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Appl Environ Microbiol, April 1998, p. 1412-1419, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Cloning and Transcriptional Regulation of
the Aspergillus nidulans xlnD Gene Encoding a
-Xylosidase
José A.
Pérez-González,1,
Noël N. M. E.
van
Peij,2
Alja
Bezoen,2
Andrew P.
Maccabe,1
Daniel
Ramón,1,* and
Leo H.
de Graaff2
Departamento de Biotecnología de los
Alimentos, Instituto de Agroquímica y Tecnología de los
Alimentos, Consejo Superior de Investigaciones Científicas,
46100 Burjassot, Valencia, Spain,1 and
Molecular Genetics of Industrial Microorganisms, Wageningen
Agricultural University, 6703 HA Wageningen, The
Netherlands2
Received 16 June 1997/Accepted 25 January 1998
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ABSTRACT |
The xlnD gene encoding the 85-kDa 
-xylosidase was
cloned from Aspergillus nidulans. The deduced primary
structure of the protein exhibits considerable similarity to the
primary structures of the Aspergillus niger and
Trichoderma reesei -xylosidases and some similarity to
the primary structures of the class 3 -glucosidases. xlnD is regulated at the transcriptional level; it is
induced by xylan and D-xylose and is repressed by
D-glucose. Glucose repression is mediated by the product of
the creA gene. Although several binding sites for the pH
regulatory protein PacC were found in the upstream regulatory region,
it was not clear from a Northern analysis whether PacC is involved in
transcriptional regulation of xlnD.
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INTRODUCTION |
Hydrolysis of xylans is of
considerable interest for various biotechnological applications (for
reviews see references 7 and 47).
Unlike cellulose, xylans are chemically heterogeneous molecules with a
characteristic backbone consisting of -(1,4)-linked D-xylosyl residues replaced with acetyl,
L-arabinosyl, and 4-O-methyl-glucuronosyl side
chains. Natural xylan degradation by microorganisms occurs through the
coordinated action of various enzymes, including the endo-(1,4)--xylanases (EC 3.2.1.8), which cleave the -(1,4) glycosidic bonds between D-xylose residues in the main
chain to produce xylooligosaccharides, and -xylosidase (EC
3.2.1.37), which cleaves xylooligosaccharides to produce
xylose.
Filamentous fungi are known to be efficient producers of xylanolytic
enzymes, and most commercial xylanolytic preparations are obtained from
fermentations of Aspergillus or Trichoderma species. Several genes encoding endo-(1,4)--xylanases from these fungal species have been characterized (10, 23, 25, 26, 39,
44), and recently genes encoding -xylosidases have been cloned
from both Aspergillus niger (46) and
Trichoderma reesei (32).
Little is currently known about the molecular mechanisms controlling
xylanase gene expression in filamentous fungi. The presence of
regulatory elements involved in xylan-specific induction in the
promoters of the Aspergillus tubingensis and T. reesei xylanase-encoding genes (10, 50) and
Cre1-mediated carbon catabolite repression of expression of the
T. reesei xln1 gene (31) are the only such data
reported so far. The ascomycete Aspergillus nidulans is a model organism for studies of gene regulation due to our extensive knowledge of its genetics and the availability of mutants (1, 9). In recent years the molecular basis of glucose repression by
the protein product of the regulatory gene creA has been
investigated; it has been found that this protein is a negatively
acting transcription factor which binds to a subset of DNA sequence
motifs conforming to the consensus sequence 5'-SYGGRG-3' (8, 24,
28). In addition, studies of mutants (5) disrupted in
their responses to external pH (alkaline growth mimic and acidic growth
mimic phenotypes) have revealed a regulatory mechanism comprising a signal transduction pathway, encoded by the pal genes, which
at alkaline ambient pH results in proteolytic conversion of the PacC transcription factor to its active form. After conversion PacC is able
to activate those genes whose expression is appropriate under alkaline
conditions and to repress those genes whose expression is suited to
acidic ambient pH (3, 12, 33, 34, 43).
When grown in media in which xylan is the only carbon source, A. nidulans produces at least three endo-(1,4)--xylanases
(17, 36) and one predominantly mycelium-bound -xylosidase
(29). These four enzymes have been purified and kinetically
characterized (15-18, 29), and the genes encoding the three
endo--(1,4)-xylanases (xlnA, xlnB, and
xlnC) have been cloned and sequenced (30, 35). In
this paper we describe the identification, cloning, and nucleotide sequence of an A. nidulans gene (xlnD) which
encodes the previously isolated -xylosidase (29).
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MATERIALS AND METHODS |
Strains, plasmids and culture conditions.
Escherichia
coli LE392 [e14-(mcrA) hsdR514 supE44
supF58 lacY1 galK2 galT22 metB1 trpR55] and DH5
[endA1
hsdR17 gyrA96 thi-1 relA1 supE44 recA1 
lacU169 (
80
lacZM15)] were used as hosts for propagation of
bacteriophage 
and plasmids, respectively. A. nidulans
biA1 (= CECT2544) was obtained from the Spanish Type Culture
Collection and was used as the wild-type strain. A. nidulans G191 (pabaA1 pyrG89 fwA1
uAY9) (4) was used as the host in transformation
experiments performed with plasmid pGW635, which contains the A. niger pyrA gene (20) for selection of transformants. A. nidulans creAd30, biA1 was a gift
from H. N. Arst, Jr., and strains palA1,
biA1, wA3 (a strain which mimics growth at acidic
pH), and pacCc14, biA1 (a strain
which mimics growth at alkaline pH) were obtained from M. A. Peñalva. A. nidulans mycelia were grown from spores in
minimal medium (MM) (37) containing various carbon sources (1%, wt/vol) as indicated below; appropriate supplements were included
when necessary. In transfer experiments, MM containing D-fructose (1%, wt/vol) and supplemented with 0.5%
(wt/vol) Casamino Acids (Difco Laboratories, Detroit, Mich.) was used
to generate mycelial biomass. Buffered media were prepared by adding
filter-sterilized sodium phosphate after autoclaving from 1 M stock
solutions having pH values of 4.1, 6.0, and 8.0 in order to obtain a
final phosphate concentration of 100 mM. In all cases the sodium ion
concentration was adjusted to 195 mM by adding 5 M NaCl. Induction
media were prepared by replacing D-fructose with
D-xylose (1%, wt/vol) from a filter-sterilized stock
solution (10%, wt/vol).
Cloning and sequencing procedures.
An A. nidulans
genomic library constructed in Charon 4A (51) was
screened by using hybridization conditions as previously described
(35). DNA manipulations were carried out by standard methods
as described by Sambrook et al. (40). DNA sequences were
determined by the dideoxynucleotide chain termination method (41) with a Sequenase sequencing kit from Amersham-USB used according to the supplier's instructions; universal, reverse, and
gene-specific oligonucleotides were used as primers. The DNA sequences
obtained were analyzed with the Genetics Computer Group sequence
analysis software package (13).
DNA isolation and RNA isolation.
Fungal
high-molecular-weight DNA was isolated as described previously
(11). Total RNA was extracted from mycelial tissue by a
procedure based on the method of Cathala et al. (6). Mycelia were harvested by filtration and rapidly press dried between sheets of
absorbent paper to remove as much liquid as possible. Each mycelial mat
was then flash frozen in liquid nitrogen and stored at 
70°C.
Approximately 100 mg of nitrogen-frozen mycelium was homogenized with a
solution containing 600 µl of lysing medium (5 M guanidinium
thiocyanate, 10 mM EDTA, 50 mM Tris [pH 7.5]) plus 48 µl of
-mercaptoethanol in a 2-ml screw-cap Eppendorf tube for 45 s at
full speed with a Mini-beadbeater (Biospec Products, Bartlesville,
Okla.) using five 2-mm-diameter steel balls. The contents reached a
temperature of about 50°C. The tube was left at room temperature for
5 min, and the contents were rehomogenized as described above. Mycelial
debris was removed by microcentrifugation at 4°C for 15 min. Five
hundred microliters of supernatant was removed and added to 1.5 ml of 4 M LiCl, and the preparation was mixed and kept on ice overnight.
Precipitated material was collected by microcentrifugation at 4°C for
15 min. The tube was thoroughly drained, and the pelleted material was
resolubilized in 500 µl of a solution containing 0.1% (wt/vol)
sodium dodecyl sulfate, 1 mM EDTA, and 10 mM Tris (pH 7.5) by
homogenization with the Mini-beadbeater for 10 s in the absence of
steel balls. The solubilized material was extracted with
phenol-chloroform, and the aqueous phase recovered was reextracted with
chloroform. Total RNA was precipitated by adding 0.1 volume of 3 M
sodium acetate (pH 4.8) and 2.5 volumes of absolute ethanol and then
mixing and incubating the preparation at 70°C for 30 to 60 min. RNA
was recovered by microcentrifugation at 4°C for 15 min. The tube was
drained, and the pellet was dissolved in 100 µl of diethyl
pyrocarbonate-treated, MilliQ-filtered water by freezing and thawing.
3-[N-Morpholino]propanesulfonic acid (MOPS) (0.6 M)-formaldehyde gels were used to perform a Northern analysis of total
RNA. A 1.9-kb DNA fragment containing the A. niger xlnD gene
was generated with oligonucleotides xylos001 and xylos004 and was used
as the xlnD-specific probe (46). An 830-bp
KpnI-NcoI fragment was used as the actin-specific
probe (19). Probes were labelled by the random
hexanucleotide primer method (14).
A. nidulans transformations.
Transformation of
A. nidulans G191 was carried out as described by Tilburn and
coworkers (42) by using plasmids pGW635 (5 µg) and pXDE1
(20 µg); the latter plasmid contained the A. nidulans xlnD
gene. Transformants were selected for growth on MM in the absence of
uridine and were clonally purified. -Xylosidase activity was
extracted and measured as described previously (29).
Immunoblot analysis.
Mycelia were grown from 2.5 × 108 spores for 14 h at 37°C and 200 rpm in 50 ml of
MM containing oat spelt xylan (1%, wt/vol), yeast extract (1%,
wt/vol), and Casamino Acids (0.5%, wt/vol) and then extracted with 25 ml of phosphate-buffered saline containing 0.05% Triton X-100 for
24 h at 30°C and 200 rpm. Portions (30 µl) of the mycelial
extracts were subjected to sodium dodecyl sulfate-10% polyacrylamide
gel electrophoresis and electroblotted onto nitrocellulose membranes.
Immunostaining was carried out by the Bio-Rad procedure by using a
1:400 dilution of the A. nidulans -xylosidase antibody
(29), followed by incubation with a 1:3,000 dilution of
anti-rabbit immunoglobulin G.
Nucleotide sequence accession number.
The A. nidulans
xlnD gene sequence has been deposited in the EMBL nucleotide
sequence database under accession no. Y13568.
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RESULTS |
Cloning of an A. nidulans gene coding for a
-xylosidase.
An A. nidulans genomic library
(51) was screened by heterologous hybridization by using a
DNA probe corresponding to the A. niger xlnD gene, as
described above. After two rounds of screening, a positive plaque was
detected and purified. The results of a Southern analysis of the phage
insert performed with different restriction enzymes correlated well
with signals detected previously in Southern blot profiles of
restricted A. nidulans wild-type genomic DNA (data not
shown). A 4.3-kb EcoRI DNA fragment that hybridized to the
A. niger xlnD probe was isolated and subcloned into pUC18,
yielding plasmid pXDE1.
Nucleotide sequence of the cloned gene.
The sequence of the
A. nidulans DNA insert (4,243 bp) cloned in plasmid pXDE1
was determined on both strands. A comparison of this sequence with the
A. niger xlnD gene sequence revealed a 2,406-bp
uninterrupted open reading frame (Fig.
1). The
deduced amino acid sequence of the A. nidulans xlnD gene
product exhibited high degrees of similarity to the primary structures
of the A. niger and T. reesei -xylosidases
(64.3 and 61.9% identity, respectively) (Fig.
2) and also exhibited significant levels
of similarity to the primary structures of -glucosidases belonging
to glycosidase family 3 (22). There was a predicted signal
peptide cleavage site (49) between amino acid residues 17 and 18. Thus, the mature protein is 785 amino acids long and has a
predicted molecular mass of 85,320 Da and an isoelectric point of 4.17.
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FIG. 1.
Nucleotide sequence of the A. nidulans xlnD
gene and the deduced amino acid sequence of the gene product. Putative
CreA and PacC binding sites are underlined and double underlined,
respectively. Potential N-glycosylation sites are in italics.
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FIG. 2.
Alignment of the -xylosidase amino acid sequences of
A. nidulans, A. niger (46), and
T. reesei (34a). Asterisks indicate identical
amino acids, and dots indicate conservative changes.
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|
Analysis of the 5' noncoding sequence of the
A. nidulans
xlnD gene revealed the presence of sequence elements (Fig.
1) that
could be involved in transcriptional initiation (
21,
45).
One TATA box is present 75 bp upstream of the ATG codon, and two
CAAT
boxes are present 85 and 100 bp upstream of the ATG codon.
The start
codon is preceded by the sequence TCACC, which resembles
the consensus
CCPuCC-ATG sequence found in higher eukaryotes (
27).
In
addition, consensus binding target sequences for the
A. nidulans wide domain regulators CreA (
8) and PacC
(
43) are present.
An AATAAA polyadenylation motif
is present 120 bp downstream of
the proposed stop codon
(
38).
-Xylosidase overproduction in A. nidulans.
Plasmid
pXDE1, which contains the A. nidulans xlnD gene plus 1,660 bp of upstream sequence, was introduced into A. nidulans G191 by cotransformation with plasmid pGW635. Uridine prototrophs were
selected and analyzed by Southern hybridization. Five cotransformants were tested for -xylosidase overexpression by direct growth in MM
containing oat spelt xylan as the carbon source. Samples (5 ml) were
collected in duplicate after 14, 24, and 36 h of incubation, and
the -xylosidase activities in extracted mycelia were measured (Table
1). In all of the strains analyzed,
-xylosidase activity was lower in the 36-h samples than in the 14-h
samples due to the presence of protease activity (data not shown). All
of the cotransformants exhibited higher levels of -xylosidase
activity than the nontransformed strain. -Xylosidase overexpression
was greatest in cotransformants TXD1.4 and TXD1.10. These two
cotransformants and A. nidulans G191 were also grown for
17 h in MM containing D-fructose and Casamino Acids as
carbon and nitrogen sources, respectively, and mycelia were then
transferred to D-xylose-containing induction medium.
Samples (5 ml) were collected in duplicate after 2, 4, and 8 h of
posttransfer incubation, and the -xylosidase activities in extracted
mycelia were measured (Table 2). Activity in the nontransformed strain was detected 2 h after transfer and then decreased, and the minimum activity was reached after 8 h. The cotransformants had similar activity profiles but higher absolute levels at all time points after transfer.
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TABLE 1.
Time course of -xylosidase production during growth of
A. nidulans G191 and five xlnD cotransformants on
MM containing 1% oat spelt xylan as the sole carbon source
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TABLE 2.
Time course of -xylosidase production by A. nidulans G191 and two xlnD cotransformants during
incubation of washed, D-fructose-grown mycelia in
D-xylose-containing induction media
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Western blot analysis of the overproduced -xylosidase.
In
previous work in our laboratory Kumar and Ramón isolated and
characterized an 85-kDa -xylosidase from A. nidulans
(29). Antibodies raised against this enzyme (29)
were used to probe a Western blot of mycelial extracts prepared from
G191 (the untransformed strain) and the overexpressing transformants
TXD1.4 and TXD1.10. Figure 3 shows that
in the extracts from the overproducing transformants there were
increased levels of a specific band detected by the A. nidulans -xylosidase antibody whose mobility was identical to
the mobility of a band found in the untransformed strain extract. The intensities of staining of the bands corresponded to the levels of
-xylosidase activity measured in the mycelial extracts.
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FIG. 3.
Western blot. Mycelial extracts of G191 (an
untransformed strain) and transformed -xylosidase overproducers
TXD1.4 and TXD1.10 were probed with an antibody raised against the
A. nidulans 85-kDa -xylosidase (29). The
numbers in parentheses are the relative (compared to nontransformed
extract) -xylosidase activities in the mycelial extracts.
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Transcriptional regulation of xlnD.
The levels of
expression of xlnD were investigated by performing a
Northern blot analysis of transfer cultures and in all cases were
compared to the levels of actin transcripts as an internal control.
Mycelial biomass was grown from spores for 17 h in
D-fructose-containing MM supplemented with Casamino Acids
and then transferred to D-xylose-containing induction
medium. Mycelial samples taken 1, 2, 4, and 6 h after the transfer
were used to prepare total RNA. No xlnD expression was
detected after growth of the A. nidulans wild-type strain in
D-fructose-containing medium. However, within 1 h of
transfer to inducing conditions a strong xlnD transcript
signal was detected, the level of which remained high throughout the
time course analyzed (Fig. 4A, lanes X).
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FIG. 4.
Northern blots of total RNAs extracted from wild-type
mycelia (A) and the creAd30 mutant (B) after
growth in the presence of D-fructose (lanes F) for 17 h and transfer to inducing conditions (1% D-xylose) (lanes
X) and inducing-repressing conditions (1% D-xylose plus
1% D-glucose) (lanes XG).
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The carbon catabolite repressibility of
xlnD expression was
investigated with the severe carbon catabolite repression mutant
creAd30 (
2). Mycelial biomasses from
the wild type and a
creAd30 mutant were each
divided into two halves and transferred to
induction medium and
induction medium supplemented with
D-glucose.
In the
wild-type mycelial samples,
xlnD transcript levels were
considerably reduced at the early time points in the presence
of
D-glucose (Fig.
4A, lanes XG). In the case of
creAd30, elevated levels of the
xlnD
transcript were observed in the
presence of glucose at all of the time
points examined, although
the transcript levels never attained the
levels seen in the samples
that were induced and derepressed (samples
containing
D-xylose
but not
D-glucose) (Fig.
4B).
The influence of pH on
xlnD expression was investigated by
the following two techniques: (i) by examining the consequences
of
transferring wild-type mycelial biomass to acidic (pH 4), neutral
(pH
6), and alkaline (pH 8) buffered induction media, and (ii)
by analyzing
the expression of
xlnD in
A. nidulans pH
regulatory
mutants. Transfer of
D-fructose-grown wild-type
mycelium to sodium
phosphate-buffered induction media revealed no
apparent influence
of pH at any of the time points analyzed (Fig.
5A). The pH of
each medium was measured
at the time of harvest to ensure that
the buffering capacity was
adequate throughout the experiment,
and no significant pH shifts were
detected (data not shown).
xlnD expression in both the acid
mimic mutant
palA1 and the alkaline
mimic mutant
pacCc14 exhibited induction patterns similar to
the pattern observed
for the wild-type strain after mycelial biomass
was transferred
to nonbuffered induction media, although induction in
the
pacCc14 mutant seemed to be partially
reduced (Fig.
5B). The transcript
levels appeared to decrease at later
time points in the mutants,
whereas the transcript level remained
essentially the same in
the wild type.
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FIG. 5.
(A) Northern blots of total RNA extracted from wild-type
mycelia after growth in the presence of D-fructose (lane F)
for 17 h and transfer to inducing conditions in media buffered
with sodium phosphate to pH 4, 6, and 8. (B) Similar experiment
performed with mycelia from the wild type (wt) and the
pacCc14 and palA1 mutants after
fructose-grown biomass was transferred to nonbuffered inducing
conditions (1% D-xylose) for 1, 2, 4, and 6 h.
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DISCUSSION |
An A. nidulans 4.3-kb EcoRI genomic DNA
fragment subcloned in pXDE1 harbors a functional -xylosidase gene
(designated xlnD) since A. nidulans multicopy
transformants exhibit significant -xylosidase overexpression (10- to
100-fold greater expression) compared to the wild type. Western blot
analysis performed with a polyclonal antibody raised against the
A. nidulans 85-kDa -xylosidase revealed elevated levels
of production of this enzyme in overexpressing transformants. Taken
together, these data show that the cloned sequences present in pXDE1
encode the previously characterized (29) 85-kDa
-xylosidase.
The nucleotide sequence of xlnD has been determined. The
coding region of the gene consists of an uninterrupted 2,406-bp open reading frame which encodes an 802-amino-acid protein. The deduced molecular mass of the mature protein (85.3 kDa) corresponds closely to
the molecular mass previously determined for the purified A. nidulans -xylosidase (29). This implies that
although a number of potential N-glycosylation sites occur within the
primary structure, the protein is either not glycosylated to a high
degree or not glycosylated at all. The vast majority of enzymic
activity (>90%) appears to be cell wall associated (data not shown).
The basis of this association might be a consequence of the enzyme's
molecular size since the purified activity has been found to be a dimer (29) and this might result in its capture within the cell
wall, a situation analogous to the situation which has been described for the A. niger glucose oxidase (48). The
deduced primary structure of the cloned -xylosidase exhibits a high
degree of similarity to the primary structures of other previously
characterized fungal -xylosidases (Fig. 2), confirming that the
cloned gene encodes the A. nidulans 85-kDa -xylosidase.
As in A. niger, expression of xlnD is
specifically induced by oat spelt xylan, as well as by
D-xylose (46). Transcription of xlnD
does not appear to be influenced by the external pH. No significant
differences were found in xlnD transcript levels after D-xylose induction at pH 4, 6, and 8. This finding is
consistent with the expression data obtained with pH regulatory
mutants, although the level of transcription in the
pacCc14 alkaline mimic mutant seems to be
somewhat lower than the level of transcription in the wild type. In
contrast, xlnD expression is subject to carbon catabolite
repression by D-glucose, indicating that the glucose
repression of -xylosidase activity observed previously
(29) occurs at the level of mRNA transcription; a transcript
is observed upon induction by D-xylose but not in the presence of D-glucose. xlnD is, however,
transcribed in the presence of D-glucose in the
creAd30 mutant, from which it can be concluded
that carbon catabolite repression of the gene is, at least in part,
controlled by CreA. Carbon catabolite repression mediated by CreA has
also been observed in other fungal genes encoding xylanolytic enzymes
(10, 31, 36). Nine putative CreA binding sites are located
in the xlnD upstream sequences. Deletion analysis of these
sites is in progress in order to determine their in vivo function.
| |
ACKNOWLEDGMENTS |
We thank H. N. Arst, Jr., and M. A. Peñalva for
kindly providing the A. nidulans mutant strains used in this
work and M. Penttilä for providing the revised sequence of the
T. reesei -xylosidase.
This work was supported by grant BIOTECH BIO2-CT93-0174 from D.G. XII
of the European Commission and by grant CICYT ALI93-0809 from the
Spanish Government. A.P.M. was the recipient of EC Biotechnology Programme fellowship BIO2-CT94-8136.
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FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Biotecnología de los Alimentos, Instituto de
Agroquímica y Tecnología de los Alimentos, Apartado
Postal 73, 46100 Burjassot, Valencia, Spain. Phone: 34-6-3900022. Fax:
34-6-3636301. E-mail: dramon{at}iata.csic.es.
Deceased 9 August 1997.
| |
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Appl Environ Microbiol, April 1998, p. 1412-1419, Vol. 64, No. 4
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
