INTRODUCTION

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The major route of ammonium assimilation in Aspergillus nidulans is through the action of NADP-linked glutamate dehydrogenase (NADP-GDH), encoded by the gdhA gene, to yield glutamate (4, 22, 23). In addition, ammonium can be assimilated to glutamate by the combined action of glutamine synthetase (GS) and glutamate synthase (GOGAT). First, GS, encoded by the glnA gene, synthesizes glutamine from glutamate and ammonium, and then GOGAT, encoded by the gltA gene, converts glutamine and 2-oxoglutarate to two molecules of glutamate (10, 29). Mutants lacking NADP-GDH show leaky growth on ammonium, whereas mutants lacking both NADP-GDH and GOGAT are strict glutamate auxotrophs (29). As mutants lacking gltA function alone grow well on ammonium, this cycle is not critical for ammonium assimilation or glutamate production in cells able to synthesize an active NADP-GDH enzyme. In contrast, previously described A. nidulans glnA mutants require glutamine supplementation for growth (10).

Glutamine, as well as being an essential amino acid, has been implicated as a key effector for nitrogen metabolite repression. Nitrogen metabolite repression is a global regulatory control mechanism that ensures the preferential utilization of simple nitrogen sources over more complex sources (30). When wild-type cells are grown on nitrogen-sufficient media containing ammonium or glutamine, the expression of a wide range of catabolic enzymes required for the utilization of secondary nitrogen sources, such as nitrate or acetamide, is repressed. Nitrogen catabolic enzyme expression under nitrogen-limiting conditions requires a transcriptional activator encoded by the areA gene (3). AreA binds via a single GATA zinc finger to the promoters of nitrogen-responsive genes (25, 41). The synthesis and activity of AreA are modulated through changes in areA mRNA transcription and stability, as well as interaction with the negatively acting NmrA protein in response to changes in the nitrogen status of the cell (2, 26, 36). The phenotypes of mutants affected in ammonium assimilation indicate that ammonium alone is not the key effector for the modulation of AreA function. gdhA mutants are partially derepressed on ammonium, suggesting that ammonium must be metabolized, possibly to glutamine, to generate the signal(s) for full nitrogen metabolite repression (4, 21, 22). Previous studies of glnA mutants in A. nidulans and gln-1 mutants in Neurospora crassa have presented conflicting evidence about the role of glutamine and have even led to the suggestion that the glutamine synthetase protein itself may have a regulatory role (10, 15, 16, 17, 28, 37, 38). To further investigate the role of this enzyme in the generation of the signal for nitrogen metabolite repression, we have isolated and characterized the A. nidulans glnA gene. By creating a glnA strain, our studies have revealed that glutamate, in addition to glutamine, may be involved in signaling nitrogen status.

	MATERIALS AND METHODS

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Molecular Methods. Molecular methods were essentially as described previously (40). Escherichia coli strain NM522 {supE thi-1 (lac-proAB) (mcrB-hsdSM)5 (r_K m_K⁺) [F' proAB lacI^qZM15]} was used for all bacterial work. DNA was prepared using the High Pure plasmid isolation kit (Boehringer Mannheim) and sequenced using the PRISM Dye Primer Cycle Sequencing Ready Reaction kit (Applied Biosystems Inc.) or by the Australian Genome Research Facility.

A. nidulans strains media, and growth conditions. A. nidulans strains used in this study were MH1 (biA1), MH50 (yA1 adE20 suA-adE20 areA102 pyroA4 riboB2), MH3018 (yA1 pabA1 argB2), MH5699 (yA1 adE20 suA-adE20 areA102 pyroA4 riboB2 areA::riboB), MH 8694 (tamA riboB2), and and MH9962 (areA102 glnA pyroA4). Gene symbols and genetic manipulations have been described previously (9).

Aspergillus transformation and enzyme assays. A. nidulans strains were transformed as described previously (1). In cotransformation experiments, transformants were selected using either the riboB⁺ selectable marker plasmid pPL3 (33) on media lacking riboflavin or the pyroA⁺ selectable marker plasmid pI4 (34) on media lacking pyridoxine. Southern blot analysis was used to confirm that cotransformants contained the plasmids of interest. -Galactosidase assays of A. nidulans protein extracts were performed as described previously (12).

Cloning of the A. nidulans glnA gene. Degenerate oligonucleotides (Tom1, 5' GARCARGARTAYACNCT 3', and Tom2, 5' RCANCCNGCNCCRTTCCA 3') were designed based on conserved regions of eukaryotic GS enzymes. These primers were used to amplify a 300-bp product from A. nidulans genomic DNA. The gel-purified product was labeled and used to probe a cosmid library of A. nidulans genomic DNA. Two cosmids, W22G7 and W3D7, were identified, and a 3-kb XbaI fragment from W3D7 was subcloned into pBluescript SK() to yield pCD5.

Construction and integration of a glnA::lacZ fusion plasmid. A glnA::lacZ fusion construct was created by subcloning a 1-kb KpnI-PstI fragment from pCD5 into pMH3863, which contains a nonfunctional argB gene, allowing selection for targeted integration of the construct at the argB gene in A. nidulans. The resulting plasmid, pMH4627, contains 0.8 kb of glnA promoter sequences and 0.2 kb of glnA coding region specifying residues 1 to 30 of GlnA fused in frame with the lacZ gene of E. coli. Following transformation of the argB2 mutant strain MH3018 by pMH4627, ArgB⁺ transformants were selected. Correct integration of a single copy of pMH4627 at the argB locus was confirmed by Southern blot analysis.

Inactivation of the A. nidulans glnA gene. A glnA inactivation construct was created by replacing an internal 0.8-kb PstI-Bg/II fragment of the glnA (3 to +827) with a 2.2-kb PstI-Bg/II fragment containing the A. nidulans riboB gene from pPL1 (33). The resulting plasmid, pSM4638, was cut with KpnI and transformed into the recipient strain, MH50. Ribo⁺ transformants were selected on medium lacking riboflavin, with 10 mM glutamine as the sole nitrogen source, and screened on medium lacking added glutamine (with 10 mM ammonium as the sole nitrogen source).

Anabaena sp. strain 7120 glnA and A. nidulans fluG constructs. For heterologous complementation of A. nidulans glnA, the Anabaena glnA gene was PCR amplified using pAN503 (20, 46) as the template and the oligonucleotides TOM4 (5'-CTT CGA TGA GCT CAA GTT TTA CTC-3') and GLN1 (5'-GTA ACA ATG AGA TCT CCA CAA GAA G-3'). A 1.6-kb BglII-SacI fragment was inserted into pMF16 (19) to yield pCD7. This results in fusion of Anabaena GlnA to the N terminus of A. nidulans AlcA and places glnA under the control of the alcA promoter. Plasmids pBN57.8, pBN68, and pBN72 encode amino acids 1 to 865, 387 to 865, and 1 to 402, respectively, of the A. nidulans FluG protein expressed from the alcA promoter (C. D'Souza, unpublished data).

	RESULTS

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A. nidulans GlnA is solely responsible for glutamine synthesis. The glnA gene of A. nidulans was isolated from a genomic cosmid library (see Materials and Methods), subcloned as a 3-kb XbaI fragment into pBluescript SK(), and sequenced. The glnA gene is predicted to contain a single intron of 167 bp and to encode a 345-amino-acid protein that is highly conserved with other GS type II enzymes from a variety of fungi and other eukaryotes (Fig. 1). The GlnA protein of A. nidulans has the highest similarity to GlnA of Schizosaccharomyces pombe (74% identity; 84% similarity).

View larger version (116K): [in this window] [in a new window]   FIG. 1.   Sequence alignment of the predicted GS enzymes of A. nidulans (accession no. AF333968), S. pombe (accession no. Z98977), Colletotrichum gloesosporoides (accession no. Q12613), Nectria haematococca (accession no. AF175498.1), S. cerevisiae (accession no. M65157), and Anabaena sp. (accession no. X00147). The sequences were aligned using CLUSTAL W (45), and identical amino acids (solid boxes) and conservative substitutions (shaded boxes) are indicated.

View larger version (112K): [in this window] [in a new window]   FIG. 2.   Growth tests of glnA mutants. Conidia of the relevant strain were inoculated onto 1% glucose-minimal medium containing 10 mM glutamine (GluN), ammonium tartrate (NH4), or alanine (Ala) as nitrogen sources as indicated. The growth of the glnA1 and glnA strains was compared to that of the glnA+ controls after 2 days of growth at 37°C.

glnA expression is not highly regulated. Northern blot analysis revealed that the glnA gene encodes an mRNA of approximately 1.5 kb, consistent with the transcript size predicted from sequence analysis. The glnA transcript was readily detected under all growth conditions tested. Levels in ammonium- and glutamine-grown mycelia of the wild-type strain were similar, and the levels increased approximately two-fold during growth on alanine or glutamate as the sole nitrogen source (Fig. 3). glnA mRNA levels were lower in strains carrying areA or tamA mutations under all conditions tested (Fig. 3). TamA is thought to function as a coactivator of AreA, and mutations in tamA lead to reduced expression of a variety of enzymes involved in nitrogen metabolism (14, 24, 42).

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Expression of the glnA gene. (A) Mycelia of the wild-type strain, MH1, were grown for 16 h on glucose-minimal medium containing 10 mM ammonium (NH4), glutamine (GluN), alanine (Ala), or glutamate (Glu) as the sole nitrogen source. Total RNAs made from these mycelia were probed with a 1.1-kb HincII fragment from the glnA gene and a 1.3-kb EcoRI fragment from the histone H3 gene (18) as loading controls. (B) Mycelia of MH1, MH8694 (tamA), and MH5699 (areA) were grown, and the RNAs were probed as described above. (C) Mycelia were grown overnight on 1% glucose-minimal medium containing the appropriate nitrogen source at a concentration of 10 mM unless otherwise indicated. The -galactosidase activities are expressed as units of activity per minute per milligram of soluble protein (+ standard errors).

Glutamine supplementation of the glnA growth defect. Besides glutamine, no other amino acid was able to allow growth of the glnA strains. Glutamine levels of 0.5 mM or above were required to supplement the glutamine auxotrophy of the glnA mutant on minimal media. However, even in the presence of 10 mM glutamine, the glnA mutant exhibited a slightly restricted colony size. When glutamine repair of the glnA mutant was tested on complete, rather than minimal, medium, it was found that the glnA mutant produced a more compact, pigmented colony with reduced conidiation. This phenotype could be due to the effects of the areA102 mutation on glutamine uptake or metabolism, because glnA was mutated in an areA102 genetic background. However, when the areA102 glnA strain was crossed with a wild-type strain, the areA⁺ glnA progeny still produced compact, pigmented colonies with poorer conidiation than those of the wild type. The inhibited growth morphology of areA⁺ glnA strains on glutamine is more extreme than that of the areA102 gln strain. In an areA⁺ background, this phenotype was evident even on minimal medium and was even more pronounced on complete medium supplemented with 10 mM glutamine (Fig. 2).

The A. nidulans glnA gene and the cyanobacterium Anabaena glnA gene can complement the glnA mutation. The phenotypes of glutamine auxotrophy and growth inhibition of strains carrying the glnA mutation were fully reversed by reintroduction of the A. nidulans glnA gene, indicating that the glutamine auxotrophy was indeed conferred by the glnA mutation. This was achieved by transforming the recipient strain, MH9962, to glutamine prototrophy with pCD5 or by cotransformation with pCD5 and the pyroA vector pI4 (34). Transformants were directly selected for complementation of glutamine auxotrophy on medium containing 10 mM ammonium as the sole nitrogen source. Pyro⁺ transformants were selected on media containing 10 mM glutamine and lacking pyridoxine. Complementing transformants were indistinguishable from glnA⁺ strains. Reintroduction of the glnA gene on pCD5 was also able to reverse the phenotypes of the glnA mutation in an areA⁺ background.

GS is an effector of nitrogen metabolite repression. Given the proposed role of glutamine as the key effector of nitrogen metabolite repression and the possibility that GS itself may have a regulatory role, the response of the glnA strains to repression was of considerable interest. A number of standard plate assays were used to assess nitrogen regulation (3, 36). Chlorate, aspartylhydroxamate, and thiourea are analogues of nitrate, asparagine, and urea, respectively. Both ammonium and glutamine are known to protect the wild-type strain from the toxicities of these analogues by repressing the synthesis of permeases and other enzymes required for their assimilation. In both areA⁺ and areA102 backgrounds, the glnA mutation led to an apparent slight sensitivity to chlorate and hypersensitivity to thiourea and aspartate hydroxymate, even in the presence of glutamine, compared to glnA⁺ controls. Similar results have been observed previously and have led to the suggestion that glutamine alone is not sufficient to signal repression of catabolic enzyme synthesis and that the GS enzyme itself may have a complex role in nitrogen regulation (28). However, interpretation of these plate tests is complicated by the finding that the growth of glnA mutants on glutamine may be affected by other compounds in the medium. Indeed, further growth tests revealed that the presence of asparagine and, to a lesser extent, nitrate or urea interfered with the growth and/or glutamine supplementation of glnA strains. Therefore, it is difficult to distinguish between direct sensitivity to toxic nitrogen source analogues, indicating derepression, and indirect effects on glutamine supplementation of the glnA mutant strains.

Glutamate as well as glutamine signals nitrogen metabolite repression. If glutamine alone were the signal for repression, it would be predicted that a glnA strain would be fully derepressed on ammonium or glutamate, as it would be unable to metabolize these compounds to glutamine. Surprisingly, it was found that the glnA mutant showed only partial derepression on ammonium (Table 1). Therefore, even in a strain lacking GS activity, ammonium was able to bring about significant repression. In addition, the effect of glutamate in the glnA mutant was similar to the effect in the glnA⁺ strain. Therefore, glutamate led to a 50% reduction in levels even though it could not metabolized to glutamine.

	DISCUSSION

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In A. nidulans, GS is absolutely required for the biosynthesis of glutamine. Previously isolated alleles have shown, to various degrees, a slight leakiness (10). Lee and Adams (27) have investigated whether the GS-like domain of FluG has sufficient activity to account for the leaky phenotype of glnA mutants. They found that a fluG glnA double mutant was no more extreme than the glnA single mutant, indicating that FluG is not the source of the residual activity. We have now shown that the leakiness is a reflection of residual GS enzymatic activity in these strains, as a complete null mutation is a strict glutamine auxotroph. We have also shown directly that FluG lacks GS activity by virtue of its inability to restore any growth to the glnA strain. Amino acid sequence comparison indicates that the GS-like region of FluG is most similar to the type I GS of prokaryotes. Type I GS enzymes form a 12-subunit complex structure and are unique to prokaryotes, while the type II GSs found in eukaryotes are octamers of a smaller polypeptide which is only distantly similar to the type I form. The presence of a type II GS in certain bacteria is thought to have arisen by lateral gene transfer (8). It is interesting that A. nidulans FluG has greater similarity to the prokaryotic type I enzyme. The lack of GS activity of this region of FluG is not simply a function of its type I similarity, as we have found that the Anabaena enzyme can function effectively in a eukaryotic context provided that the promoter is functional.

A noticeable feature of the glnA strains is their aberrant morphology on glutamine-containing media. This was particularly evident in an areA⁺ background and was partially suppressed by the areA102 mutation. MacDonald (28) noted that strains carrying the glnA1 mutation also had a slightly restricted morphology on 10 mM glutamine. The glnA1 mutant retains very low transferase and synthetase activities (10) and exhibits residual growth in the absence of glutamine, unlike the glnA strain. Therefore, the altered-morphology phenotype has been observed even with mutants that retain low levels of GS function. As high glutamine concentrations (in the millimolar range) are required to fully supplement the glutamine requirement of glnA mutants, MacDonald (28) has suggested that glutamine uptake may be limiting for A. nidulans. However, glutamine concentrations up to 20 mM were not effective in reversing the phenotype and in fact increased its severity in the glnA strains. As glnA⁺ (areA⁺ or areA102) strains grow strongly on 10 mM glutamine, glutamine uptake is not limiting for growth, and externally supplied glutamine is a potent source of nitrogen metabolite repression for both wild-type and areA102 strains.

In a glnA strain, the breakdown of glutamine to glutamate and ammonium may lead to the accumulation of glutamate within the cells. In N. crassa, a leaky gln-1 mutant grown on glutamine as a nitrogen source accumulated more glutamate and alanine than a wild-type strain, and wild-type cells treated with an inhibitor of GS have been shown to accumulate glutamate when grown on glutamine (5, 31). It is known that glutamine synthesis occurs even in cells growing on glutamine and that nitrogen is efficiently cycled between glutamine and glutamate via the GS-GOGAT cycle (31, 47). Loss of the ability to recycle glutamate into glutamine in glnA mutants may result in excessive levels of glutamate or a derivative, leading to toxic effects within the cells. This is supported by the observation that the growth inhibition associated with the glnA mutants is abolished by a gdhA mutation. In N. crassa, a significant fraction of the ammonium derived from glutamine degradation is normally assimilated by NADP-GDH into glutamate (5). As the gdhA mutant lacks NADP-GDH, the ammonium derived from glutamine breakdown would not add to the pool of glutamate within a glnA mutant. Furthermore, areA102 glnA strains were found to be less inhibited on glutamine than glnA single mutants. As areA102 strains grow more strongly than the wild-type strain on glutamate as a sole nitrogen source, the areA102 mutation may reduce the buildup of glutamate by increased glutamate catabolism via NAD-GDH (22).

The growth-restricted morphology, without associated pigmentation, has also been observed on very low glutamine concentrations. At these levels, toxic metabolite accumulation is unlikely. Therefore, the endogenous synthesis of glutamine may be required for hyphal extension, a requirement not met by exogenous glutamine, possibly because young tips do not contain active glutamine permease(s). Cardenas and Hansberg (6, 7) found that in N. crassa, the transition from mycelial mat to aerial hyphae required high glutamine pools, and they proposed that internal glutamine serves as a nitrogen carrier to the growing aerial mycelium.

The availability of a glnA strain has allowed us to investigate the generation of the signaling metabolites for nitrogen metabolite repression. Since the mutant lacks the GS enzyme, we were able to establish that the addition of glutamine is sufficient to bring about repression of the nitrogen-regulated amdS gene. The GS enzyme itself is unlikely to have other than a biosynthetic role in generating the signal. This is also supported by the finding that expression of the Anabaena sp. strain 7120 GS simultaneously restores glutamine prototrophy and normal regulation. The finding that a wide range of compounds can interfere with the growth of glnA strains on glutamine may explain previous data leading to the suggestion that glutamine alone was not effective in glnA mutant strains. These earlier findings were repeated here with plate tests, indicating that glnA mutants were sensitive to chlorate (10, 28). The fact that nitrate could partially mimic these results suggests that they are in fact an indirect consequence of the glnA mutant phenotype rather than an indication of derepression.

The use of an amdS::lacZ reporter construct allowed us to test the response to glutamine without the need for simultaneously inducing enzyme synthesis and hence avoiding the complications of inducer exclusion. From these data it is clear that glutamine is an effective trigger for repression in the glnA strains. If glutamine alone is the required signaling molecule, it would be predicted that glnA mutants unable to synthesize glutamine would be fully derepressed on ammonium. The data clearly indicate that this is not the case and that ammonium and/or glutamate may also act as signaling molecules. By introducing the gdhA10 mutation into the glnA background, we were able to show that ammonium was effective in the glnA mutant by virtue of its conversion to glutamate. While it may be expected that growth on glutamate would result in repression in the double mutant, the data indicate that this is not so. In this mutant background, glutamate levels are not replenished from the ammonium released by glutamate catabolism by either NADP-GDH or the GS-GOGAT cycle, as described above. This correlates with the phenotype of the double mutant, which grows more poorly on glutamine than the single glnA mutant but lacks the growth inhibition phenotype that accompanies the single mutant. It is well established that NAD-GDH is responsible for breaking down glutamate (22). In the gdhA10 glnA double mutant, the inability to convert the derived ammonium back to glutamate prevents intracellular glutamate from accumulating to bring about repression. Glutamate repression has been proposed as a separate mechanism for the control of NADP-GDH synthesis (35). Interestingly, studies of nitrogen regulation in Saccharomyces cerevisiae have suggested that the two GATA factor activators GLN3 and NIL1 may respond to different signals, with GLN3, but not NIL1, active in glutamate-grown cultures (43, 44). Whether these multiple forms of repression operate via a single regulatory pathway involving AreA or whether there are distinct pathways remains to be determined. However, recent evidence suggests that glutamine and glutamate may generate distinct signals that each influence the stability of the areA mRNA through interactions in the 3' untranslated region of the areA gene (32)