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 dEnfert. Tel: +33 1 40 61 32 57. Fax: +33 1 45 68 87 90. e-mail: denfert{at}pasteur.fr
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ABSTRACT |
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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.
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INTRODUCTION |
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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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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 (dEnfert 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.
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METHODS |
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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
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 [(lac-pro) F'(lacIq1
(lacZ)M15 pro+ Tn10)] and DH5
(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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Plasmid pTPS13 was obtained by subcloning the 0·8 kb SalIClaI fragment of pTPS7 into SalIClaI-digested pTPS11P, a derivative of pTPS11 with an internal deletion of a PstI fragment. Plasmid pTPS17 was then obtained by subcloning a KspIEcoRV 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
-AfpyrG-tpsA allele, a 1462 bp fragment is expected in transformants with a tpsA-pyrG-tpsA
allele. Putative merodiploids with a tpsA-AfpyrG-tpsA
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 ClaISalI 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
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
allele to the tpsA
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 (dEnfert & 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
, 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 EcoRISalI fragment could be detected corresponding to either the wild-type allele (1·15 kb) or the mutant tpsA
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 (dEnfert et al., 1999 ). RT-PCR experiments were achieved using the Reverse Transcription System according to the manufacturers 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 suppliers instructions (Invitrogen), yielding plasmid pTPS15. Following sequencing of the cloned tpsA cDNA, the BglIINotI 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)
.
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RESULTS |
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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 SalISmaI fragment derived from plasmid pTPS4 (see Methods) and of an approximately 1·2 kb EcoRIPstI 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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tpsA is required for trehalose accumulation in response to various stress conditions
In A. nidulans, trehalose is known to accumulate during conidiogenesis (dEnfert & 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
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
strain (Fig. 4a
). Furthermore, T6P and trehalose were undetectable in the mycelium of a tpsA
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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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) on various media and at different temperatures. The tpsA
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
mutant also showed reduced growth on media containing sublethal doses (12 mM) of H2O2 (Fig. 5a
).
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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 1012 h of germination (Fig. 6b
).
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DISCUSSION |
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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. dEnfert & 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. dEnfert, 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 (dEnfert & 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 (dEnfert 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. dEnfert, 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. dEnfert, unpublished results). Interestingly, the thermosensitive growth defect of the A. nidulans tpsA
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.
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ACKNOWLEDGEMENTS |
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Received 29 December 2000;
revised 15 March 2001;
accepted 29 March 2001.