(Received for publication, September 11, 1996, and in revised form, November 6, 1996)
From the Section of Microbial Biochemistry, Institute of Biochemical Technology and Microbiology, University of Technology of Vienna, Getreidemarkt 9/172-5, A-1060 Wien, Austria
Two genes encoding trehalose-6-phosphate synthase
were cloned from Aspergillus niger. tpsA was cloned using
the Saccharomyces cerevisiae GGS1/TPS1 gene as a probe. It
encodes a 517-amino acid polypeptide with 64-70% similarity to
trehalose-6-phosphate synthase of S. cerevisiae,
Kluyveromyces lactis, and Schizosaccharomyces pombe. Its transcription occurs constitutively and is enhanced on
carbon-derepressing carbon sources, coinciding with the presence of a
CreA-binding nucleotide motif in the 5-noncoding region of
tpsA. Disruption of tpsA only weakly reduces
growth on glucose, and neither influences the glucose induction of a
low affinity glucose permease nor interferes with the catabolite
repression of a pectinase; it causes reduced the heat tolerance of
conidia. tpsB was cloned by a polymerase chain
reaction-based strategy. Its 480 amino acid sequence showed 76.5%
identity to tpsA. Its transcription was hardly detectable
at ambient temperatures but was enhanced strongly upon heat shock,
which agrees with the presence of several copies of a C4T
stress-responsive element in its 5
-upstream sequences. Hence the
function of yeast GGS1/TPS1 has been split into two
differentially regulated genes in A. niger, of which none
appears to be involved in glucose sensing.
Trehalose (-glucosido-1,1-glucose) is a nonreducing
disaccharide found in such diverse organisms as bacteria, fungi, algae, plants, invertebrates, and insects (1). In fungi, trehalose is
accumulated by mycelia during the stationary phase and by conidia, and
it is metabolized rapidly once growth is resumed or germination initiated. For a long time, therefore, trehalose has been considered a
reserve carbohydrate (2). However, an additional role of trehalose as a
protectant against various conditions of physical stress such as heat,
dehydration, and hyperosmotic shock has emerged more recently
(3-5).
The biosynthesis of trehalose from UDP-glucose and glucose 6-phosphate involves two enzymatic steps catalyzed by a multienzyme complex (6). In the yeast Saccharomyces cerevisiae, at least three proteins participate in this complex: trehalose-6-phosphate synthase (EC 2.4.1.15), trehalose-6-phosphate phosphatase (EC 3.1.3.12), and a regulatory protein (7). Interestingly, S. cerevisiae and Kluyveromyces lactis mutants in trehalose-6-phosphate synthase exhibit a pleiotropic phenotype; they are unable to grow on glucose or other rapidly fermentable sugars, exhibit strongly reduced sporulation, and display improper control of glucose-induced signaling pathways (8). Thevelein and co-workers (8) proposed that the trehalose-6-phosphate synthase protein may act as a component of a "glucose sensor" that transduces the glucose signal to the various signaling pathways. However, trehalose-6-phosphate synthase does not have such a role in the yeast Schizosaccharomyces pombe (9), and these authors suggest that the effects observed in the GGS1/TPS1/tps1 mutants are the result of a different susceptibility of hexokinase to inhibition by trehalose-6-phosphate in S. pombe and S. cerevisiae (9), which in S. cerevisiae GGS1/TPS1 mutants would lead to an unrestricted flux through hexokinase and consequently inhibition of growth.
Aspergillus niger is a filamentous fungus that has been used for decades for the industrial production of enzymes and organic acids (10, 11). Both product accumulations are strongly, yet divergently, influenced by the presence of glucose (12-14). Several enzymes have been shown to take part in the control of glycolysis in A. niger (15), but under citric acid-producing conditions a major control point occurs at the level of hexokinase (16). Interestingly, the hexokinase of A. niger is inhibited only weakly by trehalose-6-phosphate (17). We have therefore investigated the role of trehalose-6-phosphate synthase in the growth of and glucose metabolism by A. niger.
A. niger ATCC 11414 was used throughout this study. Conditions for strain maintenance and preparation of conidia were described previously (18). Growth tests were performed on minimal medium (19), containing 2.0% (w/v) agar without biotin. Liquid cultures of the fungus were grown at 30 °C in 1-liter flasks containing 400 ml of complete medium, consisting of minimal medium plus 0.5% (w/v) yeast extract and 0.5% (w/v) peptone, and a carbon source (3% (w/v)). Flasks were inoculated with 2 × 108 conidia and incubated on a rotary shaker at 250 rpm for 14-18 h.
To induce heat stress response, A. niger cultures were pregrown in complete medium for 18 h, and aliquots of 20 ml of culture broth in 100-ml flasks were then transferred to a shaking water bath and incubated at 40 °C for 60 min and 120 rpm.
Escherichia coli strain DH5a was used for propagation of plasmids and grown under standard conditions (20).
Plasmids and VectorsA plasmid containing a 1.9-kb1 BamHI/XbaI fragment of the S. cerevisiae GGS1/TPS1 gene locus cloned into YEPlac 181 (8) was obtained from J. Thevelein, Leuven, Belgium. pAN7-1 (21) was obtained from P. Punt, Rijswijk, The Netherlands.
The vectors pGEM-5fZ(+), pGEM-7fZ(+) (Promega, Madison, WI) and pBluescript SK+ (Stratagene) were used for cloning.
Cloning of the tpsA GeneA BamHI/XbaI fragment of GGS1/TPS1 was labeled with [32P]dCTP by random priming and used to screen 40,000 plaques of the genomic library. Hybridization was carried out at 60 °C. The final washing was done in 2 ×SSC, 0.1% SDS at the same temperature. Inserts of hybridizing clones were analyzed by restriction mapping and subcloned.
Cloning of the tpsB GeneUsing sequence similarities of the
GGS1/TPS1/tps1 genes from S. cerevisiae (8),
K. lactis (22), S. pombe (9), and A. niger
tpsA (see "Results") two degenerated primers were designed for
amplification: ggsdegI (5-GGGTNCAYGAYTAYCAYYTNATG-3
) and ggsdegII
(5
-GNGGNACNCCYTTDATRTARTC-3
). PCRs were performed in a total volume
of 50 µl containing 10 mM Tris-HCl, pH 8.8, 50 mM KCl, 4 mM MgCl2, 1% Triton
X-100, 50 ng of genomic DNA, 25 pmol of each primer, 200 µM dNTPs, and 0.5 units of Taq polymerase (Biomedica). The program for amplification consisted of a 1-min incubation at 95 °C followed by 35 cycles of 1 min at 95 °C, 1.5 min at 55 °C and 1 min at 72 °C and was terminated by a final extension for 7 min at 72 °C. Protruding 3
-termini of PCR products were removed with Vent polymerase (New England Biolabs) according to
the manufacturer's instructions and were analyzed by restriction cleavage with TaqI. A 450-bp fragment, verified as a
fragment of tpsB by sequencing, was consequently used as a
probe to screen 40,000 plaques of the genomic library. Hybridization
was done at 64 °C. The final washing was performed in 0.2 × SSC, 0.1% SDS at the same temperature. Appropriate fragments of
hybridizing clones were subloned, subjected to restriction mapping, and
sequenced.
Sequencing was performed with the Sequenase version 2.0 system (U. S. Biochemical Corp.) and by means of an automatic sequencer (Applied Biosystems), using both universal primers as well as a series of primers specific for both strands of the tpsA locus. Sequence similarities were investigated using the BLAST server (23). Alignments were performed using the program MACAW (24) and improved by visual inspection.
Gene DisruptionA 4.5-kb NdeI/NotI
fragment of the tpsA locus containing the whole
tpsA structural gene and 2.3 kb and 0.38 kb of its 5- and
3
-noncoding sequences, respectively, was cloned into pBluescript SK+
(previously cut with SalI and NotI) to yield
pGARP10. To disrupt the tpsA locus of A. niger, a
2.6-kb fragment of the E. coli hph gene flanked by the
Aspergillus nidulans gpdA promoter and the trpC terminator was released from pAN7-1 (21) by
restriction cleavage with SacI and HindIII.
Protruding 5
- and 3
-termini were filled or removed, respectively,
with Klenow enzyme, and the fragment was ligated into the unique
EcoRV site of pGARP10, located within the tpsA
coding sequences (395 bp) to yield pGARP11 (see Fig. 4A).
The NotI/XhoI insert of pGARP11 was introduced into A. niger protoplasts by transformation as described by
Yelton et al. (25). They were regenerated in liquid medium
for 3 h at 30 °C containing 2% (w/v) glycerol, 0.5% (w/v)
yeast extract, and 0.5% (w/v) peptone for 3 h at 30 °C
followed by plating on solid minimal medium containing 300 µg/ml
hygromycin B (Calbiochem) and glycerol as a carbon source.
Standard Molecular Biological Techniques
Plasmid
constructions, E. coli transformation, plasmid isolations
from E. coli, restriction enzyme cleavage, isolation of chromosomal DNA, Southern and Northern blotting, preparation of 32P-labeled DNA probes, hybridization of blots, and
construction of the gene library in EMBL3 were performed according
to standard protocols (20). Isolation of DNA fragments from agarose
gels was performed with a Quiaex II kit (Quiagen) according to the manufacturer's protocols. RNA was isolated as described by Chomczynski and Sacchi (26) Poly(A)+ RNA was isolated from total RNA
using the FastTrack 2.0 system (Invitrogen, San Diego, CA) according to
the manufacturer's instructions.
A
ScaI/HpaII fragment of the 5-noncoding sequences
of tpsA was prepared and radioactively labeled with
[32P]dCTP and appropriate dNTPs by filling in with the
Klenow fragment of DNA polymerase. A CreA::GST fusion protein
was obtained by overexpression of a NcoI fragment (from
nucleotides 103-722) of the A. nidulans creA gene (27)
containing the two C2H2 zinc fingers and an
alanine-rich region as a GST fusion in E. coli, contained in
vector pGEX-CreA, following the expression and purification protocol of
Kulmburg et al. (28). Polyacrylamide gel shifts and
methylation protection footprinting were performed as described previously (29).
Cell-free extracts were prepared by grinding mycelium in liquid nitrogen, suspending the powder in 20 mM HEPES, pH 7.1, containing 20% (w/v) glycerol, 2 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, and centrifugation (15 min, 5,000 × g, 4 °C). Trehalose-6-phosphate synthase was measured as described by Cabib and Leloir (6) with the modifications introduced by Vandercammen et al. (30). Assay of glucose transport was carried out as described previously (32). Protein concentration was determined by the dye binding method (31).
Determination of TrehaloseMycelia were thereafter harvested by filtration, ground under liquid nitrogen, and trehalose extracted by their suspension in distilled water at 95 °C for 30 min. After centrifugation (15 min, 12,000 × g, 18 °C), the extracts were passed through a 0.45-µm filter. Trehalose was determined using the method in Ref. 33 except that commercial porcine kidney trehalase (Sigma) was used.
Electrophoretic TechniquesFor demonstration of individual pectinases in the supernatant, samples from the culture broth were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting to nitrocellulose and immunological detection as described previously (34). A polyclonal antiserum was used to detect pectinase PL II (35).
To clone the A. niger equivalent of GGS1/TPS1 we screened a genomic
EMBL3 library of A. niger by heterologous hybridization with the S. cerevisiae GGS1/TPS1 gene and obtained seven
positive clones. Restriction mapping and Southern hybridization showed that all clones were part of the same gene locus (data not shown). Hybridizing DNA fragments were subcloned into pGEM-5Zf(+) and sequenced. The clones revealed the presence of an ORF of 1,825 bp (Fig.
1), which was interrupted by four introns (identified by
the presence of consensus sequences of splicing signals for filamentous
fungi; (36)). High stringency Southern hybridization of A. niger genomic DNA cleaved with XhoI, which does not cut within the gene, with a tpsA-specific probe yielded a single
band, suggesting that tpsA occurs as a single copy in the
A. niger genome. The predicted 517-amino acid sequence shows
high identity to the GGS1/TPS1 gene products from S. cerevisiae (64.0%), K. lactis (64.3%), and S. pombe (69.5%).
The 5-noncoding region of tpsA contains two putative CreA
binding sites (consensus 5
-SYGGRG-3
; (28, 37)) at positions
169 and
177, organized as a tandem repeat. Binding sites for the yeast carbon
catabolite repressor protein MIG1, whose DNA binding domain shows high
similarity to that of A. nidulans and A. niger
CreA (27, 38), are also found in the promoter of GGS1/TPS1
from S. cerevisiae (8) and K. lactis (22). Using a CreA::GST fusion protein, binding to a tpsA
restriction fragment resembling bp
58 to
265
(ScaI-HpaII) was shown by gel retardation assays
and by methylation protection footprinting in vitro (Fig. 2). To investigate whether transcription of
tpsA was indeed regulated by carbon catabolite repression,
Northern analysis of mRNA, harvested from A. niger grown
on various carbon sources, was carried out (Fig.
3A). tpsA expression was observed
on all carbon sources tested, but the ratio between the tpsA
and the actA (control) transcripts was higher during growth
on citrate and arabinose than on glucose and lactose, which would be
consistent with partial regulation by carbon catabolite.
Transcription of the S. cerevisiae and the S. pombe
GGS1/TPS1 is known to be inducible by heat stress (8, 9). We did not find any of the consensus sequences for binding of heat
shock-regulating proteins in the tpsA promoter. However, a
single copy of a C4T motif (consensus core sequence CCCCT),
which mediates transcription in response to various conditions of
stress in S. cerevisiae (39), was present at 386. Northern
analysis of A. niger cultures subjected to 40 °C heat
shock showed that tpsA transcription declined immediately upon transfer to 40 °C (Fig. 3B). We conclude that
tpsA transcription is not triggered by heat shock. Sequences
resembling the consensus for binding of yeast GCR1 (CCTTC (40)) were
found at
249 and
457, but those for binding of other activators of
glycolytic genes were not found.
To prove that tpsA
encodes a trehalose-6-phosphate synthase of A. niger and to
study its function in this fungus, we replaced it with a
tpsA derivative into which the E. coli hph gene
from pAN7-1 had been inserted by transformation (Fig.
4A). Transformants, in which the
tpsA gene had been replaced by the disrupted gene, were
identified by the presence of a 9.6-kb DNA fragment, which replaced the
wild type 7.0-kb band (Fig. 4B). One of these, strain A. niger tpsA1-3, was selected for further analysis.
When grown at 30 °C on glucose or glycerol its trehalose-6-phosphate
synthase activity was below the detection limit (Fig.
5), which proves that the gene encodes
trehalose-6-phosphate synthase. Interestingly, the mycelial trehalose
content at 30 °C was reduced strongly compared with the parental
strain, but it was not depleted completely. In view of the clear
reduction of trehalose-6-phosphate synthase activity, this suggests the
presence of alternative enzymes/pathways for trehalose biosynthesis in
A. niger. This assumption was supported strongly by the
findings that very high trehalose-6-phosphate synthase activity was
observed in the disruptant strain upon growth of A. niger at
40 °C, which was virtually similar to that of the parent strain
where it represented a 6- and 15-fold increase (on glucose and glycerol
as carbon source, respectively). This was also reflected in drastically
increased mycelial trehalose concentrations. Interestingly, although
this trehalose-6-phosphate synthase activity was twice as high on
glycerol as the carbon source, the trehalose content on this carbon
source was 30-40% lower, which may be due to a lower
glucose-6-phosphate supply and/or a different regulation of
trehalose-6-phosphatase by glycerol and glucose.
Disruption of GGS1/TPS1 in S. cerevisiae and
K. lactis results in an impairment of growth on glucose,
fructose, and other rapidly fermentable sugars (8, 22). To see whether
there was a similar phenotype in A. niger tpsA1-3, the
wild type as well as the disruptant were pregrown for 24 h at
30 °C on solid minimal medium under derepressing conditions (0.1%
glycerol) and then transferred to fresh medium containing different
concentrations of glucose; the increase in colony diameter was measured
over a period of 5 days. Virtually the same growth rate on glucose was
observed with the wild type strain and strain tpsA
1-3 at 30 °C,
but the disruptant grew slightly lower at 37 and at 40 °C. Similar
results were also obtained with glycerol as a carbon source. No
significant differences in the morphology of the growing hyphae were
observed between the parent strain and the disruptant at either
temperature.
In contrast to S. cerevisiae and K. lactis, the
only effect of disruption of tsp1 in S. pombe was
a failure to germinate in a medium with glucose as a carbon source (9).
A. niger tpsA1-3 showed no retardation or even
deficiency in germination on glucose, however (data not shown).
The data described above suggest a role of trehalose-6-phosphate
synthase A for mycelial growth at higher temperature. To study the
possibility that the reduced trehalose content in A. niger
tpsA1-3 also affects its viability at temperatures where growth no
longer takes place, we incubated conidiospores of the wild type and the
disruptant strain at 50 and 55 °C, respectively, and thereafter
analyzed their ability to germinate at 30 °C. At 50 °C
germination of the disruptant exhibited a 27% reduction compared with
the parent strain (94 versus 70% of the control), and this
effect was even more dramatic at 55 °C (7.4 versus 2.6% of the control; i.e. 32%). Considering the fact that the
trehalose content in A. niger tpsA
1-3 is reduced by
56%, these data support the assumption that trehalose contributes to
the heat stability of A. niger conidiospores.
To learn whether the functional impairment of tpsA leads to
glucose derepression (as has been shown for yeast invertase and -glucosidase (8)), A. niger tpsA
1-3 and its parent
strain were grown on glucose as a carbon source, and the formation of pectinase A (which is subject to glucose repression (35)) was determined by Western blotting and immunostaining. Under these conditions, pectinase A remained below the limit of detection in both
strains, whereas it was demonstrated clearly during growth on pectin
(data not shown). We conclude that a disruption of tpsA in
A. niger does not lead to carbon catabolite
derepression.
A. niger has been shown to respond to the presence of elevated concentrations of glucose or sucrose (>5%, w/v), by increasing the glycolytic flux and citric acid accumulation (14, 18). This effect is, among others, reflected in the induction of a low affinity glucose permease by elevated glucose concentrations (32). However, in the presence of 10% glucose the low affinity transporter was formed in the wild type as well as the disruptant strain, and both its Km (4.1 mM) as well as Vmax (0.14 µmol/min/mg dry weight) were virtually the same. We therefore conclude that tpsA is not involved in the signaling of high glucose concentrations to A. niger.
Cloning and Transcriptional Analysis of the A. niger tpsB GeneThe fact that A. niger tpsA was only expressed
constitutively, yet the disruptant strain still contained high
trehalose-6-phosphate synthase activity at 40 °C, suggested the
presence of a second, heat-inducible trehalose-6-phosphate
synthase-encoding gene in A. niger. Using the predicted
protein sequences of the three GGS1/TPS1 genes from S. cerevisiae, K. lactis, and S. pombe as well as of tpsA, a pair of degenerated oligonucleotide primers was
designed which corresponded to the nucleotide sequences encoding amino acids 149-156 and 190-198 in the tpsA polypeptide. PCR
amplified a single nucleotide fragment of 0.5 kb. Restriction fragment
length polymorphism analysis with HinfI and TaqI
showed that this fragment corresponded to two different amplicons and
that the restriction fragments of one of them were in accordance with
those predicted from the tpsA sequence. The other amplicon
was unique. Using a 450-bp fragment of this amplicon as a probe, the
A. niger library was screened under stringent conditions and
three positive clones were obtained. Subcloning and sequencing showed
that the tpsB gene is contained in an ORF of 1,732 bp (Fig.
6). Four putative introns occur at the same relative
position as in tpsA. The predicted 480-amino acid sequence
shows 76.5% identity to tpsA, and 64.0, 64.0, and 65.1%
identity to the GGS1/TPS1 gene products from S. cerevisiae, K. lactis, and S. pombe,
respectively (Fig. 7).
In contrast to tpsA, the 5-nontranscribed sequences of
tspB did not contain nucleotide sequences homologous to CreA
binding sites. In analogy with tpsA, sequences resembling
the consensus for binding of yeast GCR1 (CCTTC (40)) were also present
at
17,
152,
300,
375, and
412. Interestingly, the
C4T motif identified in tpsA occurred in five
copies in tpsB, i.e. at
14,
103,
224,
230, and
343, respectively. Probing the Northern blots of Fig. 3
with the tpsB gene revealed only a low level of transcript
at 30 °C, whose level was even lower on arabinose and citrate, but
the transcript increased strongly upon transfer to 40 °C (Fig.
8). These findings are consistent with the assumption that tpsB encodes the heat-induced trehalose-6-phosphate
synthase activity of A. niger.
We have isolated from A. niger two different genes encoding trehalose-6-phosphate synthase (tpsA and tpsB), as indicated by the fact that the genes have roughly 65% identity at the amino acid level with the GGS1/TPS1 genes of S. cerevisiae (8) and K. lactis (22) and the TPS1 gene of S. pombe (9). Furthermore, disruption of tpsA virtually reduces the trehalose-6-phosphate synthase activity under conditions where this gene is selectively expressed (e.g. growth on glucose as a carbon source) to zero.
Since tpsA is expressed more strongly during vegetative growth on a variety of carbon sources at ambient temperatures than tpsB we considered it a more likely candidate for exerting a regulatory effect on glucose metabolism similar to that of GGS1/TPS1 in yeast. The phenotype of tpsA gene disruption in A. niger, however, was quite different from that produced by the disruption of the GGS1/TPS1 genes in S. cerevisiae and K. lactis and resembled that observed in S. pombe in some aspects. In agreement with the latter but in contrast to S. cerevisiae and K. lactis, a disruption of tpsA did not influence the capacity of A. niger to grow on glucose. Blázquez et al. (9) attributed this difference between S. cerevisiae (and K. lactis) and S. pombe to the different behavior of the hexokinases of these three yeasts toward trehalose 6-phosphate, a strong inhibitor of S. cerevisiae and K. lactis hexokinases, but not that of S. pombe (9). Such an explanation also may be valid for A. niger, whose hexokinase has a rather low Ki for trehalose 6-phosphate (17) and is virtually not inhibited by glucose 6-phosphate (41). The low Ki for trehalose 6-phosphate is reflected by the findings that disruption of tpsA leads to an increase in glycolytic flux at very high sugar concentrations (>5%, w/v) only, apparently because an intracellular concentration of trehalose 6-phosphate, sufficient for inhibiting hexokinase, can only be accumulated under these conditions (17). All of these findings argue against a regulatory role of tspA in A. niger glycolysis during vegetative growth on conventional media containing low carbon source concentrations.
Trehalose has been attributed a dual role as a reserve carbohydrate (2) and as a protectant of proteins against denaturation by dehydration (5). Consistent with the first role, disruption of tps1 in S. pombe prevented germination of spores (9). In contrast, disruption of tpsA in A. niger had no effect on the germination of conidia. These differences may be because disruption of tpsA does not produce a null phenotype of trehalose-6-phosphate synthase activity because of the presence of tpsB. Interestingly, tpsA disruption and the accompanying reduction of the conidial trehalose concentration lead to a reduction in the heat stability of the conidia. This suggests that tpsA contributes to trehalose formation during conidiation and that its lack cannot be fully compensated by tpsB.
In contrast to tpsA, the tpsB transcript was
hardly detectable during vegetative growth of A. niger but
accumulated strongly during heat shock. A similar behavior has been
described for S. cerevisiae GGS1/TPS1 (8) and S. pombe
tps1 (9) gene expression. This heat shock-triggered gene
expression is consistent with the presence of an element with the
sequence AAGGGGAT in the 5-noncoding sequences of GGS1/TPS1
(8), which confers regulation of certain S. cerevisiae genes
by heat shock, nitrogen starvation, and the RAS-cAMP pathway (39, 42).
Several copies of this motif are also present in the 5
-upstream
sequences of tpsB, and it is tempting to speculate that this
motif confers regulation by heat shock also in A. niger.
However, as the functionality of this motif in Aspergillus
and other filamentous fungi has not yet been demonstrated, this
assumption must still be treated cautiously. The fact that a single
AAGGGGAT motif also occurs in the 5
-upstream regions of
tpsA, whose expression is not triggered by heat shock,
indicates that its mere presence in A. niger does not imply
functionality and suggests at least that they may be subject to
position effects. Kobayashi and McEntee (42) reported that a single
copy of the C4T motif is active in S. cerevisiae, yet the presence of two such motifs evokes a more than
additive response.
We have shown here for the first time that the biological function of a gene that is present in a single copy in several yeasts (S. cerevisiae, K. lactis, S. pombe) is fulfilled by two genes in the filamentous fungus A. niger, which encode highly similar polypeptides but are regulated differently. A comparison of the amino acid sequences of the tpsA and tpsB gene products does not provide a clue as to the reason for this. Interestingly, while this paper was prepared for publication, Borgia et al. (43) reported on the presence of two trehalose-6-phosphate phosphatases in A. nidulans but did not investigate whether the two gene products may have different functions. It will be interesting to see if this is a general feature of multicellular fungi or a feature unique to aspergilli.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U07184[GenBank] (Fig. 1) and U63416[GenBank] (Fig. 6).
We are grateful to Dr. Johan Thevelein, Luyven, Belgium, for interest in our study, for the generous gift of GGS1/TPS1, and for disclosing his papers prior to publication. We also thank B. Felenbok, Orsay, France, for the gift of pGEX-CreA and Jaap Visser, Wageningen, Netherlands, for the provision of a polyclonal antiserum against PL II. /