Molecular Microbiology (1999) 32(3), 471–483
Neutral trehalases catalyse intracellular trehalose breakdown in the filamentous fungi Aspergillus nidulans and Neurospora crassa Christophe d’Enfert,1†* Beatriz M. Bonini,2‡ Pio D. A. Zapella,3‡ Thierry Fontaine,1 Aline M. da Silva3 and He´ctor F. Terenzi 2 1 Laboratoire des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. 2 Departamento de Biologia, Faculdade de Filosofia, Ciencias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, 14040–901 Ribeira˜o Preto, SP, Brazil. 3 Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, 005508–900 Sa˜o Paulo, SP, Brazil. Summary A cAMP-activatable Ca 2þ-dependent neutral trehalase was identified in germinating conidia of Aspergillus nidulans and Neurospora crassa . Using a PCR approach, A. nidulans and N. crassa genes encoding homologues of the neutral trehalases found in several yeasts were cloned and sequenced. Disruption of the AntreB gene encoding A. nidulans neutral trehalase revealed that it is responsible for intracellular trehalose mobilization at the onset of conidial germination, and that this phenomenon is partially involved in the transient accumulation of glycerol in the germinating conidia. Although trehalose mobilization is not essential for the completion of spore germination and filamentous growth in A. nidulans , it is required to achieve wild-type germination rates under carbon limitation, suggesting that intracellular trehalose can partially contribute the energy requirements of spore germination. Furthermore, it was shown that trehalose accumulation in A. nidulans can protect germinating conidia against an otherwise lethal heat shock. Because transcription of the treB genes is not increased after a heat shock but induced upon heat shock recovery, it is proposed that, in filamentous fungi, mobilization of trehalose during the return to appropriate growth is promoted by transcriptional and post-translational Received 11 September, 1998; revised 22 December, 1998; accepted 31 December, 1998. †Present address: Unite´ de Physiologie Cellulaire, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. ‡These authors contributed equally to this work and are therefore listed in alphabetical order. *For correspondence. E-mail
[email protected]; Tel. (þ33) 1 40 61 32 57; Fax (þ33) 1 45 68 87 90. 䊚 1999 Blackwell Science Ltd
regulatory mechanisms, in particular cAMP-dependent protein kinase-mediated phosphorylation. Introduction Trehalases are enzymes that hydrolyse the non-reducing disaccharide of glucose, trehalose (␣-D-glucopyranosyl (1,1)-␣-D-glucopyranoside). Two types of trehalases have been identified and characterized at the molecular level in fungi (Thevelein, 1984; Jorge et al ., 1997). These enzymes have been referred to as acid trehalase and neutral trehalase according to their pH optimum, and they appear to play very distinct roles in the fungal cell. Acid trehalases are secretory proteins that have been localized to the vacuole in Saccharomyces cerevisiae (Mittenbu¨hler and Holzer, 1988) and to the cell wall in filamentous fungi (Hecker and Sussman, 1973a; d’Enfert and Fontaine, 1997). The study of knockout mutants of the acid trehalase genes in both S. cerevisiae and Aspergillus nidulans, and chemical mutants in Neurospora crassa , has revealed that these enzymes are involved in the assimilation of extracellular trehalose as a carbon source (Sussman et al ., 1971; Nwaka et al ., 1996; d’Enfert and Fontaine, 1997). Furthermore, analysis of the primary structure of S. cerevisiae and A. nidulans acid trehalases shows that these enzymes do not share any homology with other prokaryotic and eukaryotic trehalases, including fungal neutral trehalases (Destruelle et al ., 1995; d’Enfert and Fontaine, 1997). Neutral trehalases (also referred to as regulatory trehalases) have been identified in ascomycete species that are able to grow in a unicellular yeast form and in zygomycetes (Thevelein et al ., 1983; Dewerchin and Van Laere, 1984; Londesborough and Varimo, 1984; De Virgilio et al ., 1991; Amaral et al ., 1997; Eck et al ., 1997). The activity of neutral trehalases is tightly controlled by phosphorylation of the enzyme and is also dependent of the presence of divalent ions (Londesborough and Varimo, 1984; Thevelein, 1988; App and Holzer, 1989; Amaral et al ., 1997). In S. cerevisiae , activation of the enzyme can be triggered by different signals (glucose, nitrogen sources, heat shock) and is mediated by cAMP-dependent and -independent phosphorylation (Thevelein, 1988). cAMP-dependent protein kinase (cAPK)-mediated phosphorylation is likely to be of general relevance because consensus cAPK
472 C. d’Enfert et al. phosphorylation sites have been identified in all neutral trehalases characterized at the molecular level to date (Kopp et al ., 1993; Nwaka et al ., 1995a; Amaral et al ., 1997; Eck et al ., 1997; Cansado et al ., 1998). Analysis of genetically engineered S. cerevisiae mutants that lack the Nth1 neutral trehalase has revealed that this enzyme is required for the mobilization of cytoplasmic trehalose in response to various environmental stimuli (Kopp et al ., 1993). Similar results have been obtained in Kluyveromyces lactis , Schizosaccharomyces pombe and Candida albicans (Amaral et al ., 1997; Eck et al ., 1997; Cansado et al ., 1998). However, inactivation of the neutral trehalase genes in any of these species did not affect growth, suggesting that breakdown of intracellular trehalose does not support the energy requirements during specific transitions in the life cycle at least in the laboratory environment. In contrast, mutants lacking neutral trehalase activity display reduced or elevated sensitivity to heat shock or osmotic stress, suggesting that cytoplasmic trehalose participates in the protection of the fungal cell against various environmental stresses and that its persistence may interfere with growth resumption (Nwaka et al ., 1995b; Cansado et al ., 1998). The stress protection role of trehalose is corroborated by the phenotypes of S. cerevisiae and S. pombe mutants that lack trehalose synthase activity (De Virgilio et al ., 1994; Ribeiro et al ., 1998). Despite intense efforts, the biochemical demonstration of a regulatory trehalase activity in filamentous ascomycetes has never been achieved. However, several lines of evidence point to the occurrence of such enzymes: (1) filamentous fungi accumulate large quantities of trehalose during spore formation or in response to heat shock, and this pool is rapidly degraded during growth resumption (Thevelein, 1984; d’Enfert, 1997); (2) A. nidulans and N. crassa mutants that lack or have reduced acid trehalase activity retain their ability to mobilize intracellular trehalose in response to various environmental stimuli (Bonini et al ., 1995; d’Enfert and Fontaine, 1997); and (3) a neutral trehalase and a gene encoding a protein with significant amino acid identity to the yeast neutral trehalases have been identified in Fusarium oxysporum and Magnaporthe grisea , respectively, but their involvement in the mobilization of intracellular trehalose remains to be established (Amaral et al ., 1995; Sweigard et al ., 1998). In order to be precise about the role of trehalose in filamentous fungi, we have investigated the occurrence of regulatory trehalases in two filamentous ascomycetes, A. nidulans and N. crassa . In this study, we have taken advantage of mutants that lack acid trehalase activity to reveal a Ca2þdependent cAMP-inducible neutral trehalase activity in the conidia of these two fungi. The AntreB and NctreB genes corresponding to these enzymes were cloned and encode proteins with ⬇58% amino-acid identity to the S. cerevisiae neutral trehalases. Genetically engineered strains of
A. nidulans lacking neutral trehalase activity show a complete deficiency in the mobilization of trehalose during conidial germination. Analysis of these mutant strains suggests that trehalose mobilization can contribute some of the energy requirements of spore germination under limiting external carbon, and that trehalose can act as a thermoprotectant in conidia of filamentous fungi. Results
Neutral trehalase activity in germinating conidia of A. nidulans and N. crassa Previous attempts to identify a neutral trehalase activity in filamentous fungi have failed (Thevelein, 1984; Bonini et al., 1995; d’Enfert and Fontaine, 1997). We reasoned that this failure might be due to: (1) the high background resulting from acid trehalase activity at neutral pH and from the presence of glucose in the cultures used to prepare extracts (Horikoshi and Ikeda, 1966; C. d’Enfert, unpublished); (2) the requirement for an activation step either in vivo or in vitro ; (3) the lysis procedure used to prepare extracts; or (4) the poor sensitivity of the assays used previously to detect glucose formed from trehalose in vitro . To circumvent these limitations, two sets of experiments were implemented using A. nidulans or N. crassa conidia respectively. First, A. nidulans conidia of strain CEA53 that lacks acid trehalase (d’Enfert and Fontaine, 1997) were germinated in minimal fructose medium for 2 h at 37⬚C. At this stage, the internal trehalose pool is almost fully degraded (data not shown), but the trehalase responsible for this degradation is likely to be active. A crude extract was prepared and assayed for trehalase activity in the presence of 5 mM CaCl2 and using a glucose oxidase/peroxidase assay for the detection of the glucose formed from in vitro trehalose hydrolysis. Using this procedure, a maximum trehalose hydrolysis rate ranging between 1.5 and 4.0 amol min¹1 trehalose per spore was obtained at pH 7.0, which is the optimal pH for this trehalolytic activity (data not shown). This activity was reduced by two- to threefold in the absence of calcium or in the presence of 10 mM EDTA. Furthermore, a lower level of activity was detected in extracts of non-germinated conidia of strain CEA53 (0.36 amol min¹1 trehalose per spore). Activity in these extracts could be enhanced after an incubation in the presence of 2.5 M cAMP and 250 M ATP (1.8 amol min¹1 trehalose per spore; activation factor ¼ 4.9). Neutral trehalase activity in conidia of A. nidulans increases during the first hour of spore germination but remains sensitive to an incubation with cAMP/ATP, although to a lesser extent than that observed with the enzyme extracted from ungerminated spores (data not shown). Second, all further attempts, such as changes in pH, temperature, addition of different ions or metabolites to crude extracts obtained from the different phases of the 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Neutral trehalase of A. nidulans and N. crassa 473 vegetative cell cycle, to detect a neutral trehalase activity in crude extracts of N. crassa were again unsuccessful, as already mentioned in a previous report (Bonini et al ., 1995). We, thus, decided to use a nystatin-based permeabilization procedure (Ave´ret et al ., 1998) applied to intact cells. Conidia, germinated for 2 h, of the acid trehalasedeficient (tre ) N. crassa mutant strain FGSC4511 were incubated in the presence of nystatin as the permeabilizing agent (see Experimental procedures ). Only nystatintreated germlings revealed a neutral (pH 7) trehalase activity. This cryptic activity increased from 3.3 mU mg¹1 protein to about 30 mU mg¹1 protein after incubation with 2.5 M cAMP and 250 M ATP, and it was reduced by 30–80% if assayed in the absence of 10 mM CaCl2 or in the presence of 5.0 mM EDTA. Without the nystatin treatment, only the residual periplasmic acid trehalase activity of the tre mutant (5–6 mU mg¹1 protein) was detected. This residual activity was not affected by incubation with cAMP/ATP or addition of either CaCl2 or EDTA. Neutral trehalase activity was undetectable in nystatin-treated ungerminated N. crassa spores, regardless of activation with cAMP/ATP. After 30 min of germination, this activity appeared and increased steadily up to 90 min of incubation. In all cases, the activity was strongly dependent upon incubation in the presence of cAMP/ATP. Taken together, these data show that A. nidulans and N. crassa have neutral trehalase activities with features similar to the yeast neutral trehalases, i.e. cAMP-dependent activation and Ca 2þ requirement for activity (Thevelein, 1984).
Isolation of the AntreB and NctreB genes encoding neutral trehalases To investigate the role of the A. nidulans and N. crassa neutral trehalases, we set out to identify genes encoding these proteins using a two-step strategy. First, two degenerate primers treBF (sense primer; Table 2) and treBB (antisense primer; Table 2) were designed that correspond, respectively, to (1) a region conserved in yeast trehalases and in a Magnaporthe grisea protein that shows significant similarity to yeast trehalases (Amaral et al., 1997; Sweigard et al ., 1998) and (2) a region conserved in bacterial, yeast and higher eukaryotes trehalases (Kopp et al ., 1993; Amaral et al ., 1997). Using these primers, 636 bp and 654 bp fragments were amplified by PCR from genomic DNA of A. nidulans strain FGSC28 and N. crassa strain FGSC424WT respectively. The nucleotide sequences of both PCR products were determined, and the deduced amino-acid sequences revealed significant identity to corresponding regions of S. cerevisiae neutral trehalases (58–62% identical amino acids). In addition, analysis of the N. crassa product revealed a putative 75 bp intron, which was later confirmed by cDNA sequencing. These results suggested that these PCR 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
products correspond to A. nidulans and N. crassa neutral trehalase-encoding genes referred to as AntreB and NctreB respectively. In a second step, the PCR products were used to probe chromosome-specific libraries of A. nidulans genomic DNA (Brody et al ., 1991) and a cDNA library of N. crassa (Brunelli and Pall, 1983) respectively. Using the A. nidulans probe, four positive cosmids, L04C12, L15H07, L24G01 and L25F08, were identified. These cosmids have been assigned to the same region of A. nidulans chromosome V (Prade et al ., 1997), suggesting that the AntreB gene is located on this chromosome. Using the N. crassa probe, positive cDNAs were identified and shown to correspond to the NctreB gene.
Sequencing of the AntreB gene and NctreB cDNA Southern hybridization of cosmid L04C12 DNA that had been digested by several restriction enzymes, using the A. nidulans PCR probe described above, revealed a ⬇3.8 kb Eco RI fragment and a ⬇2.9 kb Bam HI fragment that overlapped each other by ⬇700 bp (Fig. 1). These two fragments were subcloned into pBSLNþ (d’Enfert, 1996), and the nucleotide sequence of a 3475 bp fragment was determined (GenBank accession number AF043229; data not shown). Analysis of this DNA sequence revealed an open reading frame (ORF) of 2244 bp interrupted by four putative introns of 65, 57, 64 and 55 bp. The location of these introns was corroborated by comparison with the nucleotide sequence of the NctreB cDNA (GenBank accession number AF044218, data not shown), which contains an ORF of 2184 bp with 66.5% identity to the AntreB ORF. The A. nidulans 2244 bp and N. crassa 2184 bp ORFs
Fig. 1. Restriction maps of cosmid L04C12 and plasmids used in this study. A restriction map of a region of cosmid L04C12 is shown together with the location of the treB gene (solid box) and orientation of transcription (arrow). Inserts of the different plasmids obtained by subcloning DNA fragments of L04C12 into pBLSNþ are shown as thin lines. The direct repeats with the neo gene of transposon Tn5 and the A. niger DNA carrying the pyrG gene that constitutes the pyrG blaster used to replace the Pma CI/ Sma I fragment of pNTH9 are shown as open-boxed arrows and solid lines respectively. The region of cosmid L04C12, the nucleotide sequence of which has been deposited in the GenBank (AF043229), is shown as an arrow. B, Bam HI; E, Eco RI; G, Bgl II; H, Hin dIII; P, Pma CI; S, Sma I.
474 C. d’Enfert et al. that were identified in the cloned DNA regions encode 748 amino acids and 728 amino acids proteins, respectively, with respective molecular masses of 86176 Da and 84121 Da (Fig. 2). These two proteins share 68.0% identical
amino acids and 74.4% similar amino acids. Furthermore, they display significant similarity to previously described trehalases (e.g. 55.6% or 59.0% identical amino acids and 64.0% or 66.8% similar amino acids to the S. cerevisiae
Fig. 2. Alignments for maximal amino-acid similarities of the A. nidulans and N. crassa neutral trehalases (antreB and nctreB respectively) with S. cerevisiae (scnth1; Kopp et al ., 1993), Kluyveromyces lactis (klnth1; Amaral et al ., 1997) and Candida albicans (canth1; Eck et al ., 1997) neutral trehalases, E. coli periplasmic (ectreA; Guttierrez et al ., 1989) and cytoplasmic (ectreF; Horlacher et al ., 1996) trehalases, and Tenebrio molitor (tntreA; Su et al ., 1993), Bombyx morii (bmtreA; Su et al ., 1993) and rabbit (ratreA; Ruf et al ., 1990) trehalases. A. An alignment of the amino-terminal regions of fungal neutral trehalases. B. An alignment of the carboxy-terminal regions of prokaryotic and eukaryotic trehalases. Identical (upper case letters) and similar (lower case letters) have a black background. cAMP dependent-phosphorylation consensus sites (Kemp and Pearson, 1991) are boxed and the putative Ca 2þ-binding site (Geiser et al ., 1991) is underlined. This alignment was produced using the PILEUP program of the UWGCG package version 8 (Devereux et al ., 1984). 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Neutral trehalase of A. nidulans and N. crassa 475 Nth1 neutral trehalase respectively; Kopp et al ., 1993; Nwaka et al ., 1995a), with the exception of the S. cerevisiae and A. nidulans acid trehalases (Destruelle et al ., 1995; d’Enfert and Fontaine, 1997). As previously described for S. cerevisiae and K. lactis neutral trehalases (Amaral et al ., 1997), two domains could be distinguished in the A. nidulans and N. crassa proteins: a carboxy-terminal domain of ⬇450 amino acids that displays significant homology to eukaryotic and prokaryotic trehalases (Fig. 2B) and that is likely to correspond to the catalytic core of the enzyme; an amino-terminal domain of ⬇280 amino acids that is found only in fungal neutral trehalases and that is likely to correspond to the regulatory domain of these enzymes, as shown by the occurrence of putative cAMP-dependent protein kinase phosphorylation sites (Kemp and Pearson, 1991) and calcium binding sites (Geiser et al ., 1991) (Fig. 2A). This suggests that AntreB and NctreB encode regulatory neutral trehalases.
the stationary phase of growth (Fig. 3A, lane 6) or during conidiogenesis (Fig. 3A, lanes 8–11). As a consequence, a RT-PCR product corresponding to the AntreB transcript could not be detected in non-germinating conidia of A. nidulans (Fig. 3A, lane 1). Results presented in Fig. 3B indicate that the transcription of the NctreB gene follows a pattern strictly similar to that of AntreB during growth in liquid medium and during conidiation (Fig. 3B, lanes 1–12). It is barely detected in ungerminated conidia, and increases upon germination and during exponential growth (Fig. 3B, lanes 2–5) and declines thereafter as well as during conidiogenesis (Fig. 3B, lanes 6–7 and 8–12). When 4 h germlings of A. nidulans or N. crassa were exposed to a heat shock at 45⬚C, the expression of AntreB decreased (Fig. 3A, lanes 12–14), whereas that of NctreB was unaffected (Fig. 3B, lanes 13–15). However, expression of AntreB and NctreB increased rapidly during the recovery period at 30⬚C (Fig. 3A, lanes 15–16 and Fig. 3B, lanes 16–17).
Regulation of the AntreB and NctreB genes Expression of the AntreB and NctreB genes under different culture conditions was monitored using semiquantitative RT-PCR. Results presented in Fig. 3A show that transcription of the AntreB gene occurs during spore germination in rich medium (Fig. 3A, lane 2), is maximal during exponential growth (Fig. 3A, lanes 3–5 and 7) and is reduced during
Disruption of the AntreB gene As a preliminary step to introduce a null mutation into the A. nidulans treB gene, plasmid pNTH9 was constructed by inserting a 4.2 kb Hin dIII/BglII fragment of cosmid L04C12 into pBLSNþ (Fig. 1). An 8.6 kb Hpa I fragment of plasmid pCDA14 containing a pyrG blaster (d’Enfert, 1996) was
Fig. 3. Expression of the AntreB and NctreB genes in different culture conditions. A. Southern analysis of RT-PCR products corresponding to the A. nidulans treB transcript. Lanes 1–6 correspond to A. nidulans pabaA1 RNA obtained from liquid cultures incubated at 37⬚C for 0, 3, 8, 24, 48 or 64 h respectively. Lanes 7–11 correspond to A. nidulans pabaA1 RNA obtained from a 16 h liquid culture that had been induced to conidiate for 0, 4, 8, 16 or 24 h respectively. Lanes 12–16 correspond to A. nidulans FGSC773 RNA obtained from a 4 h liquid culture incubated at 30⬚C (lane 12) and submitted to a 45⬚C heat shock for 10 or 30 min (lanes 13 and 14 respectively) and allowed to recover at 30⬚C for 10 or 30 min (lanes 15 and 16 respectively). Lane G corresponds to a PCR product obtained using primers nth6 and nth10, but using A. nidulans genomic DNA as a template instead of a reverse transcription product. This PCR product was used as a probe. B. Southern analysis of RT-PCR products corresponding to the N. crassa treB transcript. Lanes 1–7 correspond to N. crassa FGSC424 RNA obtained from liquid cultures incubated at 37⬚C for 0, 1, 2, 4, 8, 24 or 72 h respectively. Lanes 8–12 correspond to N. crassa FGSC424 RNA obtained from an 18 h liquid culture that had been induced to conidiate for 0, 2, 4, 8 or 24 h respectively. Lanes 13–17 correspond to N. crassa FGSC424 RNA obtained from a 4 h liquid culture incubated at 30⬚C (lane 13) and submitted to a 45⬚C heat shock for 10 or 30 min (lanes 14 and 15 respectively) and allowed to recover at 30⬚C for 10 or 30 min (lanes 16 and 17 respectively). Lane G corresponds to a PCR product obtained using primers Ncnth1 and Ncnth2, but using N. crassa genomic DNA as a template instead of a reverse transcription product. The size of the RT-PCR products is given in base pairs. This PCR product was used as a probe. 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
476 C. d’Enfert et al. used to replace an internal Pma CI– Sma I fragment of pNTH9, yielding pNTH10 (Fig. 1). The resulting treB allele lacks the region encoding amino acids 1–258 of the AnTreB protein as well as 708 bp of the 5⬘ untranslated region of the AntreB gene. Not I-digested pNTH10 was used to transform an A. nidulans pyrG89 strain (FGSC773) and an A. nidulans pyrG89 treA::neo strain (CEA54) that lacks the acid trehalase. In both cases, transformants were identified that had occurred by gene replacement at the AntreB locus, as demonstrated by PCR and Southern analysis (data not shown). Strain CEA61 is a derivative of FGSC773 that lacks the neutral trehalase-encoding gene, whereas strain CEA103 is a derivative of CEA54 that lacks both the neutral and acid trehalase-encoding genes. Because similar results were obtained for CEA61 and CEA103 with respect to growth and intracellular trehalose mobilization, a comparison of strains CEA53 (pyrG89; wA3; pyroA4; treA::neo-pyrG-neo ) and CEA103 (pyrG89; wA3; pyroA4; treB::neo-pyrG-neo; treA::neo ) is reported below.
AntreB encodes a neutral trehalase required for intracellular trehalose breakdown during germination Results presented above supported the occurrence of a neutral trehalase activity in germinating conidia of A. nidulans . To test the contribution of the AntreB gene product to this activity, crude extracts of germinating A. nidulans strain CEA103 were assayed for neutral trehalase activity. No activity could be detected in CEA103 germinating spores, suggesting that the AnTreB protein is responsible for the neutral trehalase activity observed in treB þ conidia. The role of the AnTreB neutral trehalase in the degradation of the trehalose pool during germination was investigated. For this purpose, sugars and polyols were monitored in germinating conidia of A. nidulans strain CEA103. Results presented in Fig. 4 show that although the pool of trehalose is fully mobilized within 120 min of germination at 30⬚C in complete medium in a A. nidulans strain that
has a wild-type treB gene it remains intact in germinating conidia of strain CEA103. Furthermore, this defect is accompanied by a reproducible reduction in the levels of glycerol that are accumulated in the germinating conidia. Similar results were obtained using pyrGþ or pyrG¹ strains and were irrespective of the ability of the strain to produce the TreA acid trehalase (data not shown). Therefore, we conclude that the A. nidulans treB gene encodes a neutral trehalase responsible for trehalose breakdown during germination, a process that is in part involved in the transient accumulation of an internal glycerol pool.
Disruption of AntreB delays spore germination under external carbon limitation No difference could be observed between A. nidulans strains CEA53 and CEA103 with respect to colony diameter and morphology (data not shown). However, because trehalose breakdown is an early event of spore germination, we investigated whether its inactivation would affect the kinetics of germ tube formation. The kinetics of germination of conidia from strain CEA53 and CEA103 were similar when germination was monitored on solid minimal medium containing 50 mM D-glucose (data not shown). In contrast, germ tube formation was significantly delayed when conidia of strain CEA103 were germinated at 37⬚C on solid minimal medium containing trace amounts of carbon (variance analysis: P ⱕ 10¹4, F ¼ 110); a delay of ⬇ 80 min was observed between the population of wild-type conidia that reached 50% of germination by 8 h and the population of treB conidia (data not shown). These results suggest that intracellular trehalose mobilization can contribute to some extent to the energy requirements of spore germination.
Reduced heat shock sensitivity of the A. nidulans treB mutant Trehalose has been proposed to act as a protectant against various environmental stresses, in particular high temperatures (Wiemken, 1990). The availability of the A. nidulans Fig. 4. Soluble sugars and polyols in mature and germinating conidia of A. nidulans treB þ and treB ¹ strains. A. Conidia of A. nidulans strain CEA53 were grown in complete medium containing 0.1% Tween 20 at 30⬚C for differing times and collected by filtration. Soluble sugars and polyols were extracted and analysed by HPLC on an ion exclusion column. W, trehalose; B, mannitol; A, glycerol. B. As A, but for conidia of A. nidulans strain CEA103.
䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Neutral trehalase of A. nidulans and N. crassa 477
Fig. 5. Differential sensitivity of A. nidulans treB þ and treB ¹ strains to a thermal shock. Conidia of A. nidulans strains CEA53 (X) and CEA103 (W) were grown for 2 h in complete medium containing 0.1% Tween 20 at 30⬚C and shifted to 50⬚C for the indicated times. Viability was calculated as a percentage of the surviving cells relative to the original cell count. Results represent mean values ⫾ standard deviations of two independent experiments for which several cell counts were made at each time point.
treB mutant provided us with a convenient way to test this hypothesis in filamentous fungi. Conidia of A. nidulans strains CEA53 (treB þ ) and CEA103 (treB ¹ ) were germinated for 2 h at 30⬚C in complete medium and subjected to a 50⬚C heat shock for 10–40 min. Although CEA53 spores contained roughly no trehalose when heat shocked, CEA103 spores had trehalose levels similar to those found in non-germinating spores (Fig. 4). Results presented in Fig. 5 show that although treB þ germinating spores are highly sensitive to heat shock (more than 85% killing within 40 min), treB ¹ germinating spores are quite insensitive to heat shock (less than 15% killing within 40 min). Therefore, we conclude that trehalose contributes to the thermoprotection in A. nidulans conidia.
Discussion Results presented in this report show that conidia of A. nidulans and N. crassa , in addition to the previously characterized acid trehalase (Horikoshi and Ikeda, 1966; Hecker and Sussman, 1973b; d’Enfert and Fontaine, 1997), contain a neutral trehalase. The A. nidulans enzyme was shown to catalyse the mobilization of intracellular trehalose during conidial germination, and is most probably involved at other growth resumption stages when trehalose breakdown is observed. This confirms our previous assumptions that were based on the study of A. nidulans and N. crassa mutants that lack acid trehalase (Bonini et al ., 1995; d’Enfert and Fontaine, 1997). The deduced amino-acid 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
sequences of the A. nidulans and N. crassa neutral trehalases show a high level of identity to the previously characterized neutral trehalases of various yeasts and to an M. grisea gene product that was proposed to be a neutral trehalase (Sweigard et al ., 1998). It is, therefore, likely that the process of neutral trehalase-mediated trehalose mobilization is of general significance to ascomycetous fungi, if not to all fungi. Comparison of the different amino-acid sequences that are available for prokaryotic and eukaryotic trehalases confirms the occurrence of three families of trehalases, as previously suggested by Amaral et al . (1997). The first family is composed of fungal acid trehalases and possibly of bacterial enzymes (Destruelle et al ., 1995; Blattner et al ., 1997; d’Enfert and Fontaine, 1997; Kunst et al ., 1997). Strikingly, these enzymes do not share any homology with other known trehalases, as previously observed from the comparison of the S. cerevisiae acid and neutral trehalases (Destruelle et al ., 1995) and as can be seen from the comparison of the A. nidulans acid and neutral trehalases (d’Enfert and Fontaine, 1997; this study). The second group of trehalases is formed from prokaryotic and higher eukaryotes acid trehalases, whereas the third family only contains fungal neutral trehalases. These two families share a common carboxy-terminal domain (Fig. 2B) that is likely to be the catalytic core of the enzyme, as proposed by Amaral et al . (1997). Amino-acid differences that are observed in this domain might in particular reflect the different pH optima of the enzymes. A typical feature of the fungal neutral trehalases is the occurrence of a large amino-terminal domain (⬇250 amino acids; Fig. 2A) that has been proposed to form a regulatory domain because of the occurrence of cAMP-dependent phosphorylation sites and Ca 2þ-binding sites (Kopp et al ., 1993; Amaral et al ., 1997). This amino-terminal domain is also present in the A. nidulans and N. crassa neutral trehalases as well as in the M. grisea protein. The occurrence of two candidate cAMP-dependent phosphorylation sites and a candidate Ca 2þ-binding site in this domain of the A. nidulans and N. crassa neutral trehalase is consistent with our data showing that the A. nidulans and N. crassa enzymes can be activated in vitro by cAMP-dependent phosphorylation or by Ca2þ ions. However, the functionality of these sites remains to be demonstrated. Given that trehalose mobilization mediated by the AnTreB trehalase is rapidly initiated after the induction of spore germination and that neutral trehalase activity increases steadily in germinating conidia of A. nidulans and N. crassa , it is likely that a cAMPdependent signalling pathway is involved during early germination in these fungi. To our knowledge, accumulation of cAMP upon induction of conidial germination has never been reported for Aspergillus or for Neurospora . However, several lines of evidence, for example the effect of cAPK inhibitors or of a mutation in the cAPK regulatory subunit
478 C. d’Enfert et al. on germ tube formation in F. oxysporum or N. crassa respectively (Ruan et al ., 1995; Bruno et al ., 1996), suggest an involvement of the cAMP-dependent protein kinase in the control of conidial germination in filamentous fungi (for a review, see d’Enfert, 1997). The availability of the neutral trehalase gene and consequently of tools to study putative post-translational modifications of the enzyme at the onset of conidial germination may now serve as a handle for the identification of signalling pathways that control spore germination. Using an A. nidulans mutant that has a null mutation in the treB gene, we have observed that a block in intracellular trehalose mobilization results in a decrease in the levels of glycerol that are transiently accumulated in the germinating conidia (Fig. 4). This confirms previous data showing the production of 14C-labelled glycerol during the germination of conidia that had been radiolabelled with 14C-glucose and hence contained 14C-labelled trehalose (d’Enfert and Fontaine, 1997). However, our data show that glycerol is not only produced from the mobilization of intracellular trehalose but also from another carbon source. This may be either the carbon source used to induce germination or a second reserve carbohydrate that remains to be identified. d’Enfert and Fontaine (1997) have proposed that glycerol accumulation may contribute to the isotropic growth of the spore by providing a significant increase in the turgor of the spore. Although we cannot exclude that a slower rate in the mobilization of intracellular mannitol and the persistence of intracellular trehalose may compensate for the reduced glycerol accumulation (see Fig. 4), our data suggest that a reduction by two- to threefold of the intracellular glycerol levels is not sufficient to prevent germination nor modify the kinetics of conidial germination in A. nidulans , at least when external carbon is not limiting (see below). A detailed characterization of the glycerol biosynthesis pathway and of its interplay with other polyols metabolism will be required to probe the function of glycerol during germination. Analysis of the A. nidulans treB mutant revealed only a subtle growth phenotype when conidia were germinated on a medium that contains limiting amounts of carbon. This suggests that a function of trehalose mobilization could be in supplying energy during specific transitions of the life cycle, at least in an adverse environment. Interestingly, the M. grisea neutral trehalase gene was identified in a mutant hunt for non-pathogenic strains after insertional mutagenesis (Sweigard et al ., 1998). As the conidia of strains that carry a null mutation in this gene age, they lose their ability to cause disease (J. Sweigard, personal communication). This may, therefore, reflect a role for trehalose breakdown in the persistence of M. grisea spores or in their ability to germinate in adverse environments (a droplet of water on a plant leaf in the case of M. grisea conidia) after a long period of dormancy.
The A. nidulans treB mutant allowed us to probe the role of trehalose as a thermoprotectant in A. nidulans . When the sensitivity to a heat shock was compared between germinating wild-type conidia that did not contain trehalose and treB conidia that had intracellular trehalose at a level similar to that observed in dormant conidia, a significant decrease (five- to 10-fold) in the sensitivity of the treB mutant was observed (Fig. 5). This result suggests that trehalose contributes to the resistance of the conidia of A. nidulans to environmental stresses and might therefore be an important determinant of their survival. Although a similar role cannot be confirmed unambiguously in S. cerevisiae because of the pleiotropic effects of a deletion of the trehalose-6-phosphate (T6P) synthase-encoding gene (De Virgilio et al ., 1994; Thevelein and Hohmann, 1995), results obtained with S. pombe mutants defective in the synthesis of either the T6P synthase or the neutral trehalase suggest a similar role in unicellular fungi (Blazquez et al ., 1994; Cansado et al ., 1998; Ribeiro et al ., 1998). In contrast to what has been previously observed for the S. cerevisiae NTH1 and NTH2 genes and for the S. pombe ntp1 þ gene, our data did not reveal any transcriptional activation of the AntreB and NctreB gene after a heat shock (Fig. 3B). However, a modest but significant increase in the transcription of the AntreB and NctreB genes was observed during heat shock recovery, suggesting that both a transcriptional and a post-translational regulation may account for an increase in neutral trehalase activity and the subsequent trehalose mobilization observed during heat shock recovery. A more detailed analysis of the transcriptional status of the AntreB and NctreB genes in response to and during the recovery from various stresses will nevertheless be needed to reach a better understanding of how the fungal cell controls trehalose levels in an adverse environment. In particular, the functional significance of the three STRE elements (5⬘-CCCCT-3⬘ or 5⬘GGGGA-3⬘) that we have identified in the 5⬘ untranslated region of the AntreB gene will have to be investigated. STRE elements have been shown to mediate transcriptional activation in response to various stresses in S. cerevisiae and presumably perform a similar function in other fungi (Siderius and Mager, 1997). We have observed a decrease in the expression of the AntreB and NctreB genes during the stationary phase of growth and during conidiogenesis, when trehalose is known to accumulate. Similar results have been obtained for the A. nidulans tpsA gene encoding T6P synthase (C. d’Enfert, unpublished). This suggests that accumulation in the conidia of trehalose and of the enzyme responsible for its subsequent mobilization results from pre-existing mRNA or enzymes. The mechanisms that contribute to the accumulation of the neutral trehalase in the conidia remain to be investigated. 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Neutral trehalase of A. nidulans and N. crassa 479 Table 1. Aspergillus nidulans and Neurospora crassa strains used in this study.
Strain
A. nidulans pabaA1 FGSC773 CEA53 CEA54 CEA61 CEA62 CEA103 CEA100
N. crassa FGSC424 FGSC4511
Experimental procedures
Strains and growth conditions A. nidulans and N. crassa strains used in this study are listed in Table 1. A. nidulans and N. crassa FGSC strains were obtained from the Fungal Genetics Stock Centre (University of Kansas, Kansas City, KS, USA). Growth conditions for A. nidulans strains have been described previously (d’Enfert and Fontaine, 1997). Cultures of A. nidulans strains for the assay of trehalase activity were grown for 2 h at 37⬚C in minimal medium supplemented with 100 mM fructose and 0.25 mM pyridoxine.HCl, and were inoculated at 107 ml¹1 conidia. Cultures of A. nidulans strains for the assay of heat shock sensitivity were grown for 2 h at 30⬚C in complete medium containing 0.1% Tween 20 and were inoculated at 2 × 107 ml¹1 conidia. After heat shock (0–40 min at 50⬚C), two aliquots of each culture were withdrawn and were serially diluted in duplicate in PBS–Tween 20 (0.1%). Heat-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 day incubation at 37⬚C. Cultures for the preparation of total RNA from A. nidulans pabaA1 or FGSC773 were in YG. Synchronized conidiogenesis of A. nidulans pabaA1 was as described previously (d’Enfert and Fontaine, 1997). Conidiospore germination was monitored by microscopic examination of slides coated with minimal medium supplemented with or without 50 mM D-glucose and 0.25 mM pyridoxineⴢHCl, spot inoculated with ⬇104 freshly harvested conidiospores of strains CEA53 and CEA103, and incubated at 37⬚C. The percentage of germinated spores was recorded at different times. Variance analysis of the data generated in two separate experiments using two separate growths of conidia and three independent measurements of the percentage of germinated conidia at each time point was carried out using the SuperANOVA software (Abacus Concepts, Berkeley, CA, USA). N. crassa strains were handled and cultivated as described elsewhere (Bonini et al ., 1995). Conidia used for the assay of trehalase activity were germinated for 2 h in minimal Vogel’s (Vogel, 1964) liquid medium (2 × 107 ml¹1 conidia) supplemented with 2% glucose and auxotrophy requirements as 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Genotype
Origin
pabaA1 pyrG89 ; wA3 ; pyroA4 pyrG89 ; wA3 ; pyroA4 ; treA::(neo-A. niger pyrG-neo) pyrG89 ; wA3 ; pyroA4 ; treA::neo pyrG89 ; wA3 ; pyroA4 ; treB::(neo-A. niger pyrG-neo) pyrG89 ; wA3 ; pyroA4 ; treB::neo pyrG89 ; wA3 ; pyroA4 ; treB::(neo-A. niger pyrG-neo); treA::neo pyrG89 ; wA3 ; pyroA4 ; treB::neo; treA::neo
Laboratory collection FGSC d’Enfert and Fontaine (1997)
A wild type A tre (allele 1931 ); inl (allele 89601 )
FGSC FGSC
d’Enfert and Fontaine (1997) This study This study This study This study
needed. At that time, the germlings were harvested by filtration, thoroughly rinsed with chilled distilled water, and used as described below (neutral trehalase assay). Induction of conidiation in N. crassa was carried out essentially as described by Urey (1971) under high-humidity conditions. Instead of using stationary-phase (68 h) mycelium, in which the expression of the NctreB gene is already very low, log-phase mycelium (18 h) exhibiting high expression of NctreB was used. E. coli strain PAP105 [⌬(lac-pro ) F⬘(lacI q1 ) ⌬(lacZ )M15 proþ Tn10 ] was used for plasmid propagation. The -lactam antibiotic carbenicillin (100 g ml¹1 ), kanamycin (50 g ml¹1 ) and tetracycline (15 g ml¹1 ) were added to the growth medium when required.
Neutral trehalase assay and soluble sugar and polyol analysis Non-germinating and germinating A. nidulans conidia were collected by centrifugation or filtration, washed with distilled water, resuspended in water or 50 mM potassium phosphate, pH 7, at a final concentration of 2.5 × 109 ml¹1 conidia, and disrupted by mechanical agitation in the presence of glass beads (0.5 mm) using a vortex. After washing of the beads, the resulting lysate was optionally subjected to an in vitro activation step according to the method of Londesborough and Varimo (1984): three volumes of the crude extract were combined with one volume of a freshly prepared activation mix (10 M cAMP, 1 mM ATP, 5 mM theophyllin, 50 mM NaF, 9 mM Table 2. Oligonucleotides used in this study. Oligonucleotide
Sequence
treBF treBB nth2 nth6 nth9 nth10 tps4 tps5 Ncnth1 Ncnth2
5⬘-YAYCARATYACYATYGARGA-3⬘ 5⬘-AT/ARTANGARTCCCARCCRTA-3⬘ 5⬘-GGAAACGAGCGAGAACCAGA-3⬘ 5⬘-GCACTGAAGAGTCTCGTGGT-3⬘ 5⬘-CCCAGCATTTCATGGAAGTC-3⬘ 5⬘-TTTCCAGGCCGTAAACGTAG-3⬘ 5⬘-GTTGCGAGCCAAGTTCAG-3⬘ 5⬘-CCCTGGAATTCTATCCCA-3⬘ 5⬘-AGGACCGCCATCACAACCAC-3⬘ 5⬘-AGTCGGTTCACGGGGTTCTC-3⬘
480 C. d’Enfert et al. MgSO4 , 50 mM potassium phosphate, pH 7.0) and incubated for 15 min at 30⬚C. Trehalase was assayed by adjusting samples to 50 mM potassium citrate (pH 4–6), potassium phosphate (pH 6–8) or tris-HCl (pH 7–9) and adding trehalose and CaCl2 to give final concentrations of 100 mM and 5 mM, respectively. One-millilitre reaction mixtures were incubated at 30⬚C, and at different times 250 l aliquots were removed and subjected to a 13 000 × g centrifugation for 5 min. Glucose in 200 l of the supernatant was assayed using the Glucose Assay Kit according to the supplier’s instructions (Sigma). Neutral trehalase activity was routinely assayed in 50 mM potassium phosphate, pH 7. Trehalase activity in N. crassa was assayed in permeabilized cells. Briefly, conidia germinated for 2 h were resuspended (74 mg wet weight ml¹1 ) in 50 mM MES buffer, pH 7.0, containing 20 g ml¹1 nystatin (Squibb and Sons), and incubated for 15 min at 30⬚C with gentle agitation. At that time, three volumes of the cell suspension were combined with one volume of a freshly prepared activation mix (10 M cAMP, 1 mM ATP, 5 mM theophyllin, 50 mM NaF, 9 mM MgSO4 , 50 mM potassium phosphate, pH 7.0, 20 g ml¹1 nystatin) and further incubated for 15 min at 30⬚C. Trehalase was assayed by adding one volume of trehalose and CaCl2 in 50 mM MES buffer, pH 7.0, to give final concentrations of 100 mM and 5 mM respectively. The reaction was stopped by boiling the samples for 10 min, the samples were centrifuged and the glucose in the supernatant was assayed using the Glucose Assay Kit according to the supplier’s instructions (Labtest, Brazil). Soluble sugars and polyols were analysed using previously described methods (d’Enfert and Fontaine, 1997).
PCR amplification of a segment of the A. nidulans and N. crassa treB genes The genomic DNA of A. nidulans strain pabaA1 prepared according to the method of Girardin et al . (1993) and N. crassa strain FGSC424 prepared according to the method of Raeder and Broda (1985) were used as templates to amplify a segment of genes potentially encoding a neutral trehalase. A sense primer (treBF, Table 2) was based on an amino-acid sequence (MQITIED) conserved in several yeast neutral trehalases and in an M. grisea protein with strong homology to yeast neutral trehalases (Kopp et al ., 1993; Nwaka et al ., 1995a; Amaral et al ., 1997; Sweigard et al ., 1998). The antisense primer (TreBB, Table 2) was derived from a conserved amino acid sequence (YGWDSYM/F) found in the E. coli TreA, S. cerevisiae Nth1 and Nth2, K. lactis Nth1 and rabbit trehalases (Guttierrez et al ., 1989; Ruf et al ., 1990; Kopp et al ., 1993; Nwaka et al ., 1995a; Amaral et al ., 1997). The amplification protocol consisted of a denaturation step at 93.5⬚C for 5 min, followed by 35 cycles of the following steps: denaturation at 93.5⬚C for 30 s, annealing at 55⬚C for 1 min, extension at 71⬚C for 3 min. A last elongation step was carried out at 71.5⬚C for 5 min. Amplification products were subcloned using the TA cloning kit according to the supplier’s instructions (Invitrogen).
DNA 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 previously (Hanahan et al ., 1991). Oligonucleotides used in this study were obtained from Genset (Paris, France) and are listed in Table 2. PCR products obtained using either A. nidulans or N. crassa genomic DNA as template were labelled with [␣-32P]-dCTP using the Megaprime kit (Amersham). The A. nidulans fragment was used to probe replicas of 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). pNTH3 and pNTH4 are derivatives of pBLSNþ (d’Enfert, 1996) and they carry a 3.8 kb Eco RI fragment of cosmid L04C12 in opposite orientations. pNTH7 and pNTH8 are pBLSNþ derivatives that carry a 2.9 kb Bam HI fragment of cosmid L04C12 in opposite orientations. DNA sequencing was performed by the dideoxy chain termination method (Sanger et al ., 1977) on double-stranded plasmids derived from pNTH3, pNTH4, pNTH7 and pNTH8 by internal restriction enzyme-mediated deletions and using a set of appropriate oligonucleotide primers. The sequence of a 3475 bp fragment was read at least twice on each strand and is deposited at the GenBank nucleotide sequence data library under accession number AF043229. The N. crassa PCR product was used to probe a AD5NC N. crassa cDNA library (Brunelli and Pall, 1993), obtained from the Fungal Genetic Stock Center. Positive clones were characterized by restriction analysis and the largest (2.9 kb) was subjected to plasmid excision through transformation of E. coli BNN132. The excised plasmid was used for sequencing of the cDNA insert at the Core Facility for Protein/DNA Chemistry, Queen’s University at Kingston, Canada. The sequence for both strands was obtained and is available at the GenBank nucleotide sequence data library under accession number AF044218. Plasmid pNTH10 was obtained by first subcloning the 4.2 kb Hin dIII/ Bgl II fragment of cosmid L04C12 into Hin dIII/ Bam HI-digested pBLSNþ, yielding pNTH9, and then by subcloning the 8.6 kb Hpa I fragment of pCDA14 (d’Enfert, 1996) into Pma CI/ Sma I-digested pNTH9. Not I-digested pNTH10 was used to transform protoplasts of A. nidulans strains FGSC773 and CEA54 as described previously (Osmani et al ., 1987). Prototrophic transformants were first screened by a spore PCR assay using a set of four primers: nth2 and nth9 (Table 2) yield a 298 bp product that is only present in treB þ transformants, whereas tps4 and tps5 (Table 2) yield a 363 bp product that is present in treB þ and treB ¹ transformants. Spore extracts were prepared according to Xu and Hamer (1995) and were subjected to the following amplification protocol. A denaturation step at 93.5⬚C for 5 min followed by 35 cycles of the following steps: denaturation at 93.5⬚C for 30 s, annealing at 60⬚C for 1 min, and extension at 71⬚C for 1.5 min. A last elongation step was carried out at 71.5⬚C for 5 min. Putative treB mutants were further analysed by Southern analysis of Bam HI- or Bgl II-digested genomic DNA prepared according to Girardin et al . (1993) and probed with a 1.4 kb Eco RI fragment of pNTH7 that had been labelled with the Rediprime labelling kit (Amersham). Washed membranes were exposed to X-omat films (Kodak). Conversion of a 2.8 kb Bam HI fragment to a 6.9 kb Bam HI fragment and of a 7.8 kb Bgl II fragment to a 3.0 kb Bgl II fragment 䊚 1999 Blackwell Science Ltd, Molecular Microbiology, 32, 471–483
Neutral trehalase of A. nidulans and N. crassa 481 was indicative of a gene replacement at the treB locus. Conversion of the treB::(neo-A. niger pyrG-neo) allele to the treB::neo allele was obtained by in vivo excision of the A. niger pyrG gene as previously described (d’Enfert and Fontaine, 1997). Excision of the A. niger pyrG gene was confirmed by Southern analysis as described above. In this case, the 6.9 kb Bam HI fragment was converted to a 7.8 kb Bam HI fragment, whereas the 3.0 kb Bgl II fragment remained intact.
Ncnth2 oligonucleotides are sense and antisense primers located on both sides of at least two introns, one of 75 bp found in the PCR-amplified probe, and yields a ⬇600 bp product from genomic DNA, and a 422 bp product from reverse-transcribed mRNA. RT-PCR products were analysed by Southern analysis using the product of the amplification of A. nidulans DNA with primers nth6 and nth9 or N. crassa DNA with primers Ncnth1 and Ncnth2 as probes.
RNA preparation and analysis
Acknowledgements
Total RNA from A. nidulans strain pabaA1 was prepared according to the method of Chomczynski and Sacchi (1987). Conidia, germinating conidia, mycelia and developing cultures of A. nidulans were collected by filtration, frozen in liquid nitrogen, disrupted by manual grinding in the presence of liquid nitrogen and resuspended in 1 ml denaturation solution (4 M guanidium isothiocyanate, 20 mM sodium acetate, pH 5.2, 0.1 mM DTT, 0.5% sarkosyl adjusted to pH 6.5 with NaOH) with 0.1 g cells. After homogenization and addition of 0.1 volume 2 M sodium acetate, pH 4.0, one volume watersaturated phenol and 0.2 volume chloroform/isoamylalcohol (49:1), the mixture was incubated for 15 min on ice and centrifuged. RNA in the aqueous phase was precipitated twice with isopropanol, washed with 75% ethanol, dried and resuspended in DEPC-treated water. RNA quantification in the different samples was achieved both by UV adsorption and by running an aliquot on MOPS/formaldehyde gel (Ausubel et al ., 1992). Samples were normalized for similar levels of 28S and 18S rRNA. Alternatively, total RNA from A . nidulans FGSC773 was prepared using the Rneasy Plant mini kit according to the supplier’s instructions (Qiagen). Total RNA from N. crassa strain FGSC424 was prepared according to Jones et al . (1985). Lyophilized conidia, germinating conidia and mycelia were resuspended in 4.5 ml of 0.1 M NaCl, 10 mM tris-HCl, pH 7.5, 1 mM EDTA, 1% SDS and 3 ml of water-saturated phenol/ chloroform/isoamylalcohol (25:24:1). After homogenization in a Vortex, the aqueous phase was precipitated twice with two volumes of frozen 100% ethanol plus 0.1 volume of 2 M sodium acetate, washed with 75% ethanol, dried and resuspended in DEPC-treated water containing 2 M lithium acetate. After 2 days at ¹20⬚C, the pellet was resuspended in DEPCtreated water. RT-PCR experiments were carried out using the Reverse Transcription System according to the manufacturer’s instructions (Promega). Approximately 1 g of 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 nth6 and nth10 (A. nidulans reversetranscribed mRNAs) or Ncnth1 and Ncnth2 (N. crassa reversetranscribed mRNAs) (Table 2). First, 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 cycles in order to remain in a linear range and therefore produce semiquantitative data. The nth6 and nth10 oligonucleotides are sense and antisense primers, respectively, that are located on both sides of an intron in the AntreB gene. Therefore, amplification from genomic DNA yields a 391 bp product whereas amplification from reverse-transcribed mRNA yields a 336 bp product. Similarly, the Ncnth1 and
We would like to thank J. Thevelein for suggesting the use of nystatin-permeabilized cells, M. Rigoulet for the nystatin permeabilizaton procedure, J. Sweigard for communicating data before publication, and J.-P. Latge´ for statistical analysis of the data generated during the course of this work. Expert assistance of M. Cormier in typing this manuscript was particularly appreciated as was the encouragement of the members of the Laboratoire des Aspergillus . This work was supported in part by grants from FAPESP (96/2902-4 and 96/1428-7) and CNPq to A.M.S. and to H.F.T. B.M.B. and P.D.A.Z. received predoctoral fellowships from CAPES and FAPESP respectively.
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