes encoding
the main xylanolytic enzymes (endoxylanases B and C and 
-xylosidase,
respectively). Also, the transcription of the genes encoding the
accessory enzymes involved in xylan degradation, including
-glucuronidase A, acetylxylan esterase A, arabinoxylan
arabinofuranohydrolase A, and feruloyl esterase A, was found to be
controlled by XlnR. In addition, XlnR also activates transcription of
two endoglucanase-encoding genes, eglA and
eglB, indicating that transcriptional regulation by XlnR goes beyond the genes encoding xylanolytic enzymes and includes regulation of two endoglucanase-encoding genes.
| |
INTRODUCTION |
The two most abundant structural
polysaccharides in nature are cellulose and the hemicellulose xylan,
which are closely associated in plant cell walls (4).
Filamentous fungi, particularly Aspergillus and
Trichoderma species, are well-known and efficient
producers of both cellulolytic and hemicellulolytic enzymes. The
cellulase degradation system of these organisms consists of three
classes of enzymes (2): endoglucanases (EC 3.2.1.4),
cellobiohydrolases (EC 3.2.1.91), and -glucosidases (EC 3.2.1.21).
Members of all of these classes are necessary to degrade cellulose, a
homopolymer of -1,4-linked D-glucose. Xylan, however, is
a heterogeneous polymer with a backbone consisting of -1,4-linked
D-xylose residues, which can be substituted at the C-2 and
C-3 positions with various residues, such as acetic acid,
-L-arabinofuranose, (4-o-methyl)glucuronic acid, ferulic acid, and p-coumaric acid
(5). Due to this heterogeneous composition, a
more complex set of enzymes is required for xylan degradation.
The following enzymes have been found to be necessary during the
cooperative process of xylan breakdown: endoxylanase (EC 3.2.1.8),
-xylosidase (EC 3.2.1.37), acetylxylan esterase (EC 3.1.1.72), -L-arabinofuranosidase (EC
3.2.1.55), arabinoxylan arabinofuranohydrolase,
-glucuronidase (EC 3.2.1.139), feruloyl esterase, and
p-coumaroyl esterase (3).
The expression of cellulose- and xylan-degrading enzymes by
Aspergillus and Trichoderma species has been
studied extensively at the cellular level (1, 20, 21, 25).
It has been shown that xylanase- and cellulase-encoding genes are
regulated at the transcriptional level (10, 23, 31, 43). In
the presence of D-glucose the genes are not expressed, and
it has been shown that the carbon catabolite repressor protein CreA is
involved in transcriptional repression of xylanase-encoding
(10) and arabinase-encoding (38) genes in
Aspergillus species. It has been demonstrated that in
Trichoderma reesei the CreA counterpart Cre1 causes
repression of transcription of cellulase-encoding (22, 23)
and xylanase-encoding (30, 31) genes. However, far less is
known about the mechanism by which cellulase- and xylanase-encoding
genes are induced. The inducing abilities of various saccharides have
been tested, and some saccharides induce the synthesis of both
xylanases and cellulases (10, 21, 31, 37, 48). Nevertheless,
on the basis of biochemical data (1, 20, 21) and mRNA
expression analysis data (23, 31), a separate induction
mechanism has been proposed for these systems in both
Aspergillus and Trichoderma.
Recently, a selection system was developed to isolate
Aspergillus niger strains having mutations in a
transcription factor involved in induction of expression of
xylanolytic genes. Complementation of such a mutation by
transformation with a plasmid library led to the isolation of the
A. niger xlnR gene, which encodes a transcriptional activator of the A. niger xylanolytic system
(44). This xlnR gene encodes a zinc binuclear
cluster protein, which is a member of the GAL4 family of transcription
factors. Isolation of both the xlnR gene and A. niger xlnR loss-of-function mutants provided an opportunity to
study the spectrum of genes that are controlled by XlnR at the
transcriptional level.
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MATERIALS AND METHODS |
Aspergillus strains, transformation, and culture
conditions.
All of the A. niger strains used
were derived from wild-type strain N400 (= CBS 120.49). The strains
used were A. niger N402 (cspA1), a
short-conidiophore derivative, NW205::130 [argB13 cspA1 nicA1 pyrA6 UAS(xlnA)-pyrA], NXA1-4
[argB13 cspA1 nicA1 pyrA6 UAS(xlnA)-pyrA
xlnR1] (strains NW205::130 and NXA1-4 are described more extensively in reference 44), and N902
(argB15 cspA1 fwnA1 metB10 pyrA5).
Strain N902::230-25.12 (argB15 cspA1 fwnA1 metB10),
which contains approximately 20 additional copies of xlnR,
as determined by a phosphorimager analysis of Southern blots, was
obtained by cotransformation of A. niger N902. The
cotransforming plasmids were pIM230 (44) and pGW635
(19), which contain the functional xlnR gene
(EMBL accession no. AJ001909) and the pyrA gene (EMBL accession no. X96734), respectively. Transformation was carried out as
described previously (27).
All media were based on
Aspergillus minimal medium
(
36). The media contained the carbon sources indicated
below, and the
starting pH of each medium was 6. Spores were inoculated
at a
concentration of 10
6 spores ml
1. In
transfer experiments the first culture containing
D-fructose
was supplemented with 0.2% (wt/vol) Casamino
Acids and 0.1% (wt/vol)
yeast extract. After overnight growth, mycelia
were recovered
by filtration and washed with saline. These mycelia were
transferred
to media containing
D-xylose or xylan as a
carbon source and 0.05%
(wt/vol) Casamino Acids. The xylan used was
birchwood xylan (Roth-7500).
Expression cloning of A. niger glucanases in
Escherichia coli.
A xylan-induced cDNA library of
A. niger (43) was screened for expression of
endoglucanases by using a modified procedure (6, 46, 47).
The plates contained 20 ml of 2× TY, 0.2% carboxymethyl cellulose
(CMC) (Sigma), 1.5% agar, and 100 µg of ampicillin per ml. E. coli cells were plated in an overlay consisting of 5 ml of the
same medium containing about 300 colonies per plate, and the plates
were incubated for 48 h at 37°C. Next, 5 ml of 0.1% Congo red
(Aldrich) was poured onto each plate. After it was stained for 1 to
2 h, each plate was destained with 5 ml of 5 M NaCl for 0.5 to
1 h. About 12,000 colonies from the A. niger cDNA
library were plated. Screening on CMC resulted in 89 colonies that had
halos after staining with Congo red. None of these colonies produced a
halo when it was screened with Remazol brilliant blue-modified xylan.
All colonies contained a full-length cDNA copy, which appeared to
originate from two different genes. Both of the enzymes encoded were
active on CMC and on -glucan (unpublished data). The corresponding genes, eglA and eglB, were cloned by using these
cDNA fragments.
Northern blot analysis.
Total RNA was isolated from powdered
mycelia by using TRIzol reagent (Life Technologies) according to the
supplier's instructions. For Northern blot analysis 10 µg of total
RNA was glyoxylated and separated on a 1.6% (wt/vol) agarose gel
(39). After capillary blotting onto Hybond-N filters
(Amersham), the amounts of RNA were checked by staining the rRNA on the
Hybond filters with a 0.2% (wt/vol) methylene blue solution. The
filters were hybridized at 42°C in a solution containing 50%
(vol/vol) formamide, 10% (wt/vol) dextran sulfate, 0.9 M NaCl, 90 mM
trisodium citrate, 0.2% (wt/vol) Ficoll, 0.2% (wt/vol)
polyvinylpyrrolidone, 0.2% (wt/vol) bovine serum albumin, 0.1%
(wt/vol) sodium dodecyl sulfate, and 100 µg of single-stranded
herring sperm DNA per ml. Washing was done under homologous
hybridization conditions with a solution containing 30 mM NaCl, 3 mM
trisodium citrate, and 0.1% (wt/vol) sodium dodecyl sulfate at 68°C.
The 32P-labelled DNA probes used were either cDNA or
genomic fragments, as shown in Table 1.
A 1-kb
-glucosidase cDNA fragment of
A. niger, as
determined by sequence analysis, was isolated from a xylan-induced cDNA
library (
43) by using PCR with degenerate oligonucleotides
based
on the
Aspergillus kawachii (EMBL accession no.
AB003470) and
Aspergillus aculeatus (EMBL accession no.
P48825) sequences
for
-glucosidase and cloned into pGEM-T (Promega).
Nucleotide sequence accession numbers.
The eglA
and eglB sequences have been deposited in the GenBank-EMBL
sequence database under accession no. AJ224451 and AJ224452,
respectively.
| |
RESULTS AND DISCUSSION |
Induction of the xylanolytic system.
An A. niger mutant having a loss-of-function mutation in the
xylanolytic transcriptional activator gene xlnR lacks
transcription of the endoxylanase B- and -xylosidase-encoding genes
xlnB and xlnD (44). To
investigate the spectrum of genes which are under control of the
transcriptional activator xlnR, expression in an A. niger wild-type strain and expression in the strain
with the xlnR loss-of-function mutation were analyzed by
Northern blot analysis. To do this, we used fragments of genes cloned
from A. niger encoding enzymes which are
potentially involved in the breakdown of xylan (Table 1). A. niger NW205::130 (wild type) and NXA1-4 (a xlnR
mutant) were precultured and subsequently transferred to media
containing 1% birchwood xylan and to media containing 1%
D-xylose (both birchwood xylan and D-xylose
are known to be carbon sources that induce the xylanolytic system in
A. niger) (10). Northern blot analysis of
RNA obtained from the wild-type strain showed that xylanolytic,
arabinanolytic, and cellulolytic genes were induced when the organism
was grown on xylan, which is the natural substrate, and on
D-xylose (Fig. 1A). High
levels of expression were obtained in most cases after 6 h of
growth on the polymeric carbon source xylan, although the patterns of expression for individual genes differed. Whereas some genes, including
xlnD and aguA, had a high transcription level
during the early phase of induction, other genes, including
axeA and eglB, were highly transcribed at a later
stage. The level of induction on D-xylose was usually lower
than the level of induction on xylan, but some genes, including
xlnD and xlnB, had a relatively high level of
expression on D-xylose. As expected for extracellular enzyme systems under the control of carbon catabolite repression (10, 38), none of the genes was expressed on
D-fructose.
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FIG. 1.
Northern blot analysis of expression of A. niger genes encoding cellulose- and xylan-degrading
enzymes. (A) Time course of induction in A. niger
NW205::130 (wt) and NXA1-4 (xlnR1) (a loss-of-function
mutant). Both strains were cultured for 18 h in medium containing
3% D-fructose (Fr), and mycelia were subsequently
transferred to medium containing 1% xylan (Xa) or 1%
D-xylose (Xo) and incubated for the times indicated.
Each lane contained 10 µg of total RNA, which was checked by
hybridization with the 18S rRNA probe. Blots were hybridized with
gene-specific probes as indicated. (B) Comparison of expression of
genes encoding cellulose- and xylan-degrading enzymes in A. niger N902 (wt), NXA1-4 (xlnR1), and N902-pIM230-25.12
(XlnR+) (N902 with multiple copies of xlnR) upon
transfer to medium containing 1% D-xylose for 6 h
after growth for 18 h in medium containing 1%
D-fructose. A Northern blot analysis was performed
exactly as described above. The signal intensities of the
different blots cannot be compared to each other due to the unknown
specific activities of the probes used and the different
exposure times used for the various blots.
|
|
Effect of the xlnR loss-of-function mutation on
expression.
An analysis of transcription in the
xlnR loss-of-function mutant NXA1-4 revealed
expression of only abfB and bglA upon growth on
D-xylose and xylan (Fig. 1A). Mutant NXA1-4 lacks the
ability to induce transcription of genes encoding xylanolytic enzymes which are involved in the degradation of the polyxylose backbone of
xylan. Also, transcription of genes encoding accessory enzymes is
absent in this mutant. These findings explain the previously described impaired growth of NXA mutants on xylan (44).
Strains with an xlnR loss-of-function mutation are not able
to express the xylanolytic enzymes, which results in impaired
release of saccharides (and therefore carbon source) from the polymeric
xylan. Although arabinofuranosidase B is expressed in
these mutants, apparently the L-arabinose released from
arabinoxylan by this enzyme is not sufficient to allow normal growth of
the fungus. The inability of the xlnR loss-of-function
mutants to degrade xylan also affects the release of inducer from the
polymeric substrate. Induction by D-xylose, however, is
independent of the presence of the xylanolytic enzyme system.
Therefore, gene expression was reexamined in a second experiment
by using mutant NXA1-4, wild-type strain N902, and xlnR
multicopy strain N902::230-25.12; D-xylose was used as
the inducing carbon source in this experiment.
Effect of multiple copies of xlnR.
In the wild-type
strain all of the genes tested were induced on D-xylose
(Fig. 1B), whereas in strain NXA1-4 only abfB and bglA transcription was observed. In xlnR
multicopy strain N902::230-25.12 all of the genes were also
expressed. Some genes (for example, aguA and
faeA) had equal transcript levels in both the wild-type and
the xlnR multicopy strain, whereas other genes (for
example, abfB, axhA, bglA,
xlnB, and xlnC) had increased transcription levels in the xlnR multicopy strain compared to that in the
wild-type strain. From this finding we concluded that the
transcriptional activator XlnR regulates the transcription of the
xlnB, xlnC, and xlnD genes encoding
the main xylanolytic enzymes (endoxylanases B and C and -xylosidase,
respectively). In addition, the aguA, axeA,
axhA, and faeA genes encoding accessory
enzymes (-glucuronidase A, acetylxylan esterase A,
arabinoxylan arabinofuranohydrolase A, and feruloyl esterase A) are
controlled by the transcriptional regulator XlnR. The transcriptional
activator XlnR also activates transcription of the eglA and
eglB genes, which encode endoglucanases A and B. This
indicates that regulation by the transcriptional activator XlnR goes
beyond regulation of the genes encoding xylanolytic enzymes and also
includes regulation of at least two endoglucanase-encoding genes.
All of the genes that were found to be controlled by the
transcriptional activator XlnR exhibited differences in their levels
of
expression in response to increased
xlnR gene copies. The
differences
in the responses to the
xlnR gene copy number
might originate
from differences in the XlnR binding sites in the
various xylanolytic
promoters. The sequence 5'-GGCTAAA-3'
has been suggested previously
to be a consensus binding site for
the XlnR protein; this suggestion
was based on the results of a
comparison of a limited number of
mainly endoxylanase promoters of
different
Aspergillus species
(
44). The results
presented here show that expression of at
least nine genes in
A. niger is controlled by XlnR. The
axeA,
axhA,
eglA,
eglB, and
xlnC
genes, for which an increased
xlnR copy number has a
positive effect on the level of transcription,
all have a
nucleotide other than adenine at the last position.
Transcription
of
xlnB, which has an adenine at the last position,
however,
is also positively influenced. A comparison of the sequences
of these
nine promoters (Table
2) suggests,
therefore, that the
last nucleotide in the proposed consensus
sequence is less important
in the binding site and that
5'-GGCTAA-3' is a more appropriate
consensus sequence, but
the seventh nucleotide could play a role
in XlnR binding.
All of the
A. niger genes for which XlnR
transcriptional control has been demonstrated contain one or more
copies of this
consensus sequence in the promoter region. The different
genes
vary in the number of putative XlnR binding sites
present, as
four genes have two putative sites. Also, the
orientation varies,
as some genes have the opposite orientation or both
orientations
are present. The differences found in the effect of the
xlnR copy
number and the level of transcription of the
individual genes
cannot be explained by the differences in the
presumed XlnR binding
sites, since the mode of binding of
XlnR is not known (
44).
The context in which the sites are located in the promoter region may
also play an important role. The putative XlnR binding
site is
not a direct or inverted repeat, while most zinc binuclear
cluster
proteins have a dimeric nature and bind to symmetric sites.
However,
some proteins (for example, the AlcR protein of
Aspergillus nidulans) are thought to act as monomers. Two molecules of AlcR
can simultaneously bind to symmetric sites, whereas only one molecule
occupies a direct repeat (
28).
It has been proposed that repression by CreA of the xylanolytic genes
(
10,
44) is analogous to the double lock mechanism
described
for the ethanol regulon in
A. nidulans (
13,
26).
In this model CreA represses both the positive and
autoregulated
trans-acting gene
alcR and
structural genes, such as
alcA and
aldA (
14,
26,
28,
32). Some CreA binding sites in the
alcA and
alcR upstream region are close to or overlap the AlcR
targets (
13,
26). Therefore, it has been suggested that
competition
between the AlcR and CreA proteins for the same region is a
mechanism
in the regulation of the ethanol regulon genes. This is also
the
case in the regulation of expression of
amdS by the
trans-acting
factors AmdR, FacB, AmdA, AmdX, AreA, and CreA
(
8,
29,
34,
35). The overlap of AmdX binding sites with CreA
and AmdA binding
sites suggests that there is competition for binding
sites by
multiple factors (
34). The repressor protein CreA
has been shown
to also have a function in xylanolytic gene expression
in
A. niger (
10,
17). However, putative CreA
sites in the XlnR-controlled
genes in
A. niger are
generally at distances of more than 40 bp
from the putative XlnR
binding sites; an exception is the 1-bp
distance in the
xlnD
upstream region (
43). Thus, XlnR-CreA competition
for all
xylanolytic promoters is unlikely. Besides the
trans-acting
factors XlnR and CreA, other
trans-acting factors may
have a function
in modulating the transcription of the various
XlnR-controlled
genes.
Transcription of the
-glucosidase-encoding gene
bglA
is under separate control, and therefore not all genes
encoding cellulolytic
enzymes are controlled by XlnR. Of the
genes involved in xylan
degradation, the
abfB gene is
the only gene whose transcription
is not controlled by XlnR.
The encoded enzyme, however, is involved
in hydrolysis of
L-arabinofuranosyl residues not only from arabinoxylan
but
also from arabinan (
41) and pectin (
40).
abfB gene expression
is under coordinate control with
expression of the arabinofuranosidase
A-encoding
abfA gene
and the endoarabinase-encoding
abnA gene
(
16).
Although it is clear from the results presented here that
the
abfB and
bglA genes are not controlled by XlnR,
the level
of transcription is increased in both the NXA1-4 mutant and
the
xlnR multicopy transformant. The promoter sequence of
the
A. niger bglA gene is not available, and the
abfB gene does not contain
the XlnR binding site. The
increase in the levels of expression
of
abfB and
bglA on
D-xylose may be an indirect effect of
the
xlnR loss-of-function mutation and gene dosage. For
example, there
could be an effect on pentose catabolism
(
45), thereby influencing
the
L-arabitol
concentration, which is the inducer of the
abfB gene
(
42).
The use of a loss-of-function mutation in the transcriptional activator
XlnR is a powerful tool for understanding the fungal
strategy for
degrading the variety of xylan structures which occur
in nature. The
fact that expression of the xylanolytic enzymes
and expression of some
cellulolytic enzymes are coordinately regulated
at the molecular level
provides new insight into the regulation
of expression of both enzyme
systems. The findings presented here
strengthen the hypothesis that
there is an evolutionary relationship
between some of the xylanolytic
and cellulolytic enzyme systems.
Xylanases and cellulases have
been shown to be related at various
levels. The
three-dimensional structures of, for example, family
11 endoxylanases and family 12 endoglucanases are similar
(
7).
Also, there are similarities in the primary structures
of, for
example,
-xylosidase XlnD and
-glucosidase BglA, both of
which
are members of the family 3 glycosyl hydrolases
(
43). Here we
provide evidence that there is
coordination in the regulation
of xylanases and some cellulases.
| |
ACKNOWLEDGMENT |
We appreciate financial support from grant BIO2-CT93-0174 from
the BIOTECH Programme of the European Commission to J.V.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section
Molecular Genetics of Industrial Microorganisms, Wageningen
Agricultural University, Dreijenlaan 2, NL-6703 HA Wageningen, The
Netherlands. Phone: 31 317 484691. Fax: 31 317 484011. E-mail:
office{at}algemeen.mgim.wau.nl.
| |
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Applied and Environmental Microbiology, October 1998, p. 3615-3619, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
