Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans

Sabine Fillinger1, Marie-Kim Chaveroche1, Patrick van Dijck2, Ronald de Vries3, George Ruijter3, Johan Thevelein2 and Christophe d’Enfert1

Unité Microbiologie et Environnement, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Flanders Interuniversity Institute for Biotechnology, VIB and Laboratory of Molecular Cell Biology, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium2
Molecular Genetics of Industrial Micro-organisms, Wageningen University, Dreijenlaan 2, 6703HA Wageningen, The Netherlands3

Author for correspondence: Christophe d’Enfert. Tel: +33 1 40 61 32 57. Fax: +33 1 45 68 87 90. e-mail: denfert{at}pasteur.fr


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Trehalose is a non-reducing disaccharide found at high concentrations in Aspergillus nidulans conidia and rapidly degraded upon induction of conidial germination. Furthermore, trehalose is accumulated in response to a heat shock or to an oxidative shock. The authors have characterized the A. nidulans tpsA gene encoding trehalose-6-phosphate synthase, which catalyses the first step in trehalose biosynthesis. Expression of tpsA in a Saccharomyces cerevisiae tps1 mutant revealed that the tpsA gene product is a functional equivalent of the yeast Tps1 trehalose-6-phosphate synthase. The A. nidulans tpsA-null mutant does not produce trehalose during conidiation or in response to various stress conditions. While germlings of the tpsA mutant show an increased sensitivity to moderate stress conditions (growth at 45 °C or in the presence of 2 mM H2O2), they display a response to severe stress (60 min at 50 °C or in the presence of 100 mM H2O2) similar to that of wild-type germlings. Furthermore, conidia of the tpsA mutant show a rapid loss of viability upon storage. These results are consistent with a role of trehalose in the acquisition of stress tolerance. Inactivation of the tpsA gene also results in increased steady-state levels of sugar phosphates but does not prevent growth on rapidly metabolizable carbon sources (glucose, fructose) as seen in Saccharomyces cerevisiae. This suggests that trehalose 6-phosphate is a physiological inhibitor of hexokinase but that this control is not essential for proper glycolytic flux in A. nidulans. Interestingly, tpsA transcription is not induced in response to heat shock or during conidiation, indicating that trehalose accumulation is probably due to a post-translational activation process of the trehalose 6-phosphate synthase.

Keywords: trehalose 6-phosphate synthase, spore germination, glycolysis, heat stress, oxidative stress, hexokinase

Abbreviations: EST, expressed sequence tag; 5-FOA, 5-fluoro-orotic acid; T6P, trehalose 6-phosphate; T6PP, trehalose-6-phosphate phosphatase; T6PS, trehalose-6-phosphate synthase

The GenBank accession number for the sequence reported in this paper is AF043230.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Trehalose ({alpha}-D-glucopyranosyl-{alpha}-D-glucopyranoside) is a non-reducing disaccharide found in a wide variety of organisms (bacteria, fungi, protozoa, plants). In lower eukaryotes, it constitutes up to 15% of the dry weight of stationary-phase cells or spores (Elbein, 1974 ). The universal mobilization of trehalose during growth resumption from resting stages supports a role as a storage carbohydrate (Arguelles, 2000 ; Thevelein, 1984 , 1996 ). However, because of a number of physico-chemical properties, including high hydrophilicity and chemical stability, non-hygroscopic glass formation and the absence of internal hydrogen bond formation, trehalose is apparently able to serve a unique role as stress metabolite (Arguelles, 2000 ; Thevelein, 1996 ).

In the yeast Saccharomyces cerevisiae, biosynthesis of trehalose is mediated by a multi-protein complex that contains a trehalose-6-phosphate synthase (T6PS; Fig. 1) encoded by TPS1 and a trehalose-6-phosphate phosphatase (T6PP; Fig. 1) encoded by TPS2 (Bell et al., 1992 , 1998 ; de Virgilio et al., 1993 ). This multi-protein complex contains regulatory subunits, the products of the redundant TSL1 and TPS3 genes, which share a conserved amino-terminal domain with Tps1 and Tps2 (Bell et al., 1998 ; Reinders et al., 1997 ; Vuorio et al., 1993 ). Mobilization of the trehalose pool in response to various stimuli is mediated by a neutral trehalase encoded by NTH1 (Kopp et al., 1993 ) while the role of a second neutral trehalase encoded by NTH2 remains uncertain (Nwaka et al., 1995 ). Studies using mutants in the different genes of trehalose metabolism in S. cerevisiae and in other yeasts support the protective role of trehalose and more specifically a role in the acquisition of stress tolerance (Arguelles, 2000 ). However, additional functions have been proposed for trehalose 6-phosphate (T6P), which is the first intermediate in the biosynthesis of trehalose (Fig. 1). In particular, analysis of S. cerevisiae mutants impaired in the biosynthesis of T6P has indicated a role for T6P and the T6PS in the control of the influx of glucose into glycolysis (Blazquez et al., 1993 ; Bonini et al., 2000 ; Hohmann et al., 1996 ; Thevelein & Hohmann, 1995 ).



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Fig. 1. Trehalose metabolism in fungi. ScTps1 and AnTpsA, trehalose-6-phosphate synthase; ScTps2 and AnOrlA, trehalose-6-phosphate phosphatase; ScNth1, ScNth2 and AnTreB, neutral trehalase; Sc, S. cerevisiae; An, A. nidulans.

 
Several studies have been conducted to determine the role of trehalose and T6P in filamentous fungi but they have remained inconclusive. Two genes encoding T6PS have been identified in Aspergillus niger (Wolschek & Kubicek, 1997 ). However, disruption of only one of these genes has been achieved and therefore an unambiguous role for T6P or trehalose in filamentous fungi could not be deduced from this study (Wolschek & Kubicek, 1997 ). Arisan-Atac et al. (1996) have nevertheless shown that T6P participates in the control of citrate production in A. niger. In A. nidulans, T6PP is encoded by the orlA gene, the inactivation of which results in an osmoremediable thermosensitive growth phenotype that can be explained by an inhibitory role of T6P in the control of chitin biosynthesis (Borgia et al., 1996 ). However, orlA mutants produce normal levels of trehalose and are therefore not suited to determine the role of trehalose in filamentous fungi (Borgia et al., 1996 ). T6P is a potent inhibitor of hexokinase also in A. nidulans and A. niger, suggesting a role in the control of glycolytic flux in these species (Panneman et al., 1998 ; Ruijter et al., 1996 ). Finally, neutral trehalases have also been identified in filamentous fungi and a role in the control of trehalose mobilization has been demonstrated during growth resumption stages, in particular spore germination, in a manner similar to that observed in yeast (d’Enfert et al., 1999 ).

Interestingly, analysis of A. nidulans mutants devoid of neutral trehalase has suggested that trehalose has a minor role as a storage carbohydrate and could function in the protection of germinating conidia against thermal stress (d’Enfert et al., 1999 ).

Here, we report the characterization of the tpsA gene of A. nidulans, encoding a T6PS, and the construction of an A. nidulans tpsA-null mutant. This mutant fails to accumulate trehalose in response to a variety of stress conditions but this defect is not associated with an increased sensitivity to a short exposure of the same stress conditions. In contrast, this mutant is defective for growth at high temperature, shows an increased sensitivity to long exposure to sublethal doses of reactive oxygen species and has reduced spore viability, thus suggesting a role for trehalose in the resistance of A. nidulans to sustained exposure to various stress conditions, including starvation.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
A. nidulans strains FGSC28 (pabaA6 biA1) and FGSC773 (wA3; pyroA4; pyrG89) were obtained from the Fungal Genetics Stock Centre (University of Kansas, Kansas City, KA, USA). A. nidulans strains CEA150 (wA3; pyroA4; pyrG89; tpsA-AfpyrG-tpsA{Delta}) and CEA152 (wA3; pyroA4; pyrG89; tpsA{Delta}) are derivatives of FGSC773 obtained in the course of this study. Growth conditions for A. nidulans strains have been described (d’Enfert & Fontaine, 1997 ). H2O2 was added to solid culture media in a 0–5 mM range. Cultures of A. nidulans strains for the assay of heat or oxidative-shock sensitivity were inoculated at 2x107 conidia ml-1 and grown for 3 h at 30 °C in minimal glucose medium containing 0·01% Tween-20. Following heat shock (0–60 min at 50 °C) or oxidative shock (100 mM H2O2 for 0–60 min), an aliquot of each culture was withdrawn and serially diluted in PBS (150 mM NaCl, 10 mM sodium phosphate pH 7·2)/0·1% Tween-20. Heat- or H2O2-shocked cells were then plated on complete medium containing 0·1% Triton X-100 in order to limit the growth of the colonies. Colonies were counted after a 2 d incubation at 37 °C. The same cultures were used to measure trehalose content in the conidia with previously described procedures (d’Enfert & Fontaine, 1997 ). Alternatively, trehalose, T6P, sugar phosphates and ATP were measured using extracts from mycelia grown at 30 °C in minimal glucose medium for 4 h using the procedure of Ruijter & Visser (1996) . Conidium viability was monitored using duplicates of two independent stocks of FGSC773 and CEA152 conidia stored at either 4 °C or 20 °C in PBS/0·1% Tween-20. At different time intervals, an aliquot of each stock was serially diluted and plated in duplicate on complete medium containing 0·01% Triton X-100. Colonies were counted after a 2 d incubation at 37 °C. Conidiospore germination was monitored by microscopic examination of slides coated with minimal glucose medium and spot-inoculated with approximately 104 freshly harvested conidia. The percentage of germinated spores was recorded at different times.

The S. cerevisiae strains used in this study were W303.1A (Thomas & Rothstein, 1989 ; MATa leu2-3,112 ura3-1 trp1-92 his3-11,15 ade2-1 can1-100 GAL SUC mal) and the isogenic YSH290 strain containing the tps1{Delta} mutation (Neves et al., 1995 ). Yeast cells were routinely grown on a rotary shaker at 30 °C in yeast nitrogen base medium (YNB; Sherman, 1991 ) containing 2% glucose or 2% galactose (YSH290) as carbon source.

Escherichia coli strains PAP105 [{Delta}(lac-pro) F'(lacIq1 {Delta}(lacZ)M15 pro+ Tn10)] and DH5{alpha} (Woodcock et al., 1989 ) were used for plasmid propagation. The ß-lactam antibiotic carbenicillin (100 µg ml-1) and tetracycline (15 µg ml-1) were added to the growth medium when required.

PCR amplification of a segment of the A. nidulans tpsA gene.
The genomic DNA of A. nidulans FGSC28 prepared according to Girardin et al. (1993) was used as template to amplify a segment of genes potentially encoding a T6PS. The sense and antisense primers (tps1F and tps1B, Table 1) were based on amino-acid sequences (WPLFHYH and DYIKGVP, respectively) conserved in several fungal T6PS (Bell et al., 1992 ; Blazquez et al., 1994 ; Luyten et al., 1993 ; Wolschek & Kubicek, 1997 ). The amplification protocol consisted of a denaturation step at 94 °C for 5 min followed by 35 cycles of the following steps: denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, extension at 72 °C for 2 min. A last elongation step was carried out at 72 °C for 10 min. An approximately 650 bp amplification product was gel purified and cloned in Bluescript SK+ (Stratagene) using standard cloning procedures. Two plasmids, pTPS1 and pTPS2, were obtained that carry the amplification product in opposite orientations.


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Table 1. Oligonucleotides used in this study

 
DNA and RNA manipulations.
General recombinant DNA techniques and Southern-blot analyses were essentially performed according to Sambrook et al. (1989) and Ausubel et al. (1992) . Transformation of calcium-manganese-treated E. coli was as described by Hanahan et al. (1991) . Oligonucleotides used in this study were obtained from Genset (Paris, France) and are listed in Table 1. A BamHI–EcoRV fragment corresponding to the cloned PCR product was labelled with [{alpha}-32P]dCTP using the Megaprime kit (Amersham) and used to probe a replica of a genomic library (a gift from C. Scazzochio) made in plasmid pFB39, a derivative of pUC18 carrying the A. nidulans argB gene. This screening yielded a single positive plasmid, pTPS4, which lacked the 3' end of the tpsA gene as deduced from DNA sequencing. To identify a clone carrying the entire tpsA gene, the chromosome-specific libraries of A. nidulans genomic DNA (Brody et al., 1991 ) that had been obtained from the Fungal Genetic Stock Center and transferred onto nylon membranes (ZetaProbe, Bio-Rad) were probed using a 400 bp EcoRI fragment located at the 3' end of the cloned tpsA region and labelled as above. The plasmids pTPS6 and pTPS7 are derivatives of pUC18 and pBLSN+ (d’Enfert, 1996 ), respectively, that carry a 1·75 kb SalI–SmaI fragment containing the region of tpsA carried by pTPS4. The plasmids pTPS11 and pTPS12 are derivatives of pBLSN+ which carry a 2·4 kb EcoRI fragment of cosmid L24E04 in opposite orientations. DNA sequencing was performed by the dideoxy chain termination method (Sanger et al., 1977 ) on double-stranded plasmids derived from pTPS6, pTPS7, pTPS11 and pTPS12 by internal restriction-enzyme-mediated deletions and using a set of appropriate oligonucleotide primers. The sequence of a 2699 bp fragment was read at least twice on each strand and is deposited in the GenBank database under accession number AF043230.

Plasmid pTPS13 was obtained by subcloning the 0·8 kb SalI–ClaI fragment of pTPS7 into SalI–ClaI-digested pTPS11{Delta}P, a derivative of pTPS11 with an internal deletion of a PstI fragment. Plasmid pTPS17 was then obtained by subcloning a KspI–EcoRV fragment carrying the A. fumigatus pyrG gene (Weidner et al., 1998 ) into KspI/SmaI-digested pTPS13. Plasmid pTPS17 was used to transform protoplasts of A. nidulans strains (Osmani et al., 1987 ). Genomic DNA from 15 prototrophic transformants was prepared according to Mol et al. (1998) and screened by PCR using primers tps10 and tps11 (Table 1). While a 2006 bp product is expected in transformants carrying an ectopic integration of pTPS17 or a tpsA{Delta}-AfpyrG-tpsA allele, a 1462 bp fragment is expected in transformants with a tpsA-pyrG-tpsA{Delta} allele. Putative merodiploids with a tpsA-AfpyrG-tpsA{Delta} allele were confirmed by Southern blot analysis of EcoRI/SalI-digested genomic DNA prepared according to Girardin et al. (1993) and probed with a 0·8 kb ClaI–SalI fragment of pTPS13 that had been labelled with the Rediprime labelling kit (Amersham). Washed membranes were exposed to X-omat films (Kodak). While strains carrying only a wild-type tpsA allele show a 1·15 kb hybridizing fragment, tpsA-AfpyrG-tpsA{Delta} merodiploids show 1·15 kb and 0·8 kb hybridizing fragments corresponding to the wild-type and mutant allele respectively. Conversion of the tpsA-AfpyrG-tpsA{Delta} allele to the tpsA{Delta} allele was obtained by plating conidia of strain CEA150 on minimal glucose plates containing 1 mg ml-1 5-fluoro-orotic acid (5-FOA), uridine, uracil and pyridoxine.HCl, thus promoting the excision of the A. fumigatus pyrG gene through recombination between the two tpsA alleles (d’Enfert & Fontaine, 1997 ). The nature of the tpsA allele in 5-FOA-resistant clones was checked by PCR using primers tps10 and tps11 (Table 1), which can discriminate between wild-type tpsA and tpsA{Delta}, and with primers tps4 and tps5 (Table 1), which yield only a 363 bp product if the wild-type tpsA gene is present. Excision of the A. fumigatus pyrG gene was confirmed by Southern blot analysis as described above. In this case a single EcoRI–SalI fragment could be detected corresponding to either the wild-type allele (1·15 kb) or the mutant tpsA{Delta} allele (0·8 kb).

Preparation of total RNA from conidia, germinating conidia, heat-shocked germinating conidia, mycelia and developing cultures of A. nidulans strain pabaA1 and FGSC773 has been previously reported (d’Enfert et al., 1999 ). RT-PCR experiments were achieved using the Reverse Transcription System according to the manufacturer’s instructions (Promega). Approximately 1 µg total RNA was used for each oligo-dT primed reverse transcription. An aliquot of the reaction was then subjected to the following amplification protocol using primers tps4 and tps5 (Table 1): a denaturation step at 93·5 °C for 5 min followed by 20 cycles of the following steps: denaturation at 93·5 °C for 30 s, annealing at 58 °C for 1 min, extension at 71 °C for 1 min. Amplification was limited to 20 or 25 cycles in order to remain in a linear range and therefore produce semi-quantitative data. The tps4 and tps5 oligonucleotides are sense and anti-sense primers, respectively, that are located on both sides of an intron in the tpsA gene. Therefore amplification from genomic DNA yields a 363 bp product while amplification from reverse-transcribed mRNA yields a 308 bp product. Alternatively, primers tps4 and tps9 were used that yield a 282 bp fragment corresponding only to reverse-transcribed mRNA because tps9 overlaps with an intron in tpsA.

Expression of tpsA in S. cerevisiae.
To obtain a cDNA encompassing the full tpsA ORF, total RNA prepared from the mycelium of a A. nidulans pabaA1 strain was reverse transcribed as described above. Reverse transcription products were then amplified using primers tps5'Bgl and tps3'Not (Table 1) and the following amplification procedure. A denaturation step at 93 °C for 5 min, 30 cycles of the following steps: denaturation at 93 °C for 30 s, annealing at 54 °C for 1 min, extension at 72 °C for 5 min and a final extension step of 10 min at 72 °C. The amplification product was subcloned using the TA cloning kit according to the supplier’s instructions (Invitrogen), yielding plasmid pTPS15. Following sequencing of the cloned tpsA cDNA, the BglII–NotI fragment of pTPS15 was subcloned into the yeast expression vector pCM190L (Llorente et al., 1999 ) cut by BamHI and NotI. Controlled expression in yeast is achieved by a tetracycline-repressible promoter. Furthermore, the protein is produced as a fusion with an HA-epitope and a (His)6 tail that allows quantification of protein production. Two independent recombinants, pTPS16-1 and pTPS16-2, were selected for transformation into the S. cerevisiae strain YSH290 along with pCM190L and pCM190L::X, a derivative of pCM190L carrying a S. cerevisiae ORF unlinked to trehalose metabolism. Yeast transformation was performed using the one-step method (Chen et al., 1992 ). Trehalose levels in the transformants were measured using cells that had been grown into stationary phase on galactose as described by Neves et al. (1994) .


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of the A. nidulans tpsA gene encoding a T6PS
To investigate the role of trehalose in A. nidulans, we set out to identify genes encoding T6PS using a two-step strategy. First, two degenerate primers, tps1F (sense primer; Table 1) and tps1B (antisense primer; Table 1), were designed that correspond to two regions conserved in the T6PS of yeast (Bell et al., 1992 ; Blazquez et al., 1994 ; Luyten et al., 1993 ) and in the A. niger TpsA T6PS (Wolschek & Kubicek, 1997 ). Using these primers, a 624 bp fragment was amplified by PCR from genomic DNA of A. nidulans strain FGSC28. The nucleotide sequence of this PCR product was determined and the deduced amino acid sequence revealed significant identity to corresponding regions of A. niger and S. cerevisiae T6PS (94·2% and 66·8% identical amino acids, respectively). These results suggested that the PCR product corresponded to an A. nidulans T6PS-encoding gene referred to as tpsA.

In a second step, the PCR product was used to probe different libraries of A. nidulans genomic DNA. Using chromosome-specific libraries (Brody et al., 1991 ), three positive cosmids, L09H05, L14G06 and L24E04, were identified. These cosmids have been assigned to the same region of A. nidulans chromosome V (Prade et al., 1997 ), suggesting that the tpsA gene is located on this chromosome.

Sequencing of the A. nidulans tpsA gene
DNA sequencing of an approximately 1·8 kb SalI–SmaI fragment derived from plasmid pTPS4 (see Methods) and of an approximately 1·2 kb EcoRI–PstI fragment derived from cosmid L24E04 yielded a nucleotide sequence of 2699 bp (GenBank accession number AF043230; data not shown). Analysis of this DNA sequence revealed an ORF of 1512 bp interrupted by four putative introns of 63, 55, 48 and 55 bp, respectively. The location of these introns was confirmed by sequencing a cDNA of tpsA obtained by amplification of reverse-transcribed mRNAs using primers tps5'Bgl and tps3'Not (Table 1). Furthermore, analysis of several A. nidulans-expressed sequence tags (c3e03, m7e02, c5f08) determined within the A. nidulans EST sequencing program (D. Kupfer & B. Roe, http://www.genome.ou.edu/fungal.html) confirmed the location of these introns and revealed the occurrence of an additional intron of 200 bp located in the 5'-untranslated region of the gene and extending from position -302 to -103 relative to the tpsA start codon (data not shown).

The A. nidulans 1512 bp ORF identified in the cloned DNA region encodes a 504 amino acid protein with a molecular mass of 56·8 kDa. This protein shares a minimum of 62·2% identical amino acids and 74·1% similar amino acids with known fungal T6PSs and its closest known homologue is the A. niger TpsA protein (87·4% identical amino acids and 90·1% similar amino acids; Fig. 2).



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Fig. 2. Alignments for maximal amino acid similarities of the A. nidulans TpsA protein (antpsA) with the S. cerevisiae (sctps1; Bell et al., 1992 ), K. lactis (klggs1; Luyten et al., 1993 ), Schiz. pombe (sptps1; Blazquez et al., 1994 ) and A. niger (AgtpsA and AgtpsB; Wolschek & Kubicek, 1997 ) T6PSs. This alignment was produced using the PILEUP program of the UWGCG package version 9 (Devereux et al., 1984 ). Conserved residues (identical, upper case letters; similar, lower case letters) in all six proteins have a black background.

 
Regulation of the tpsA gene
Expression of the A. nidulans tpsA gene under different culture conditions was monitored using semi-quantitative RT-PCR. Results presented in Fig. 3 show that transcription of the tpsA gene is induced during spore germination in rich medium (Fig. 3, lane 2), is maximal during exponential growth (Fig. 3, lanes 3–5 and lane 7) and decreases during the stationary phase of growth (Fig. 3, lane 6) and during conidiogenesis (Fig. 3, lanes 8–11). As a consequence, an RT-PCR product corresponding to the tpsA transcript could not be detected in non-germinating conidia of A. nidulans (Fig. 3, lane 1). When 4 h germlings of A. nidulans were exposed to a heat shock at 45 °C (Fig. 3, lanes 12–14) expression of tpsA was not altered.



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Fig. 3. Expression of the tpsA gene under different growth conditions. RNA was prepared from A. nidulans cultures grown under the following conditions: rich liquid medium for 0–64 h (samples 1–6); rich liquid medium for 16 h and transfer to rich solid medium for 0–24 h (samples 7–11); minimal glucose medium at 30 °C for 4 h and transfer to 45 °C for 10 and 30 min (samples 12–14). Total RNA was reverse transcribed and subjected to an amplification using oligonucleotides that yield a PCR product specific to the tpsA transcript. Amplification was reduced to 20 cycles in order to obtain semi-quantitative data.

 
Expression of the tpsA gene complements the various defects of a S. cerevisiae tps1{Delta} mutant
S. cerevisiae strains lacking the TPS1 gene are deficient in the synthesis of trehalose in response to various stress conditions and are unable to grow on glucose or other rapidly fermentable sugars because of an uncontrolled influx of sugar into glycolysis, causing rapid ATP depletion (Van Aelst et al., 1993 ). To test whether tpsA could functionally complement the various defects of a S. cerevisiae tps1{Delta} strain, the tpsA ORF was amplified and cloned into the yeast expression vector pCM190L (Llorente et al., 1999 ), yielding plasmids pTPS16-1 and pTPS16-2. After transformation of the plasmids into the yeast tps1{Delta} mutant, growth of the transformants on glucose and galactose media was monitored. Expression of the A. nidulans tpsA gene fully restored growth of the S. cerevisiae tps1{Delta} mutant on glucose (data not shown). Furthermore, trehalose production in the S. cerevisiae transformants expressing A. nidulans tpsA was restored to wild-type levels (Table 2). These results indicate that the A. nidulans TpsA protein can fulfil all of the functions of the yeast Tps1 T6PS, including the control of hexokinase function.


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Table 2. Trehalose levels in transformants of the S. cerevisiae tps1{Delta} strain

 
Disruption of the tpsA gene in A. nidulans
A pop-in/pop-out strategy was used to inactivate the tpsA gene. First, a strain carrying both the wild-type tpsA gene and a mutant allele separated by the A. fumigatus pyrG gene was constructed (CEA150: wA3 pyroA4 pyrG89 tpsA-pyrG-tpsA{Delta}) by transformation of A. nidulans strain FGSC773 with plasmid pTPS17. The tpsA{Delta} mutant allele removes 146 internal amino acids (90–235) in the 504 residue TpsA polypeptide, including stretches of amino acids that are highly conserved within fungal T6PS (Fig. 2). Excision of the A. fumigatus pyrG gene by recombination between the two tpsA alleles was then forced in the presence of 5-FOA. PCR analysis of random 5-FOA-resistant clones using primers tps10 and tps11 or tps4 and tps5 (Table 1), which yield products of different sizes according to the tpsA allele, revealed that segregation between the wild-type and mutant alleles occurred randomly under these conditions (data not shown). Interestingly, all segregants with a tpsA{Delta} allele were thermosenitive for growth while the CEA150 parental strain and tpsA+ progenies were not, suggesting that thermosensitive growth resulted from the inactivation of tpsA (see below). The occurrence of the tpsA{Delta} allele in two of the 5-FOA-resistant clones was confirmed by Southern analysis of EcoRI/SalI-digested genomic DNA. One of the clones carrying the tpsA{Delta} allele was designated CEA152. A comparison of strains FGSC773 (pyrG89 wA3 pyroA4) and CEA152 (pyrG89 wA3 pyroA4 tpsA{Delta}) is reported below.

tpsA is required for trehalose accumulation in response to various stress conditions
In A. nidulans, trehalose is known to accumulate during conidiogenesis (d’Enfert & Fontaine, 1997 ) as well as in response to heat shock (Noventa-Jordao et al., 1999 ). In other fungal species, trehalose has been shown to accumulate in the stationary phase of growth and in response to an oxidative or osmotic shock (Hounsa et al., 1998 ; Lewis et al., 1995 ; Lingappa & Sussman, 1959 ; Van Laere, 1989 ; Wiemken, 1990 ). Accumulation of trehalose was therefore monitored in wild-type and tpsA{Delta} conidia as well as in germinating conidia that were subjected to a heat shock or an oxidative shock. Trehalose could not be detected in mutant conidia (data not shown). When conidia of the wild-type strain FGSC773 were germinated for 3 h at 30 °C and subsequently subjected to a 50 °C heat shock, a rapid increase in trehalose levels was observed (Fig. 4a). Similarly, addition of 100 mM H2O2 to wild-type germlings resulted in trehalose accumulation, although to a lesser extent (Fig. 4a). Addition of 1 M NaCl did not result in a significant increase in trehalose levels (data not shown). In contrast to these results, neither heat shock nor 100 mM H2O2 resulted in trehalose accumulation in germlings of the tpsA{Delta} strain (Fig. 4a). Furthermore, T6P and trehalose were undetectable in the mycelium of a tpsA{Delta} strain (Table 3). We conclude that tpsA encodes a T6PS essential for biosynthesis of T6P and trehalose in A. nidulans under various conditions, including conidiogenesis, heat shock and oxidative shock.



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Fig. 4. A. nidulans tpsA{Delta} mutants are defective for trehalose accumulation in response to a heat or oxidative stress. (a) Trehalose accumulation in response to a heat shock (30 °C->50 °C) or an oxidative shock (100 mM H2O2) in germinating conidia of A. nidulans strains FGSC773 (wA3 pyroA4 pyrG89) ({blacksquare}, heat shock; {bullet}, oxidative shock) and CEA52 (wA3 pyroA4 pyrG89 tpsA{Delta}) ({square}, heat shock; {circ}, oxidative shock). Conidia were germinated for 3 h in minimal glucose medium at 30 °C and subjected to the shock for the indicated times. (b) Conidia survival following a heat shock or an oxidative shock in germinating conidia of A. nidulans tpsA+ and tpsA{Delta} strains. Duplicates of the samples analysed in (a) were diluted and plated on complete medium containing 0·01% Triton X-100. Colonies were counted after 2 d at 37 °C. Results are representative of two independent experiments. Symbols as in (a).

 

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Table 3. Metabolites accumulated in the wild-type and tpsA{Delta} strains during vegetative growth at 30 °C

 
Inactivation of tpsA does not increase sensitivity of germinating conidia to heat or oxidative shock
It has previously been shown that viability of wild-type conidia is significantly decreased following a heat shock and that the loss of viability can be prevented by accumulated trehalose (d’Enfert et al., 1999 ). This would suggest that the inability to accumulate trehalose in response to stress may further increase the stress sensitivity of germinating conidia. To test this hypothesis, the viability of germinating wild-type or tpsA{Delta} conidia that had been subjected to a 50 °C heat shock or a 100 mM H2O2 shock (see above) was monitored. Results in Fig. 4(b) show that wild-type and tpsA{Delta} germlings display a similar sensitivity to heat and oxidative shock independent of the ability to accumulate trehalose. It therefore appears that the rapid accumulation of trehalose in response to a heat or oxidative stress does not contribute significantly to cell survival during short-term exposure to a stress.

Inactivation of tpsA results in thermosensitive growth
To investigate the consequence of the inactivation of the tpsA gene in A. nidulans, we first compared the growth of strains FGSC773 (wild-type) and CEA152 (tpsA{Delta}) on various media and at different temperatures. The tpsA{Delta} mutant was unable to form colonies at temperatures above 44 °C when glucose (Fig. 5a) or fructose (data not shown) were used as a carbon source. Thermosensitive growth was also observed when glucose was replaced by glycerol (Fig. 5a) although to a lesser extent. The tpsA{Delta} mutant also showed reduced growth on media containing sublethal doses (1–2 mM) of H2O2 (Fig. 5a).



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Fig. 5. Inactivation of tpsA results in a thermosensitive growth defect. (a) Comparison of the growth of the tpsA{Delta} mutant (CEA152, wA3 pyroA4 pyrG89 tpsA{Delta}) and the wild-type strain (FGSC773, wA3 pyroA4 pyrG89) on minimal medium with 1% glucose or 1% glycerol as carbon source in the presence or absence of 2 mM H2O2. Incubation was for 48–64 h at 30 °C or 45 °C. (b) Germination frequency of the tpsA{Delta} mutant and the wild-type strain at 30 °C or 45 °C in glucose-containing minimal medium. Results are representative of two independent experiments (c) Microscopic examination (x570) of germinated spores after 24 h at 45 °C in liquid minimal medium with the indicated carbon sources.

 
Microscopic examination of conidia grown on minimal glucose medium revealed that the inability of strain CEA152 to establish a colony at temperatures above 44 °C was due to a block of conidia germination (Fig. 5b, c). Only a few spores had produced a germ tube after 24 h of growth at 44 °C (Fig. 5c). At lower temperatures, conidia of strain CEA152 were able to produce germ tubes at a frequency similar to that observed for the wild-type strain FGSC773, although with slower kinetics (Fig. 5b). When conidia were germinated on minimal glycerol medium at 45 °C germination was only partially delayed (Fig. 5c).

Results presented in Fig. 6 show that the thermosensitive growth defect of strain CEA152 became irreversible after prolonged incubation at the non-permissive temperature and was limited to the developmental stages extending from spore germination to early filamentous growth. Indeed, when conidia of strain CEA152 were germinated at the non-permissive temperature (45 °C) and shifted to the permissive temperature (37 °C) after various times, they were only able to form a colony when incubation at the non-permissive temperature was restricted to 12 h (Fig. 6a). In contrast, transfer to the non-permissive temperature of conidia germinated at the permissive temperature did not block colony formation when the transfer was performed after 10–12 h of germination (Fig. 6b).



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Fig. 6. The thermosensitive phenotype of the tpsA{Delta} mutant is dependent on the developmental stage. Shift experiments from restrictive (45 °C) to permissive (37 °C) temperature (a) and vice versa (b). Between 100 and 200 spores of the A. nidulans wild-type (FGSC773, wA3 pyroA4 pyrG89; {blacksquare}) and tpsA{Delta} (CEA152, wA3 pyroA4 pyrG89 tpsA{Delta}; {square}) strains were inoculated on complete medium and incubated for the indicated time periods before the shift. Counts represent the percentage of the maximal count. Results are representative of two independent experiments.

 
Inactivation of tpsA results in reduced conidium viability during prolonged storage
Since the results presented above appeared to confirm a protective role of trehalose against sustained exposure of A. nidulans cells to various stress conditions, we evaluated the role of trehalose for the survival of conidia of A. nidulans during prolonged storage. Conidia of strains FGSC773, which contain trehalose, and CEA152, which are devoid of trehalose (data not shown), were maintained at 4 °C or 20 °C for several weeks and the number of viable spores was estimated at different times. Results presented in Fig. 7 show that the conidia of the tpsA{Delta} mutant strain lost viability much more rapidly than wild-type conidia when incubated at 20 °C. In contrast, no difference in viability between the wild-type and mutant conidia was observed upon incubation at 4 °C over a period of up to 7 weeks (Fig. 7). These results indicate that trehalose might play an important role in the survival of conidia during prolonged storage, either as a protective molecule or reserve carbohydrate.



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Fig. 7. Trehalose is required for conidium viability during prolonged storage. Viability of conidia from A. nidulans tpsA+ (FGSC773, wA3 pyroA4 pyrG89) or tpsA{Delta} (CEA152, wA3 pyroA4 pyrG89 tpsA{Delta}) strains upon prolonged incubation at 4 °C [tpsA+ ({bullet}) and tpsA{Delta} ({circ}), or 20 °C, tpsA+ ({blacksquare}) and tpsA{Delta} ({square})]. Conidia were resuspended in PBS/0·1% Tween-20 at a density of 109 conidia ml-1 and maintained at room temperature or 4 °C for the indicated times. Spore viability was determined by the number of c.f.u. after plating on complete medium and growth at 37 °C.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper we have reported the cloning of the A. nidulans tpsA gene and physiological characterization of the tpsA{Delta} mutant. Our results demonstrate unambiguously that the tpsA gene product is a T6PS that is essential for trehalose biosynthesis in A. nidulans. The finding that tpsA is the unique T6PS-encoding gene in A. nidulans, in contrast to the situation in A. niger, where two T6PS genes have been identified (Wolschek & Kubicek, 1997 ), and that inactivation of tpsA results in a complete loss of trehalose biosynthesis, in contrast to the inactivation of the A. nidulans T6PP-encoding orlA gene (Borgia et al., 1996 ), has allowed us for the first time to investigate the physiological role of trehalose in a filamentous fungus.

In the yeast S. cerevisiae, trehalose is synthesized by a large multi-subunit complex (Bell et al., 1998 ). The subunits of this complex share a domain which is similar to T6PS although devoid of T6PS activity. Our results show that the A. nidulans T6PS can fulfil all of the functions of its yeast counterpart, thus suggesting that it can associate in the multi-subunit trehalose synthase complex in yeast. Our reinvestigation of the sequence of the orlA gene shows that the A. nidulans T6PP is larger than previously proposed (Borgia et al., 1996 ) with an amino-terminal domain which is homologous to A. nidulans T6PS (C. d’Enfert & A. Antczak, unpublished data). Furthermore, cDNAs encoding a homologue of S. cerevisiae Tps3 have been identified in the course of expressed sequence tag (EST) sequencing of A. nidulans cDNAs (D. Kupfer & B. Roe, http://www.genome.ou.edu/fungal.html; C. d’Enfert, unpublished data) thus suggesting that the A. nidulans trehalose synthase is also present in a multi-subunit complex in A. nidulans.

In A. nidulans trehalose has been shown to accumulate under a variety of conditions including nutrient starvation, conidiospore differentiation and heat stress (d’Enfert & Fontaine, 1997 ; Noventa-Jordao et al., 1999 ; this study). Here we have shown that trehalose accumulation is also stimulated in response to an oxidative stress (Fig. 4a) consistent with the results of Noventa-Jordao et al. (1999) , who showed a link between heat shock recovery and the cellular response to oxidative stress in A. nidulans. Results presented in this paper show that trehalose accumulation is mediated by TpsA under the different conditions tested, i.e. heat stress, oxidative stress and conidiogenesis. Furthermore, TpsA is responsible for the basal levels of trehalose and T6P that are produced during mycelial growth (data not shown). Although these results would suggest that trehalose biosynthesis in response to temperature and oxidative stress could contribute to the resistance of the germlings to these stress conditions, results presented in Fig. 4(b) show that this is not always the case: wild-type and tpsA-null germlings are similarly sensitive to a heat or an oxidative shock. In contrast, the absence of trehalose and/or trehalose biosynthesis results in reduced spore viability and a reduced ability to grow upon constant exposure to sublethal stress, including prolonged exposure to high temperature and growth in the presence of reactive oxygen species. Taken together, these results suggest that trehalose is mainly involved in the resistance of A. nidulans to progressive exposure to lethal stress or prolonged exposure to sublethal stress rather than rapid exposure to lethal stress. This is in agreement with the previous observations that germlings of A. nidulans maintaining a high level of trehalose due to a defect in the TreB neutral trehalase are less sensitive to heat stress than wild-type germlings (d’Enfert et al., 1999 ) and that viability of the conidia of an A. niger tpsA mutant is reduced (Wolschek and Kubicek, 1997 ). This is also consistent with the role of trehalose in the acquisition of thermotolerance and halotolerance demonstrated in S. cerevisiae (Hounsa et al., 1998 ; Lewis et al., 1995) and in Schizosaccharomyces pombe (Ribeiro et al., 1998 ). Results presented in Fig. 6 show that this protective role of trehalose is most important during A. nidulans conidial germination as opposed to later developmental stages, including mycelial growth, suggesting that additional mechanisms of adaptation to stress operate following germ tube formation.

Analysis of the expression of the tpsA gene in response to a heat shock or during conidiogenesis, when trehalose is synthesized, did not reveal induction of the transcription of tpsA under these conditions (Fig. 3). In contrast, expression of tpsA is induced during the early stages of mycelial growth, when trehalose biosynthesis appears minimal. Our results contrast with those of Wolschek & Kubicek (1997) , who showed that the A. niger tpsA and tpsB genes are respectively down-regulated and up-regulated by a heat shock. While activation of trehalose biosynthesis in A. niger appears to be controlled in part at the transcriptional level, our data suggest that, in A. nidulans, an inactive form of T6PS is accumulated during phases of rapid growth to prepare for induction of trehalose biosynthesis in response to stress, nutrient starvation, or developmental transitions by means of post-transcriptional control mechanisms. In S. cerevisiae T6PS activation is mediated both at the transcriptional level through an STRE-dependent activation mechanism (de Virgilio et al., 1993 ; Winderickx et al., 1996 ) and at the post-translational level by the protein kinase Rim15 (Reinders et al., 1998 ). A homologue of Rim15 has been identified by systematic sequencing of A. nidulans cDNAs (D. Kupfer & B. Roe, http://www.genome.ou.edu/fungal.html; C. d’Enfert, unpublished data). It is a possible candidate for post-translational control of TpsA, allowing rapid activation of trehalose synthesis under stress conditions.

In S. cerevisiae, inactivation of the T6PS results in an inability to grow on rapidly fermentable sugars such as glucose and fructose because of an uncontrolled influx of the sugars into glycolysis, causing rapid ATP depletion (Van Aelst et al., 1993 ). Recent results suggest that both T6P inhibition of hexokinase and a direct involvement of T6PS are responsible for this phenomenon (Bonini et al., 2000 ). In A. nidulans, T6P is also known to inhibit hexokinase (Ruijter et al., 1996 ) and our results show that TpsA is able to fulfil all the functions of the yeast T6PS, including its control on glucose influx into glycolysis, thus suggesting that similar mechanisms of glucose influx could operate in yeast and A. nidulans. On the other hand, the A. nidulans tpsA-null mutant is able to grow on glucose or fructose as a carbon source. Although increased levels of sugar phosphates could be detected in the mycelium of the mutant strain grown at 30 °C, this increase was not associated with a decrease of the ATP pool (Table 3). This suggests that T6P is a physiological inhibitor of hexokinase in A. nidulans but that the increase in glycolytic flux resulting from T6P depletion has only minor consequences in this fungus compared to what has been observed in S. cerevisiae. This physiological role of T6P is also supported by the poor growth on fructose of the A. nidulans orlA mutant which accumulates T6P and consequently should have reduced hexokinase activity (S. Fillinger & C. d’Enfert, unpublished results). Interestingly, the thermosensitive growth defect of the A. nidulans tpsA{Delta} strain appeared less pronounced on minimal glycerol medium than on minimal glucose medium (Fig. 5). Although this may reflect a more stringent role of T6P on the control of glycolytic flux at high temperature or the replacement of trehalose as a stress metabolite by intracellular glycerol resulting from glycerol uptake, the thermosensitive growth defect might also be less pronounced on slowly metabolizable carbon substrate because of a slower growth rate and the resulting presence of a higher intrinsic stress resistance (see Thevelein & de Winde, 1999 , for a recent review). Further analysis of metabolic fluxes at different temperatures in wild-type and trehalose biosynthesis mutants is needed to assess precisely the role of T6P glycolytic control in filamentous fungi.

In summary, results presented in this study show that T6P, in addition to its role in the control of chitin biosynthesis (Borgia et al., 1996 ), appears to play only a minor role in the control of the glycolytic flux in A. nidulans, in contrast to what has been observed in S. cerevisiae and some phylogenetically close yeast species (Kluyveromyces lactis, Candida albicans). More importantly, our results show that trehalose is a major stress metabolite in A. nidulans and is probably involved in the acquisition of resistance to a variety of stress conditions, including heat and oxidative stress, as well as in the survival of conidia during prolonged storage.


   ACKNOWLEDGEMENTS
 
We are indebted to Jaap Visser for his constant support and interest during the course of this work. Thanks are due to Jean Paul Latgé, in whose laboratory part of this work was carried out, to Markus Wolschek and Christian P. Kubicek from Section Microbial Biochemistry, Institute for Biochemical Technology and Microbiology, TU Wien, Austria, for their contribution to the sequencing of the A. nidulans tpsA gene and to Bertrand Llorente for providing plasmid pCM190L. This work was supported by the EU Biotechnology Programme (BIO4-CT98-0268).


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Received 29 December 2000; revised 15 March 2001; accepted 29 March 2001.