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Curr Genet (1998) 34: 50–59

© Springer-Verlag 1998

O R I G I N A L PA P E R

Rosa-Elena Cardoza · Francisco-José Moralejo Santiago Gutiérrez · Javier Casqueiro Francisco Fierro · Juan F. Martín

Characterization and nitrogen-source regulation at the transcriptional level of the gdhA gene of Aspergillus awamori encoding an NADP-dependent glutamate dehydrogenase Received: 13 January / 12 May 1998

Abstract A 28.7-kb DNA region containing the gdhA gene of Aspergillus awamori was cloned from a genomic DNA library. A fragment of 2570 nucleotides was sequenced that contained ORF1, of 1380 bp, encoding a protein of 460 amino acids (Mr 49.4 kDa). The encoded protein showed high similarity to the NADP-dependent glutamate dehydrogenases of different organisms. The cloned gene was functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase to the transformants. The A. awamori gdhA gene was located by pulsed-field gel electrophoresis in a 5.5-Mb band (corresponding to a doublet of chromosomes II and III), and was transcribed as a monocistronic transcript of 1.7 kb. Transcript levels of the gdhA gene were very high during the rapid growth phase and decreased drastically after 48 h of cultivation. Very high expression levels of the gdhA gene were observed in media with ammonium or asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. These results indicate that expression of the gdhA gene is subject to a strong nitrogen regulation at the transcriptional level. Key words Glutamate dehydrogenase · Nitrogen regulation · Aspergillus awamori · Chromosome resolution · Promoter

R.-E. Cardoza · F.-J. Moralejo · S. Gutiérrez · J. Casqueiro F. Fierro · J. F. Martín Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avda. del Real, 1, E-24006 León, Spain J. F. Martín (½) Universidad de León, Facultad de Biología, Area de Microbiología, E-24071 León, Spain Tel.: +34-87-291505 Fax: +34-87-291506 e-mail: [email protected] Communicated by K. Esser

Introduction

Aspergillus awamori is widely used for the secretion of extracellular proteins, including homologous proteins [e.g. glucoamylase (Finkelstein et al. 1989), α-amylase (Korman et al. 1990), 1.4-beta-endoxylanase (Hessing et al. 1994)] and a variety of heterologous polypeptides (Verdoes et al. 1995; Gouka et al. 1996). The basic molecular genetics of A. awamori is largely unknown. This species is amenable to efficient transformation (Gouka et al. 1995) but only a few A. awamori genes have as yet been cloned and characterized (Berka et al. 1990; Thompson 1990; Adams and Royer 1990; Piddington et al. 1993; Hijarrubia et al. 1997). Nitrogen regulation of metabolism in filamentous fungi (Marzluf 1997) is particularly interesting in the context of gene expression for protein secretion. The proteins to be secreted are targeted to the endoplasmic reticulum and proceed via the Golgi system to the cytoplasmic membrane, where they are released. During protein secretion the polypeptides to be secreted are subject to “quality controls” by several mechanisms (Hammond and Helenius 1995) and those that do not fold adequately for transport through the different secretory compartments are degraded by proteases. Proteolytic processing plays an important role in protein secretion. In addition to their involvement in protein degradation, specific peptidases (e.g. the KEX protease cleaving specifically at Arg-Lys sites in polypeptides; Calmels et al. 1991) are used to release polypeptides of interest attached to carrier extracellular proteins (Gouka et al. 1997). These proteolytic systems are subject to nitrogen regulation (Cohen 1973). NADP-dependent glutamate dehydrogenase (GDH) (encoded by gdhA) has been shown to play an important role in nitrogen metabolism and regulation in Aspergillus nidulans (Kinghorn and Pateman 1973a). The gdhA mutants lack repression by ammonium of a number of enzymes and uptake systems, including extracellular proteases, nitrate reductase, xanthine dehydrogenase and glutamate uptake (Kinghorn and Pateman 1973b).

51

It was, therefore, of great interest to clone and characterize the gdhA gene of A. awamori as a tool to study nitrogen metabolism and its regulation in this fungus. In this article we report the characterization of the A. awamori gdhA gene encoding NADP-dependent glutamate dehydrogenase, the complementation of gdhA mutants, and transcriptional studies with different nitrogen sources.

Materials and methods Microorganisms and cloning vectors. A. awamori ATCC 22342 was used as the source of DNA and RNA. A. nidulans mutants A686 (gdhA1, yA2, methH2, galA1) and A699 (gdhA1, biA1) (Kinghorn and Pateman 1973a) were obtained from the Fungal Genetics Stock Center, and used for complementation studies with the gdhA gene from A. awamori. The partial glutamate auxotrophy of these two strains was confirmed by growth on media with glutamic acid, or high ammonium sulphate concentrations (100 mM), as a nitrogen source. Both gdhA mutants grow very poorly on high ammonium sulphate concentration but showed normal growth on glutamic acid. A. nidulans ATCC 28901 (Ditchburn et al. 1974) was used as a control in the complementation studies of the gdhA mutants. Escherichia coli DH5α served as the recipient strain for high-frequency plasmid transformation. E. coli WK6 was used as a host for obtaining single-stranded DNA from pBluescript plasmids, and E. coli NM539 served as a host for Lambda GEM12 (Promega Co., Wis.) phage derivatives. Media and culture conditions. Filamentous fungi were routinely maintained on solid-Power sporulation medium (Fierro et al. 1996) at 30°C for 3 days. A. awamori and A. nidulans seed cultures in CM medium (containing 20 g/l malt extract; 5 g/l yeast extract; 5 g/l glucose) were inoculated with 106 spores/ml and grown at 28°C in a rotary G10 incubator (New Brunswick Scientific, New Brunswick, N. J.) for 48 h. For gdhA transcript isolation and characterization studies, A. awamori cultures in MDFA medium (Shen et al. 1984) were incubated with a 15% seed culture and grown at 30°C for 48–72 h in a rotary shaker as described above. A. awamori genomic library. A genomic library of total DNA of A. awamori ATCC 22342 was constructed in a Lambda GEM12 phage vector. Total DNA was extracted as described by Specht et al. (1982) and partially digested with Sau3AI to obtain DNA fragments of between 17 and 23 kb. This DNA was purified by sucrose-gradient centrifugation, ligated to Lambda GEM12 phage arms, and packaged in vitro using a Gigapack III Gold packaging system (Stratagene), resulting in a total of 8×104 recombinant phages. Transformation of A. nidulans gdhA mutants. A. nidulans A686 and A699 were transformed by the method of Yelton et al. (1984) with the 7.1-kb plasmid pGDHaw that contains the A. awamori gdhA gene in a 2570-bp XbaI fragment. This fragment also contains an upstream promoter region of 740-bp and a 322-bp region downstream from ORF1 (gdhA gene). The 2570-bp XbaI fragment was inserted into the XbaI site of the fungal vector pIBRC43 that contains the phleomycin resistance marker (under the control of the A. awamori gdh promoter), giving rise to plasmid pGDHaw. Southern blotting and hybridization. DNA from positive phages and total DNA from A. awamori was digested with restriction endonucleases, electrophoresed in 0.7% agarose and blotted by standard techniques (Sambrook et al. 1989). Probes were labelled by nick translation with [32P]dCTP and hybridized by standard methods (Sambrook et al. 1989). Isolation of RNA, Northern hybridization and slot blotting. Total RNA of A. awamori was obtained by the phenol-SDS method (Au-

subel et al. 1987) from mycelia grown for 12, 24, 48, 60 or 72 h in MDFA medium (Shen et al. 1984) with 55.5 mM of glucose and 10 mM of ammonium sulphate as carbon and nitrogen sources respectively. For nitrogen-regulation studies, the MDFA base medium (without ammonium sulphate) was supplemented with different nitrogen sources including glutamic acid, L-glutamine, sodium nitrite, sodium nitrate and L-asparagine at a 10 mM final concentration. For Northern analysis, total RNA (5 µg) was run on a 1.2% agarose-formaldehyde gel. The gel was blotted onto a nylon filter (Nytran 0.45; Schleicher and Schuell) by standard methods. The RNA was fixed by UV irradiation using a UV-Stratalinker 2400 lamp (Stratagene, La Jolla, Calif.). For slot blotting, the RNA (5 µg) was loaded on a filter (Nytran 0.45), by vacuum, in a Bio-Dot SF Microfiltration apparatus (Slot Blotting, Bio-Rad). The RNA was fixed by UV irradiation as above. The filters were pre-hybridized for 3 h at 42°C in 50% formamide, 5× Denhardt’s, 5×SSPE, 0.1% SDS, 500 µg of denatured salmon-sperm DNA per ml, and hybridized in the same buffer containing 100 µg of denatured salmonsperm DNA per ml at 42°C for 18 h, using as a probe an internal fragment (0.694-kb PvuII) of the A. awamori gdhA gene. The filter was washed once in 2×SSC, 0.1% SDS at 42°C for 15 min, once in 0.1×SSC, 0.1% SDS at 42°C for 15 min, and once more in 0.1× SSC, 0.1% SDS at 55°C for 20 min and then autoradiographed with Amersham X-ray film. The mRNA was purified from total RNA by using the Poly(A) Quick mRNA-isolation kit (Stratagene, La Jolla, Calif.). cDNA synthesis and PCR amplification. cDNA was obtained from total RNA extracted as described above from mycelia grown for 48 h in MDFA medium. The first and second cDNA strands were synthetized using a cDNA synthesis kit from Stratagene (La Jolla, Calif.). This cDNA was used for PCR-amplification of the fragments containing the exon-exon junctions by the following program: 1 cycle at 94°C for 5 min, 50°C for 1 min, 72°C for 1 min followed by 30 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min and finally one cycle at 72°C for 8 min. Primer extension. Identification of the transcription start point was performed with 2 µg of mRNA obtained from mycelia grown in MDFA for 48 h using the procedure described by Ausubel et al. (1987). Extension was primed with the oligonucleotide “Pe” 5′GGGGTTCTTCTGGAAGAGGGT-3′ (see Results). DNA sequencing. DNA fragments containing sequences that hybridized with gdhA were subcloned into pBluescript SK+ in both orientations, and sequenced by generating ordered sets of deletions with the Erase-a-base system (Promega Co., Wis.) by digestion with exonuclease III from appropriate ends, followed by removal of single-stranded DNA with S1 exonuclease. Sequencing of fragments of the gdhA gene was performed by the dideoxynucleotide chain-termination method (Sanger et al. 1977). For sequencing the cDNA clones containing the exon-exon junctions, reactions were performed with 90 ng of dsDNA using the GeneAmp PCR 2400 system coupled to the ABI-PRISM 310 automatic sequencer (Perkin Elmer). Computer analysis of nucleotide and amino-acid sequences was made with the DNASTAR Programs (DNASTAR, Inc., UK). Enzyme assays. Nicotinamide adenine dinucleotide phosphate (NADP)-specific glutamate dehydrogenase (NADP-GDH) was assayed by following the reductive amination of α-ketoglutarate in the presence of ammonia and NADPH (Kinghorn and Pateman 1973a) and expressed as units of enzyme activity per mg of protein. The initial reaction velocity was estimated from the change in optical density at 340 nm in a Hitachi U-2001 spectrophotometer. One unit of glutamate dehydrogenase was defined as the activity that catalyzes the oxidation of 1 nmol of NADPH per min. Accession number. The nucleotide sequence of the A. awamori gdhA gene has been deposited in the EMBL GeneBank under the accession number Y15784.

52 Oligonucleotides. The following oligonucleotides were used as primers for introns I and II: IA 5′ ATG TCT AAC CTT CCT CAC 3′ IB 5′ ACC CTT ACC ACC ACC CAT 3′ IIA 5′ CGC TTC TGT GTT TCC TTC 3′ IIB 5′ GTA CTT GAA CTT GTT GGC 3′ Chromosome resolution by pulsed-field gel electrophoresis. Protoplasts of A. awamori were prepared by the method of Yelton et al. (1984). High-molecular-weight DNA was obtained by lysing the protoplasts in agarose blocks as described previously (Fierro et al. 1993). Resolution of the chromosomes was performed in 0.6% agarose (FastLane, FMC, Rockland, Me., USA) in 0.5× modified TBE (buffer 100 mM Tris, 100 mM boric acid; 0.2 mM NaEDTA) in a CHEF DRII apparatus (BioRad). The buffer was kept at 10°C in the electrophoresis chamber and changed every 48 h. Electrophoresis conditions and pulse times were as indicated in the legend to Fig. 4. The gels were blotted onto nylon membranes by capillarity, and hybridized with a 0.694-kb PvuII probe, internal to the gdhA gene, labelled by nick translation (Gutiérrez et al. 1991). Pre-hybridization and hybridization were made in standard buffer with 40% formamide (Sambrook et al. 1989) at 42°C.

Results

Cloning of a DNA fragment of A. awamori containing the gdhA gene A genomic library of A. awamori was constructed in the Lambda phage derivative λGem12 (Promega) as described in Materials and methods. Using as a probe the 2.6-kb BamHI fragment containing the gdhA gene of Neurospora crassa (Kinnaird et al. 1982; Kinnaird and Fincham 1983), two phages, FAN1 and FAN2, that gave a clear hybridization signal, were isolated and purified by three rounds of infection. Restriction mapping of these two phages showed that they overlap in 7.2 kb. The total DNA region cloned in the two phages extended for 28.7 kb. BamHI fragments of 1.7, 5.5 and 10 kb were subcloned in pBluescript KS+ giving rise to plasmids pB1.7, pB5.5 and pB10 (Fig. 1). Initial sequencing showed that an open reading frame (ORF1) occurred at the right end of the 5.5kb insert of pB5.5 extending into the left region of the 1.7kb BamHI fragment of pB1.7. The 5.5-kb and 1.7-kb BamHI fragments were mapped in detail. A 2.1-kb XbaIBamHI fragment corresponding to the right end of pB5.5 was subcloned in pBluescript SK+, creating plasmid pBSGh. A region of 2570 nt was sequenced in both strands by the dideoxynucleotide chain-termination method. This region contained ORF1, of 1380 bp, that started at an ATG located 740 bp downstream from the left end of the insert in pBSGh and extended until the end of the 5.5-kb BamHI fragment with 60 additional bp into the adjacent 1.7-kb fragment. ORF1 was preceeded by a 740-nucleotide region that contained the signals required for transcription initiation and regulation (see below). ORF1 contained two putative introns at positions 45–120 and 673–732 that showed lariat and 5′ and 3′ splicing sequences similar to those of other fungal introns (Ballance 1986). The presence of the two introns was confirmed by sequencing the DNA regions (corresponding to introns

Fig. 1 A restriction map of a 28.7-kb region of A. awamori DNA including the gdhA gene. pB10, pB5.5 and PB1.7 indicate the DNA fragments subcloned in the corresponding plasmids. The 3′ end of the gdhA gene was contained in the left region of the insert in pB1.7. B=BamHI, E=EcoRI, EV=EcoRV, P=PstI, S=SalI, X=XbaI. B sequenced DNA region. The arrow indicates the position of the gdhA gene

I and II) obtained by PCR from an A. awamori cDNA library using as primers oligonucleotides IA and IB for intron I, and IIA and IIB for intron II. ORF1 encodes a putative NADP-dependent glutamate dehydrogenase ORF1 encoded a protein of 460 amino acids with a deduced molecular mass of 49.4 kDa and a pI value of 5.62. Comparison of the protein encoded by ORF1 with proteins in the SWISS-PROT data base showed that the encoded protein has a high homology with the NADP-dependent glutamate dehydrogenases of A. nidulans (84.7% identical amino acids), N. crassa (74.4% identity), Saccharomyces cerevisiae (66.5% identity) and Schwanniomyces occidentalis (66.9% identity) (Fig. 2). All these proteins are NADP-dependent glutamate dehydrogenases that catalyze the reductive amination of α-ketoglutarate to form L-glutamate. The encoded protein contains nine conserved motifs when compared with other fungal and yeast glutamate dehydrogenases. One of the conserved domains (amino acids 108–121) corresponds to a region implicated in the catalytic mechanism. The consensus sequence of this region is [LIV]-X(2)-G-G-[SAG]-K-X-[GV]-X(3)-[DNS]-[PL] (PROSITE PS00074). The lysine residue K114, located in the glycine-rich region GGGK114GG, corresponds to the lysine in the active center of Glu/Leu/Phe/Val (GLFV) de-

53 Fig. 2 Alignment of the deduced amino-acid sequences of NADP-specific glutamate dehydrogenases of A. awamori (this work), A. nidulans (accession number P18819), N. crassa (P00369 ), S. cerevisiae (P07262), S. occidentalis (P29507), A. bisporus (P54387), S. typhimurium (P15111), E. coli (P00370) and C. glutamicum (P31026). Identical amino acids are shadowed. Motifs a to i with several consecutive conserved residues are overlined

hydrogenases. The gene encoded by ORF1 was, therefore, named gdhA according to standard fungal gene nomenclature. The cloned gene complements A. nidulans gdhA mutants Fourteen transformants of A. nidulans A686 with the A. awamori gdhA gene and 30 transformants of A. nidulans A699 were analyzed on minimal medium supplemented with high (10, 50 and 100 mM) ammonium sulphate as a nitrogen source and their growth was compared with that of the wild-type A. nidulans. As a control, growth was also tested on 10 mM glutamic acid. The untransformed A. nidulans mutants A686 and A699 grow very poorly in plates

with 100 mM ammonium sulphate, whereas all tested (randomly selected) transformants grow well in this medium. The residual growth of A. nidulans gdhA mutants A686 and A699 in ammonium sulphate as a nitrogen source has been described by Kinghorn and Pateman (1973b) and is due to the presence of a second glutamate dehydrogenase activity that allows partial growth of these mutants. Glutamate dehydrogenase activity and copy number of the gdhA gene in the transformants To confirm the complementation results, the NADP-dependent glutamate dehydrogenase activity was measured in the A. nidulans gdhA mutants A686 and A699, and in

54 Table 1 NADP-dependent glutamate dehydrogenase activity in the A. awamori and in the A. nidulans gdhA mutants A686 and A699, and in three transformants of each of these mutants with the A. awamori gdhA gene NADP-dependent glutamate dehydrogenase (units/mg of protein) Time (h)

Aspergillus awamori

Aspergillus nidulans

A686

A686-4

A686-6

A686-7

A699

A699-2

A699-3

A699-4

24 48 72

210 0 0

97 12.8 0

0 0 0

350 280 100

340 200 80

310 160 90

0 0 0

270 240 100

410 420 400

500 670 580

Fig. 3 Southern-blot analysis of the copy number of the A. awamori gdhA gene in different transformants of A. nidulans A686 and A699. DNAs were digested with XbaI and hybridized with a 694-bp PvuII fragment of the gdhA gene and a 905-bp NcoI-KpnI fragment of the A. nidulans β-actin gene as probes. The relative copy number of the gdhA gene in the transformants is indicated at the bottom of the figure

three randomly selected transformants complemented with the A. awamori gdhA gene. The results (Table 1) clearly indicated that the glutamate dehydrogenase activity in strains A686 and A699 was below detection level whereas significant levels of glutamate dehydrogenase activity were obtained in the transformants with the A. awamori gdhA gene, particularly at 24 and 48 h of growth. Some of the transformants, like A699-4, showed relatively high levels of glutamate dehydrogenase activity, perhaps due to the integration of more than one copy of gdhA in the genome of this transformant. To confirm this hypothesis Southern hybridizations of the transformants were made with the gdhA probe using as a control hybridizations with the β-actin gene. As shown in Fig. 3, transformants A686-4, A686-6, A686-7 and A699-2 contain one copy of the A. awamori gdhA gene, whereas transformant A699-3 contains two and transformant A699-4 contains four identical copies. The genome of A. awamori is resolved into seven bands: gdhA is located in the doublet of chromosomes II or III Identification of the chromosomal locus of gdhA was of great interest for further molecular analysis of the trans-

formants. As shown in Fig. 4, experiments using two different electrophoresis conditions allowed the resolution of seven bands that appear to contain nine chromosomes. Low-molecular-weight chromosomes were resolved using a pulse time of 156 h with ramped pulses from 35 to 50 min (gel 1, Fig. 3A). Under these conditions five bands were resolved in A. awamori and six bands in A. nidulans (as expected from previous results, Montenegro et al. 1992). The five A. awamori bands corresponded to one minichromosome of 1.4 Mb, two bands of 2.8 and 3.1 Mb, an intense band (presumably a doublet) of 4.0 Mb, and a large band of unresolved chromosomes in the upper part of the gel. In the second gel (Fig. 3B) the unresolved chromosomes were separated into three bands of 6.1 Mb, another (apparently a doublet) of 5.5 Mb, and a third of 4.7 Mb. The chromosomes have been numbered by decreasing size from chromosome I (6.1 Mb) to chromosome IX (1.4 Mb). When the PFGE gels were hybridized with a 0.694-kb PvuII probe internal to the gdhA gene, the results showed that this gene in A. awamori is located in a band of 5.5 Mb (a doublet of chromosomes II and III) (Fig. 3C). In the first gel the hybridization corresponds to the large band of unresolved high-size chromosomes (data not shown). In A. nidulans, used as a control, hybridization corresponded to a band of 3.2 Mb (a doublet of chromosomes III and VI). This results correlates well with the classical mapping of A. nidulans where the gdhA gene was located in linkage group III (Clutterbuck 1982, 1997). In both gels there was a band of small-molecular-weight DNA that presumably corresponds to mitochondrial DNA and to degraded small fragments of chromosomal DNA (Fierro et al. 1993). Characterization of the promoter region Analysis of the nucleotide sequence upstream from the ATG translation initiation codon revealed the presence of GTATA, CTATA and TCAATC sequences at positions –316, –61 and –17, respectively, with respect to the translation initiation codon that may correspond to the putative TATA and CAAT boxes involved in the regulation of gene expression. Primer extension analysis, using as a primer the oligonucleotide “Pe” 5′-GGGGTTCTTCTGGAAGAGGGT-3′ (corresponding to the nucleotide sequence 70 bp downstream from the ATG), revealed a single band in the exten-

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Fig. 4A–C Separation of the chromosomes of A. awamori by pulsed-field gel electrophoresis and location of the gdhA gene. A resolution of the smaller chromosomes of A. awamori (lane 4). Chromosomes from S. cerevisiae (lane 1), S. pombe (lane 2) and A. nidulans ATCC 12996 (lane 3) were used as molecular-weight markers to calculate the sizes of the A. awamori chromosomes. The sizes of the three large S. pombe chromosomes and the two largest ones of S. cerevisiae are indicated in megabase pairs at the left of the panel. The sizes of the six chromosomal bands of A. nidulans are 5.0, 4.5, 4.1, 3.7, 3.2 and 2.4 Mb according to Montenegro et al. (1992). The A. awamori chromosomes contained in each of the five resolved bands are indicated at the right of the panel. B resolution of the large chromosomes of A. awamori (lane 1); the upper band of panel A is separated into three different bands in this gel; one of them is a doublet of chromosomes II and III. The chromosomes assigned to each band are indicated on the left and the sizes of control S. pombe chromosomes are shown on the right (lane 2). C location of the gdhA gene in A. awamori; the hybridization signal appears on the doublet containing chromosomes II and III. Electrophoresis conditions were as follows. For gel 1 (panel A): total electrophoresis time, 156 h; pulse time, ramp from 35 min to 50 min; voltage, 40 V. For gel 2 (panel B): total electrophoresis time, 180 h; pulse time, ramp from 45 min to 90 min; voltage, 40 V

sion reaction (Fig. 5). The 5′-end of the RNA corresponds to a T located 86 bp upstream of the ATG (indicated with a bent arrow in Fig. 5). The gdhA gene is transcribed as a monocistronic transcript of 1.7-kb Northern analysis of the transcription of the gdhA gene revealed that it is strongly expressed as a 1.7-kb mRNA with a size slightly larger than that of the β-actin mRNA. Since ORF1 contains 1380 nt, this size indicates that gdhA is expressed as a monocistronic transcript. The gdhA steady state transcript levels in the cell were high, indicating that the glutamate dehydrogenase is expressed from a very efficient promoter.

gdhA is efficiently expressed during the rapid growth phase To determine the pattern of expression of the gdhA gene during the growth time-course of A. awamori, gdhA-hybridizing RNA was compared to β-actin-hybridizing RNA in MDFA medium with ammonium sulphate (Fig. 6 a) and expressed as the ratio of counts in the gdhA-hybridizing band to the β-actin hybridizing counts (Fig. 6B). The results indicate that the expression of both gdhA and the βactin genes is associated with growth: but, whereas low steady state levels of β-actin mRNA remained in the cells until 96 h, the levels of glutamate dehydrogenase mRNA decreased drastically after 48 h. The glutamate dehydrogenase activity in MDFA medium with ammonium sulphate (10 mM) as a nitrogen source at different times of culture is shown in Fig. 6C. There is a sharp decrease in glutamate dehydrogenase activity from 24 to 48 h in good agreement with the decrease in transcript levels in the culture at this time. Nitrogen regulation of expression of the gdhA gene Since glutamate dehydrogenase plays a central role in nitrogen utilization it was of interest to study if expression of gdhA was regulated by different nitrogen sources. As Table 2 NADP-dependent glutamate dehydrogenase activity in A. awamori cultures grown for 24 h in MDFA medium with different nitrogen sources NADP-dependent glutamate dehydrogenase Nitrogen source (10 mM)

Total activity (U/ml)

Specific activity (U/mg protein)

NH4+ Glutamic acid Glutamine NO2– NO3– Asparagine

1450 330 1100 990 1150 1300

800 280 600 660 1680 720

56

shown in Fig. 7, very high gdhA transcript levels were obtained in 24-h cultures in MDFA medium containing NH4+, or asparagine, as sole nitrogen sources. Glutamic acid repressed the transcription of the gdhA gene whereas intermediate levels of expression (normalized with respect to the β-actin) were observed in media with nitrate, glutamine or nitrite. These results indicate that the NADP-dependent glutamate dehydrogenase is subject to strong nitrogen regulation at the transcriptional level. The glutamate dehydrogenase in 24-h cultures grown in the different nitrogen sources (10 mM) is shown in Table 2. The highest activity per ml of culture was observed in cultures with NH4+ or asparagine as nitrogen sources. These two nitrogen sources favored a strong growth. When expressed per mg of protein in the cell extracts, the highest specific activity was observed in medium with nitrate due to the low growth in MDFA medium with nitrate as the sole nitrogen source. The lowest activity was observed in medium with glutamate as the nitrogen source, confirming the results observed at the mRNA level.

Discussion

L-glutamate dehydrogenases which use either NAD or NADPH as coenzymes occur in different organisms (Brit-

Fig. 5A, B Primer extension identification of the 5′ end of the gdhA transcript. The primer extension reaction was performed as indicated in Materials and methods. A one protected band (arrow) is observed in the extension reaction (lane Pe). The G, A, T, and C lanes correspond to the sequencing reactions of M13 phage from the –40 primer. B promoter region of the gdhA gene of A. awamori (0.74-kb fragment). The GTATA, CTATA and CAAT boxes are underlined in bold face and the initiation of the transcription is indicated by a bent arrow at the T of position –86

ton et al. 1992; Benachenhou-Lahfa et al. 1993). In A. nidulans there are two distinct glutamate dehydrogenases encoded by different structural genes. An NADP-dependent glutamate dehydrogenase (E. C.1.4.1.4), encoded by the gdhA gene (Gurr et al. 1986; Hawkins et al. 1989), catalyzes the reductive amination of α-ketoglutarate to glutamate with ammonium as the nitrogen source (Arst and

57

Fig. 7 Relative level of expression of gdhA to the β-actin gene in different nitrogen sources. Transcript levels of the A. awamori gdhA and β-actin genes from mycelia grown for 24 h in MDFA medium with different nitrogen sources were quantified by slot-blot analysis. The medium contained ammonium sulphate 10 mM as a control and glutamic acid, glutamine, sodium nitrite, sodium nitrate and asparagine as nitrogen sources at 10 mM each

Fig. 6A–C Slot-blot analysis of the transcript of the A. awamori gdhA gene during the course of the fermentation in MDFA medium with glucose 1% and ammonium sulphate 10 mM (panel A). For comparative purposes the transcript of the β-actin gene in the same RNA sample was also studied. B relative level of the expression of the gdhA to the β-actin gene. C NADP-dependent glutamate dehydrogenase activity in the same cultures from where the RNAs were extracted

McDonald 1973; Kinghorn and Pateman 1973a). A second NAD-dependent glutamate dehydrogenase, encoded by gdhB, has a de-aminating catabolic function (Kinghorn and Pateman 1976). In S. cerevisiae a third glutamate dehydrogenase isozyme, encoded by the GDH3 gene, has been recently reported (Avendaño et al. 1997). The gdhA gene of A. awamori cloned in the present work resembles the gene of A. nidulans. Both of them encode proteins of similar size with a molecular mass of 49.4 kDa for the A. awamori, as compared to 49.6 kDa for the A. nidulans, enzyme. The A. awamori NADP-GDH has

84.7% identical amino acids to the corresponding enzyme of A. nidulans (Hawkins et al. 1989), 74.4% identity to that of N. crassa (Kinaird and Fincham 1983), 66.5% with that of S. cerevisiae (Moye et al. 1985) and 66.9% with the enzyme of Schwanniomyces occidentalis (De Zoysa et al. 1991). The conservation is clearly smaller when compared with the glutamate dehydrogenases of E. coli (47.4% identical amino acids), Salmonella typhimurium (49.0%) and Corynebacterium glutamicum (45.9%). The cloned A. awamori gdhA complements the glutamate-dehydrogenase deficiency of two different A. nidulans gdhA mutants, A686 and A699. These mutants, deficient in NADP-glutamate dehydrogenase activity, grow poorly using high ammonium concentration as a nitrogen source (Kinghorn and Pateman 1973b). Their transformants with the gdhA gene grow on inorganic nitrogen as well as on organic nitrogen sources, confirming the functionality of the cloned A. awamori gdhA gene. The A. awamori gdhA gene is transcribed very efficiently giving a high steady state level of gdhA transcript as compared to the β-actin gene in the same RNA preparation. The strong expression of this gene during the growth phase correlates well with the high NADP-glutamate dehydrogenase activity required for glutamate synthesis in this fungus. The gdhA promoter region is, therefore, of great interest for over-expressing heterologous genes in A. awamori. An AATCAAT sequence, located at the nontranslated part of the gdhA mRNA, matches with 6/7 positions of the CAAT boxes found in other fungi. Knowledge of the transcription initiation site and the putative regulatory regions upstream of gdhA will facilitate the use of this promoter as a tool for gene expression purposes. Fungi can use a diverse array of compounds as nitrogen sources. Ammonium salts, glutamine and glutamate are preferentially used by fungi (Marzluf 1997). Our results indicate that expression of the A. awamori gdhA gene is prevented in the presence of glutamic acid. Regulation pro-

58

ceeds at the transcriptional level as shown by the low level of gdhA-hybridizing RNA in glutamate-grown cultures. Similar results were observed in A. nidulans (Hawkins et al. 1989). Nitrogen control of catabolic enzymes and permeases in fungi actually involves activation of the structural genes, which is prevented in the presence of preferred nitrogen souces. Positively acting global regulatory genes, such as areA, have been isolated in A. nidulans (Kudla et al. 1990; Caddick 1992), but their relationship to the transcription of gdhA has not been established. In A. nidulans the gdhA (but not the gdhB) mutants are de-repressed for a number of enzyme and uptake systems which are regulated by ammonium. Kinghorn and Pateman (1973b) proposed that the NADP-dependent glutamate dehydrogenase protein plays a role in nitrogen regulation, as part of a complex regulatory system. These authors proposed that the NADP-dependent glutamate dehydrogenase is a multi-functional protein which has catalytic activity and also plays a role in the regulation of a number of other activities involved in nitrogen metabolism. The results of the Northern analysis fit well with the general model of the role of the NADP-dependent glutamate dehydrogenase in nitrogen metabolism. The high expression levels of gdhA in a medium with ammonium as a nitrogen source suggest that the increased levels of the glutamate dehydrogenase formed may be responsible (together with other transcriptional factors) for the control of ammonium-regulated genes. The availability of the A. awamori gdhA gene will allow us to study the role of this enzyme in the nitrogen regulation of this industrially important strain. Acknowledgements This work was supported by a grant of URQUIMA, S. A. (Barcelona, Spain). We thank Francisco J. Fernández and Ana T. Marcos for helpful discussions, and B. Martín and M. Corrales for excellent technical assistance.

References Adams RA, Royer T (1990) Complete genomic sequence encoding trpC from Aspergillus niger var. awamori. Nucleic Acids Res 18:4931 Arst HNJr, MacDonald, K ( 1973) A mutant of Aspergillus nidulans lacking NADP-linked glutamate dehydrogenase. Mol Gen Genet 128:111–141 Ausubel FM, Brent R, Kingston RE, Moore D, Smith JA, Seidman JG, Struhl K (1987) Current protocols in molecular biology. John Wiley and Sons, New York Avendaño A, Deluna A, Olivera H, Valenzuela L, González A (1997) GHD3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae. J Bacteriol 179:5594–5597 Ballance DJ (1986) Sequences important for gene expression in filamentous fungi. Yeast 2:229–236 Benachenhou-Lahfa N, Forterre P, Labedan B (1993) Evolution of glutamate dehydrogenase genes: evidence for two paralogous protein families and unusual branching patterns of the archaebacteria in the universal tree of life. J Mol Evol 36:335–346 Berka RM, Ward M, Wilson LJ, Hayenga KJ, Kodama KH, Carlomagno LP, Thompson SA (1990) Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 86:153–162

Britton KL, Baker PJ, Rice DW, Stillman TJ (1992) Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases. Eur J Biochem 209:851–859 Caddick MX (1992) Characterization of a major Aspergillus regulatory gene, areA. In: Stahl U, Tudzynski P (eds) Molecular biology of filamentous fungi. VCH Press, Weinheim, Germany, pp 141–152 Calmels TPG, Martin F, Durand H, Tiraby G (1991) Proteolytic events in the processing of secreted proteins in fungi. J Biotechnol 17:51–66 Clutterbuck AJ (1982) Aspergillus nidulans (nuclear genes). In: O’Brien SJ (ed) Genetic maps, vol. 2. National Cancer Institute, Maryland, USA, pp 208–217 Clutterbuck AJ (1997) The validity of the Aspergillus nidulans linkage map. Fungal Genet Biol 21:267–277 Cohen BL (1973) The neutral and alkaline proteases of Aspergillus nidulans. J Gen Microbiol 77:521–528 De Zoysa PA, Connerton IF, Watson DC, Johnston JR (1991) Cloning, sequencing and expression of the Schwanniomyces occidentalis NADP-dependent glutamate dehydrogenase gene. Curr Genet 20:219–224 Ditchburn P, Giddings B, MacDonald KD (1974) Rapid screening for the isolation of mutants of Aspergillus nidulans with increased penicillin yields. J Appl Bacteriol 37:515–523 Fierro F, Gutiérrez S, Díez B, Martín JF (1993) Resolution of four chromosomes in penicillin-producing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome I (10.4 Mb) in Penicillium chrysogenum. Mol Gen Genet 241:573–578 Fierro F, Montenegro E, Gutiérrez S, Martín JF (1996) Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence. Appl Microbiol Biotechnol 43:597–604 Finkelstein DB, Rambosek J, Crewford MS, Soliday CL, McAda PC, Leach J (1989) Protein secretion in Aspergillus niger. In: Hershaberger CL, Queener SW, Hegeman G (eds) Genetics and molecular biology of industrial microorganisms. American Society of Microbiology. Washington, D. C., pp 295–300 Gouka RJ, Hessing JG, Stam H, Musters W, van den Hondel CA (1995) A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific intergration system for Aspergillus awamori. Curr Genet 27:536–540 Gouka RJ, Punt PJ, Hessing JG, van den Hondel CA (1996) Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains. Appl Environ Microbiol 62: 1951–1957 Gouka RJ, Punt PJ, van den Hondel CAMJJ (1997) Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 47:1–11 Gurr SJ, Hawkins AR, Drainas C, Kinghorn JR (1986) Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase. Curr Genet 10:761–766 Gutiérrez S, Díez B, Alvarez E, Barredo JL, Martín JF (1991) Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants. Mol Gen Genet 225:56–64 Hammond C, Helenius A (1995) Quality control in the secretory pathway. Curr Opin Cell Biol 7:523–529 Hawkins AR, Gutt SJ, Montague P, Kinghorn JR (1989) Nucleotide sequence and regulation of expression of the Aspergillus nidulans gdhA gene encoding NADP-dependent glutamate dehydrogenase. Mol Gen Genet 218:105–111 Hessing JG, van Rotterdam C, Verbakel JM, Roza M, Maat J, van Gorcom RF, van den Hondel CA (1994) Isolation and characterization of a 1,4-beta-endoxylanase gene of A. awamori. Curr Genet 26:228–232 Hijarrubia MJ, Casqueiro J, Gutiérrez S, Fernández FJ, Martín JF (1997) Characterization of the bip gene of Aspergillus awamori encoding a protein with an HDEL retention signal homologous to the mammalian BiP involved in polypeptide secretion. Curr Genet 32:139–146

59 Kinghorn JR, Pateman JA (1973a) NAD and NADP L-glutamate dehydrogenase activity and ammonium regulation in Aspergillus nidulans. J Gen Microbiol 78:39–46 Kinghorn JR, Pateman JA (1973b) A new class of mutants affecting ammonium regulation and methyl-ammonium resistance in Aspergillus nidulans. Heredity 31:427 Kinghorn JR, Pateman JA (1976) Mutants of Aspergillus nidulans lacking NAD-specific glutamate dehydrogenase. J Bacteriol 125: 42–47 Kinnaird JH, Fincham JRS (1983) The complete nucleotide sequence of the Neurospora crassa am (NADPH-specific glutamate dehydrogenase) gene. Gene 26:253–260 Kinnaird JH, Keighren MA, Kinsey JA, Eaton M, Fincham JRS (1982) Cloning of the am (glutamate dehydrogenase) gene of Neurospora crassa through the use of a synthetic DNA probe. Gene 20:387–396 Korman DR, Baylis FT, Barnett CC, Carmona CL, Kodama KH, Royer TJ, Thompson SA, Ward M, Wilson LJ, Berka RM (1990) Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori. Curr Genet 17:203–212 Kudla B, Caddick MX, Langdon T, Martínez-Rossi NM, Bennett CF, Sibley S, Davis RW, Arst HN (1990) The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–1364 Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev 61:17–32 Montenegro E, Fierro F, Fernández FJ, Gutiérrez S, Martín JF (1992) Resolution of chromosomes III and VI of Aspergillus nidulans by pulsed-field gel electrophoresis shows that the penicillin biosynthetic pathway genes pcbAB, pcbC, and penDE are clustered on chromosome VI (3.0 megabases). J Bacteriol 174:7063–7067

Moye WS, Amuro N, Rao JKM, Zalkin H (1985) Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase. J Biol Chem 260:8502–8508 Piddington CS, Houston CS, Paloheimo M, Cantrell M, MiettinenOinonen A, Nevalainen H, Rambosek J (1993) The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori. Gene 133:55–62 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467 Shen Y-Q, Heim J, Solomon NA, Wolfe S, Demain AL (1984) Repression of β-lactam production in Cephalosporium acremonium by nitrogen sources. J Antibiot 37:503–511 Specht CA, DiRusso CC, Novotny CP, Ullrich RC (1982) A method for extracting high-molecular-weight deoxyribonucleic acid from fungi. Anal Biochem 119:158–163 Thompson SA (1990) Molecular cloning and deletion of the gene encoding aspergillopepsin A from Aspergillus awamori. Gene 96:313 Verdoes JC, Punt PJ, van den Hondel CAMJJ (1995) Molecular genetic strain improvement for the over-production of fungal proteins by filamentous fungi. Appl Microbiol Biotechnol 43: 195–205 Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by using a trpC plasmid. Proc Natl Acad Sci USA 81:1470–1474