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1 Department of Applied Biological Sciences, Nihon University College of Bioresource Sciences, Fujisawa, Kanagawa 252-8510, Japan
2 University Research Center, Nihon University, Chiyoda-ku, Tokyo 102-8275, Japan
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Abstract |
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) mutations, and tested for growth on low ammonium and resistance to the toxic ammonium analog methylammonium. The amt1 and amt2 single mutants had different growth defects, and the amt1
amt2 double mutant displayed a much more severe growth defect on 
5 mM ammonium. All single mutants exhibited methylammonium resistance but to different extents: amt2 was the most resistant and amt3 was the least. These results suggest that the amt genes encode functional transporters with distinct uptake properties. In response to ammonium limitation, the wild-type strain isogenic to the amt mutants underwent filamentous growth underneath the surface of solid medium. No such filamentous invasive growth, however, was observed for the amt1 mutant, indicating that Amt1 transporter is required for ammonium limitation-induced filamentous invasive growth. |
Introduction |
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Transport of ammonium across the cell membrane is mediated by members of the ammonium transporter/methylammonium permease (Amt/Mep) family. Genes encoding Amt/Mep protein were first cloned from S. cerevisiae and Arabidopsis thaliana (Marini et al. 1994; Ninnemann et al. 1994), and Amt/Mep homologs have since been identified by sequence similarity in many organisms in all domains of life (Pfam accession number PF00909). The presence of multiple transporters with different properties in a single organism is common. For instance, S. cerevisiae has three Mep proteins and A. thaliana has six Amt proteins (Marini et al. 1997; von Wirén et al. 2000).
High-resolution crystal structures have recently been reported for the Escherichia coli AmtB and Archaeoglobus fulgidus Amt-1 proteins (Khademi et al. 2004; Zheng et al. 2004; Andrade et al. 2005). The Amt proteins form a trimer in which each monomer contains 11 transmembrane helices and has an NH4+ recruitment site and a narrow hydrophobic pore that allows the passage of NH3 but not NH4+, leading to the view that AmtB is a gas channel (Khademi et al. 2004). Although these structural studies have provided a molecular basis for ammonium transport, the precise mechanism of the transport is still under debate (Andrade et al. 2005).
The S. cerevisiae ammonium transporters, Mep1, Mep2, and Mep3, have been well characterized (Marini et al. 1994, 1997). Mep2 is the highest-affinity transporter, while Mep3 displays the lowest affinity for substrates. Expression of the MEP genes is subject to nitrogen catabalite repression and requires the GATA transcription factors Gln3 and Gat1 (Nil1) (Marini et al. 1994, 1997), whose nuclear localization is negatively regulated by the TOR (target of rapamycin) pathway (Cooper 2002). In fact, MEP2 is among the most up-regulated genes in response to addition of rapamycin, an inhibitor of TOR (Cardenas et al. 1999; Hardwick et al. 1999).
S. cerevisiae cells lacking the Mep proteins exhibit a growth defect at low ammonium concentrations (Marini et al. 1997), suggesting that the ammonium transporters function to take up limiting concentrations of ammonium for use as a nitrogen source. Similar growth defects have been reported for ammonium transporter mutants of E. coli and Aspergillus nidulans (Soupene et al. 1998; Monahan et al. 2006). In addition, ammonium transporters are likely to have a role in the retention of intracellular ammonium (Marini et al. 1997; Monahan et al. 2002b). Physiological roles for ammonium transporters have also been investigated in the slime mold Dictyostelium discoideum, whose development is known to be regulated at multiple stages by ammonia (Gross 1994). A null mutation of one of the three D. discoideum ammonium transporter genes causes a defect in fruiting body formation (Kirsten et al. 2005).
Interestingly, ammonium transporters appear also to play a role in differentiation in S. cerevisiae (Lorenz & Heitman 1998a,b). When nitrogen is limited, diploid S. cerevisiae cells undergo a morphological transition from a unicellular yeast form to a filamentous form called pseudohyphae, which are composed of chains of elongated cells that radiate away from the colony and penetrate the agar on which they are grown, permitting the cells to forage for nutrients (Gimeno et al. 1992). Cells lacking Mep2 are unable to differentiate into pseudohyphae in response to ammonium limitation. However, they have no defects in growth rate or ammonium uptake under ammonium-limiting conditions and they do exhibit pseudohyphal growth on media containing limiting concentrations of a different nitrogen source, such as glutamine or proline. These observations have led to the proposal that Mep2 acts as an ammonium sensor that generates a signal to induce pseudohyphal differentiation (Lorenz & Heitman 1998a). A similar requirement for ammonium transporters in filamentous growth has recently been reported for the pathogenic fungi Ustilago maydis (Smith et al. 2003) and Candida albicans (Biswas & Morschhäuser 2005). The pseudohyphal growth in S. cerevisiae is regulated by both the MAPK (mitogen-activated protein kinase) and cAMP pathways (Lengeler et al. 2000; Gagiano et al. 2002), and Mep2 has been suggested to function upstream of Gpa2 in the cAMP cascade (Lorenz & Heitman 1998a). How ammonium is sensed, however, remains unknown.
In the fission yeast Schizosaccharomyces pombe, when dividing haploid cells are deprived of ammonium, they cease proliferating, arrest in G1 phase, and undergo conjugation between cells of opposite mating type (Egel & Egel-Mitani 1974). When there is no partner available for mating, cells enter the dormant G0 state in response to ammonium deprivation (Su et al. 1996). Recently, it has been reported that, under nitrogen-limiting conditions, S. pombe can differentiate to form hyphae that invade the solid medium (Amoah-Buahin et al. 2005). The identification of ammonium transporters in S. pombe would open new avenues of investigation, not only into ammonium transport but also into ammonium sensing in these ammonium limitation-induced processes.
Although putative ammonium transporter genes have been found in the S. pombe genome (Monahan et al. 2002a; Hertz-Fowler et al. 2004), they have not yet been functionally characterized. In this study, three S. pombe ammonium transporter (amt) genes identified on the basis of sequence similarity were shown to encode functional transporters with different uptake capacities. Furthermore, one of the amt genes was found to be required for filamentous invasive growth induced by ammonium limitation.
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Results |
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To identify ammonium transporter genes of S. pombe, BLAST searches of the S. pombe genome were performed using the S. cerevisiae Mep sequences as queries. The searches yielded three S. pombe genes: SPCPB1C11.01, SPAC664.14, and SPAC2E1P3.02c, which are systematic names in the S. pombe database GeneDB (Hertz-Fowler et al. 2004; http://www.genedb.org/genedb/pombe/) and will hereafter be referred to as amt1, amt2, and amt3, respectively. The amt1, amt2, and amt3 genes are predicted to contain no introns and to encode proteins of 497, 512, and 517 amino acids with molecular masses of 53.3, 55.5, and 56.4 kDa, respectively (Fig. 1A). The Amt1 and Amt2 proteins are 44% identical to each other and they are both 34% identical to the Amt3 protein. Between the S. pombe Amt and S. cerevisiae Mep proteins, the highest identity (48%) is observed between Amt1 and Mep2, consistent with their functional similarities (see below). The C-terminal and to a lesser extent the N-terminal regions of the Amt/Mep proteins have diverged in sequence and length (Fig. 1A). The sequence alignment also revealed a 47-amino acid insertion unique to S. pombe Amt3 (Fig. 1A). Figure 1B shows a phylogenetic tree of the Amt/Mep proteins from E. coli, S. cerevisiae, and S. pombe. It is evident also from this tree that S. pombe Amt1 and Amt2 are more closely related to each other than to Amt3 and that Amt1 shows a close relationship to S. cerevisiae Mep2.
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S. cerevisiae Mep2 is glycosylated at an asparagine residue in its N-terminal region, Asn-4 (Marini & André 2000). A search of the S. pombe Amt proteins for the consensus sequence for asparagine-linked glycosylation (Asn-X-Ser/Thr) revealed two possible sites: Asn-46 of Amt2 and Asn-14 of Amt3. The latter is presumed to be located in the N-terminal extracytoplasmic region and may thus be subject to glycosylation, as is Asn-4 of Mep2. Mep2 has also been reported to contain a putative phosphorylation site for protein kinase A (Thr-288) that is required for pseudohyphal differentiation (Smith et al. 2003). The corresponding residue in the S. pombe Amt proteins is invariably serine (Fig. 1A).
Construction of a series of amt mutants
As a first step towards determining the function of the amt genes, a series of amt deletion (amt) strains in which the amt genes are deleted in all combinations were constructed as described in the Experimental procedures section. Briefly, the amt1, amt2, or amt3 gene of an h90 ura4 strain was replaced by a ura4+ pop-out cassette to yield amt::ura4+ strains, from which amt strains were obtained by selection for 5-fluoroorotic acid resistance (Fig. 2A). The homothallic strain was used as the parent because it would be advantageous for future analysis of mating. Double and triple deletion mutants were then constructed via genetic crosses. Finally, the ura4 allele in each mutant was converted to the wild-type ura4+ allele to generate prototrophic amt strains (Fig. 2B). The absence of auxotrophic mutations in these strains eliminates the need for nutritional supplements (for example, uracil) that can serve as a nitrogen source and thus could complicate or hamper analysis that requires nitrogen limitation. Three single (amt1, amt2, and amt3) mutants, three double (amt1
amt2, amt1
amt3, and amt2
amt3) mutants, and one triple (amt1
amt2
amt3) mutant, along with a wild-type strain, constitute the set of strains used in subsequent experiments (Fig. 2C). In the course of constructing the series of prototrophic amt strains, a series of ura4
amt strains were also generated, and these strains will be useful for future molecular genetic analyses.
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mutants on low ammonium
None of the amt mutants, including the amt1
amt2
amt3 triple mutant, exhibited a growth defect on the rich medium YE or on the synthetic minimal medium EMM (data not shown), despite the fact that the nitrogen source in EMM is ammonium (93 mM). Therefore, the amt mutants were next tested for growth on minimal medium containing lower concentrations of ammonium (50, 5, 0.5, or 0.1 mM NH4Cl). The amt1
amt2 and amt1
amt2
amt3 mutants displayed a severe growth defect on 5 mM ammonium (Fig. 3), indicating that at least one of the amt1 and amt2 genes is required for growth at low ammonium concentrations. The amt2 and amt2
amt3 mutants, but not the amt1 and amt1
amt3 mutants, exhibited a small but noticeable reduction in growth on 5 mM ammonium, which can be seen in Fig. 3 as slightly smaller cell patches. In other words, on 5 mM ammonium, the amt2 and amt2
amt3 mutants grew more slowly than the amt1 and amt1
amt3 mutants, respectively. A similar growth pattern was observed on 0.5 mM ammonium (Fig. 3). Interestingly, the opposite was observed on 0.1 mM ammonium; that is, the amt2 and amt2
amt3 mutants grew better than the amt1 and amt1
amt3 mutants, respectively (Fig. 3). A similar reversal in the growth pattern (but with more pronounced differences) was observed on 0.5 mM ammonium after a longer incubation of 7 days (data not shown). Reduced growth of the amt1 mutant was also observed on LNB medium, which contained 0.76 mM ammonium and was used for the invasive growth assay described below (see below). In summary, the amt1 and amt2 mutants showed different growth defects, and the amt1
amt2 double mutant displayed a much more severe defect than the single mutants. No growth defect was associated with the amt3 mutation.
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amt2
amt3 triple mutant grew normally at high concentrations of ammonium (
50 mM) (Fig. 3 and data not shown), indicating that, under these conditions, the Amt proteins are dispensable for the utilization of external ammonium as the nitrogen source. In addition, even at lower ammonium concentrations (5 mM), the amt1
amt2
amt3 mutant did grow, albeit slowly (Fig. 3). The Amt-independent growth of the amt1
amt2
amt3 mutant might be explained either by passive diffusion of NH3 or by uptake of NH4+ or NH3 by a transporter that is unrelated to the Amt proteins (see Discussion).
Methylammonium resistance of amt mutants
Methylammonium, an ammonium analog that is not metabolized in S. cerevisiae (Soupene et al. 2001), is known to inhibit the growth of yeast cells. Since methylammonium is taken up into the cell by an ammonium transporter despite lower affinity than ammonium, sensitivity to methylammonium seems to reflect the ammonium transporter function of the cell. Indeed, methylammonium-resistant mutants of S. cerevisiae are defective in ammonium uptake (Roon et al. 1975). The amt mutants were thus tested for growth on minimal proline medium containing 50 mM methylammonium. Proline was used as the nitrogen source because proline is known not to repress the expression of the MEP genes in S. cerevisiae (Marini et al. 1994, 1997). The wild-type strain showed sensitivity to methylammonium, as expected (Fig. 4). Among the amt mutants, the amt1
amt2 and amt1
amt2
amt3 mutants were the most resistant (Fig. 4), consistent with their growth defects at low ammonium concentrations (Fig. 3). The single amt mutants also exhibited methylammonium resistance but to different extents. Of the single mutants, amt2 was the most resistant; amt1 showed intermediate resistance; and amt3 was the least resistant (Fig. 4). The slight resistance conferred by the amt3 mutation was also noticeable in the amt2
amt3 and amt1
amt3 mutants, which were more resistant than the amt2 and amt1 mutants, respectively (Fig. 4). Taken together, these results suggest that Amt1, Amt2, and Amt3 are all able to transport methylammonium and probably also ammonium.
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mutants
As a more direct measure for ammonium uptake capacity, the removal of ammonium from the growth medium was examined. Cells were grown in minimal proline medium, and the concentration of ammonium in the culture medium was monitored after adding NH4Cl to a final concentration of 1 mM. The wild-type strain effectively removed ammonium from the medium, such that external ammonium was lost within 90 min (Fig. 5). In contrast, little if any reduction in the ammonium level in the medium was observed for the amt1
amt2
amt3 triple mutant: after a 120-min incubation, the ammonium concentration had decreased by only 2% (Fig. 5). These results indicate that there is no other transporter that effectively takes up ammonium under these conditions. To assess the uptake capacities of individual Amt proteins, ammonium removal was tested in the double mutants. The amt1
amt3 mutant removed ammonium effectively from the medium (Fig. 5). The amt2
amt3 mutant also removed ammonium, but less effectively than the amt1
amt3 mutant (Fig. 5). Finally, the amt1
amt2 mutant was indistinguishable from the amt1
amt2
amt3 mutant, that is, almost no uptake was observed (Fig. 5).
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amt3 and amt2
amt3 mutants exceeded the uptake by the wild type. A similar observation was made in S. cerevisiae (Marini et al. 1997). These results may be accounted for by an effect of one Amt protein on the expression or activity of another Amt protein (see Discussion).
Filamentous invasive growth of amt mutants
Fungal ammonium transporters (Mep2 in S. cerevisiae, Ump2 in U. maydis, and Mep2 in C. albicans) have been shown to be required for a switch from single-cell growth to filamentous growth in response to limitation of nitrogen (Lorenz & Heitman 1998a; Smith et al. 2003; Biswas & Morschhäuser 2005). S. pombe has recently been reported to undergo a similar morphological transition, differentiation into hyphae that penetrate the agar under nitrogen-limiting conditions, although the nitrogen-sensing mechanism itself has remained elusive (Amoah-Buahin et al. 2005). This finding prompted me to test the ability of the amt mutants to undergo the morphological transition in response to nitrogen limitation. I first examined the homothallic strain HMP94 (the wild type used throughout this study) as a control. Cells of the wild-type strain were grown at 30 °C for 14 days on LNB agar medium, which contained 0.76 mM ammonium as the nitrogen source, and then tested for invasive growth by washing cells off the agar surface with running water. The wash uncovered the presence of cells that had invaded the agar substrate (Fig. 6A). Microscopic examination revealed three-dimensional structures consisting of filamentously growing cells that were totally distinct from normal colony morphology (Fig. 6B). These results indicate that, under ammonium-limiting conditions, the wild-type strain isogenic to the amt mutants forms colonies of filamentously growing cells within the agar underneath a lawn of cells.
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single mutants were grown on LNB to determine whether S. pombe Amt plays a role in filamentous invasive growth induced by ammonium limitation. Strikingly, no invasive growth was observed for the amt1 mutant (Fig. 6A). The amt1 mutant also showed impaired growth on the surface of the agar, as can be seen in Fig. 6A as a smaller cell patch with papillae. Importantly, when grown on 1 mM glutamine, the amt1 mutant exhibited invasive growth as well as the wild type (Fig. 6A). Notably, the wild-type strain displayed very little, if any, invasive growth when the ammonium concentration in LNB was lowered from 0.76 to 0.1 mM (Fig. 6A). Under these conditions, the cells did not grow as much as they did on LNB (Fig. 6A). Thus, it appears that lowering the ammonium concentration in the medium has similar effects as the amt1 mutation. The amt2 mutant showed a clear reduction in invasive growth compared with the wild type, whereas the amt3 mutant exhibited agar invasion as well as or slightly better than the wild type (Fig. 6A). Taken together, the invasive growth assays of the amt mutants revealed that Amt1 is required for ammonium limitation-induced filamentous invasive growth, in which Amt2 also plays a positive role.
In the experiment shown in Fig. 6A, invasive growth was scored after incubation at 30 °C for 14 days, the same conditions as reported by Amoah-Buahin et al. (2005). Having observed the impaired surface growth of the amt1 mutant under these conditions, I tested the wild type and the amt1 mutant for surface and invasive growth over shorter incubation periods. Invasive growth could be observed for the wild type after incubation for 5 days (Fig. 6C). Although not visible in Fig. 6C, microscopic examination revealed that the wild-type strain had formed colonies of filamentously growing cells underneath the agar surface after 3 days of incubation (Fig. 6D). In addition, agar invasion of the wild-type cells could be seen microscopically as early as after incubation for 2 days (data not shown). These observations indicate that the wild type begins to undergo filamentous invasive growth after 2–3 days of incubation, when the difference in surface growth between the wild type and the amt1 mutant is not so pronounced as after a 14-day incubation (Fig. 6C). Again, no invasive growth was observed for the amt1 mutant (Fig. 6C).
S. pombe hyphal growth has been reported to depend on the cAMP pathway (Amoah-Buahin et al. 2005). I found that the addition of 1 mM cAMP to LNB medium strongly enhanced the invasive growth of the wild type and also suppressed the invasive growth defect of the amt1 mutant (data not shown). It has also been reported that hyphal growth in S. pombe is inhibited by addition of nutritional supplements, such as adenine, leucine, or uracil, presumably because they serve as a nitrogen source (Amoah-Buahin et al. 2005). The invasive growth assays described above therefore illustrate an advantage of the amt strains constructed in this study, which are prototrophic and thus need no nutritional supplements.
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Discussion |
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mutants have suggested that Amt1, Amt2, and Amt3 are all capable of ammonium/methylammonium uptake but have distinct uptake properties.
The amt2 mutant showed a small but noticeable growth defect on 5 mM ammonium, while the amt1 mutant displayed reduced growth at lower ammonium concentrations, particularly after long incubation. These observations suggest that Amt1 has a higher affinity for ammonium than Amt2, although kinetic analysis will be needed to confirm this prediction. Consistent with the idea that Amt1 is a high-affinity transporter, Amt1 is closely related to Mep2, the highest-affinity transporter in S. cerevisiae. S. pombe Amt1 and S. cerevisiae Mep2 are also similar in that they are required for a morphological transition induced by ammonium limitation. The amt1
amt2 double mutant had a much more severe defect in growth on 5 mM ammonium than the amt1 and amt2 single mutants, suggesting that Amt1 and Amt2 share partially redundant functions. The growth phenotype of the S. pombe amt1
amt2 double mutant reported here resembles that of a S. cerevisiae mep1
mep2
mep3 triple mutant (Marini et al. 1997).
The physiological role of Amt3 is currently unknown. Deletion of the amt3 gene had no effect on growth at any ammonium concentration tested. In addition, no ammonium uptake was observed for Amt3 in the ammonium removal assay. However, slight but detectable methylammonium resistance was found to be conferred by the amt3 mutation, suggesting that Amt3 is able to transport methylammonium at least to some extent. Amt3 is unique in containing a predicted long cytoplasmic loop between transmembrane helices 5 and 6. The presence of this long loop may account for the apparently poor uptake capacity of Amt3. Recently, an ammonium transporter that seems not to contribute to ammonium uptake has also been reported for A. nidulans (Monahan et al. 2006).
In this study, the function of individual Amt proteins was assessed by analysis of deletion mutants. The interpretation of the results may not be straightforward, considering the possibility that the expression or activity of one Amt protein may be affected by another. In A. nidulans, deletion of the low-affinity transporter gene meaA allows expression of the high-affinity transporter gene mepA in otherwise repressing nitrogen-rich conditions, and this is probably due to reduced ammonium uptake caused by the meaA deletion (Monahan et al. 2002a). Protein–protein interaction between ammonium transporters has been suggested by the fact that a missense mutation in the S. cerevisiae gene encoding Mep1 has a dominant-negative effect on the Mep3 function (Marini et al. 2000). Taken together, these observations suggest that deletion of one amt gene in S. pombe may exert an effect on another amt gene at the transcriptional or post-transcriptional level.
S. pombe cells lacking all three Amt proteins grew normally at high ammonium concentrations. This result was not surprising because similar observations have been reported for S. cerevisiae, E. coli, and A. nidulans (Marini et al. 1997; Soupene et al. 1998; Monahan et al. 2006). Two possibilities have been considered to explain growth in the absence of Amt/Mep family ammonium transporters. First, the growth may be supported by gaseous NH3, which can penetrate the cell membrane by passive diffusion. Ammonium exists as a mixture of the ion NH4+ and the gas NH3 (pKa = 9.25); the lower the pH, the lower the concentration of NH3. Growth of cells that lack Amt/Mep is impaired when the pH of the medium is low (Soupene et al. 1998; Jahn et al. 2004; Loqué et al. 2005; Monahan et al. 2006), which is in accordance with the idea that it is NH3 that supports the Amt/Mep-independent growth. Second, NH4+ or NH3 may enter the cell via an as yet unidentified low-affinity transport system. In this regard, of particular interest are recent studies suggesting that some plant and mammalian aquaporins transport ammonium (Jahn et al. 2004; Holm et al. 2005; Loqué et al. 2005; Liu et al. 2006).
I have shown that, in response to ammonium limitation, the wild-type homothallic strain used in this study forms colonies of filamentously growing cells underneath the surface of agar medium. Filamentous invasive growth within low-ammonium medium was visible by microscopy as early as after incubation for 2–3 days. Notably, Amt1 is required for filamentous invasive growth and Amt2 also seems to have a positive role. The defect in filamentous invasive growth of the amt1 mutant is reminiscent of the pseudohyphal defect of S. cerevisiae mep2 mutants. A mep2 mutation prevents pseudohyphal differentiation in response to ammonium limitation without affecting the growth rate or ammonium uptake of the cell, leading to the proposal that Mep2 acts as a sensor for ammonium (Lorenz & Heitman 1998a). An intriguing possibility is that S. pombe Amt1 also functions as an ammonium sensor. Upon ammonium binding or transport, Amt1 may interact with an effector. A mutation in the amt1 gene that either eliminates the signaling function without affecting the transport activity or constitutively activates the signaling cascade independently of the substrate, such as those identified for the S. cerevisiae glucose sensors Snf3 and Rgt2 (Özcan & Johnston 1999), would strongly support the hypothesis that S. pombe Amt1 functions as a sensor that generates a signal for filamentous invasive growth.
There is another possibility, however, suggested by the impaired growth of the amt1 mutant on low-ammonium media that becomes more pronounced with longer incubation periods. Under these conditions, the amt1 cells seem to suffer ammonium starvation, presumably because of a defect in high-affinity ammonium uptake. Thus, the invasive growth defect of the amt1 mutant may be ascribed to a defect in ammonium uptake. Interestingly, lower ammonium in the medium (0.1 mM), like the amt1 mutation, led to both reduced surface growth and a defect in invasive growth, suggesting that filamentous invasive growth may require some level of intracellular ammonium. Ammonium taken up into the cell may function not only as a nutrient source for growth but also as a signal for growth transition. In any case, Amt1-dependent filamentous invasive growth in S. pombe should provide a useful system for investigating the role of ammonium transporters in ammonium sensing.
Although the underlying mechanism remains to be determined, one of the S. pombe ammonium transporters has clearly been shown to be required for filamentous invasive growth in response to limited availability of ammonium. A question of interest is whether the S. pombe Amt proteins also play a role in other processes known to be triggered by ammonium limitation or deprivation, including G0 entry and mating. The amt strains constructed in this study are well suited for analysis of mating because they are homothallic and thus are capable of mating in a clonal culture.
Ammonium also regulates nitrogen metabolism of the cell by repressing genes required for the utilization of poor nitrogen sources such as proline. This phenomenon is known as nitrogen catabolite repression, but the nitrogen-sensing mechanism remains unclear. Ammonium transporter genes are themselves subject to nitrogen catabolite repression. Generally, a gene encoding high-affinity transporter (for example, S. cerevisiae MEP2 and A. nidulans mepA) is repressed by high concentrations of ammonium, whereas a gene for low-affinity transporter (S. cerevisiae MEP3 and A. nidulans meaA) is subject to less regulation (Marini et al. 1994, 1997; Monahan et al. 2002a, 2006). Interestingly, the expression of genes that are subject to nitrogen catabolite repression, including MEP2, is induced by addition of rapamycin, an inhibitor of TOR (Cardenas et al. 1999; Hardwick et al. 1999), implicating the TOR pathway in nitrogen catabolite repression (Cooper 2002). Thus, investigation of transcriptional regulation of ammonium transporters may provide insight into the mechanism of nitrogen sensing in nitrogen catabolite repression.
The present study identified three S. pombe genes that encode functional ammonium transporters with distinct properties. The sets of amt deletion strains (ura4+ and ura4 series) constructed in this study would serve as valuable tools for investigating the physiological roles of the S. pombe ammonium transporters.
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Experimental procedures |
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The S. pombe strains used in this study are listed in Table 1. All strains, with the exception of FY7507, were derived from the homothallic (h90) strain FY7406. FY7507 was only used as a source of ura4+, which was amplified by PCR and used to replace the ura4-D18 allele in FY7406 derivatives.
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YE and EMM media (Moreno et al. 1991) were used for routine growth of S. pombe. EMM contained 93 mM (5 g/L) NH4Cl as the nitrogen source. EMM-N, which lacks NH4Cl, was used for preparing media that contained lower concentrations of NH4Cl or 0.1% proline as the nitrogen source. The pH of EMM-N was 5.9. ME medium (Moreno et al. 1991) was used for mating in the course of strain construction. LNB, low-ammonium medium described by Amoah-Buahin et al. (2005), contained 2% glucose, the same salts and vitamins as in EMM, and 0.0067% (that is, 1% of the standard concentration) yeast nitrogen base without amino acids (Difco), which contained 0.38 mM (NH4)2SO4 (0.76 mM ammonium) as the nitrogen source. When necessary, (NH4)2SO4 in LNB was replaced by 0.1 mM NH4Cl or 1 mM glutamine by using yeast nitrogen base without amino acids and ammonium sulfate (Difco). Solid media contained 2% Bacto agar (Difco).
Construction of amt deletion strains
The amt1, amt2, and amt3 genes were disrupted by a PCR-based method (Baudin et al. 1993), in which a 2.5-kb ura4+ pop-out cassette flanked by 80-bp 5' and 3' sequences of the amt genes was PCR-amplified from pGEM3ZpBRura4+pBR (Waddell & Jenkins 1995) and used to replace the amt genes by lithium acetate transformation of the h90 ura4-D18 strain FY7406. A haploid strain was used as the host for the gene disruption with the assumption that the amt genes are dispensable for growth on ammonium-rich media such as EMM, which proved to be the case. Large Ura+ transformants among tiny background colonies were picked and tested by PCR for the correct disruption that would yield amt::ura4+ strains. In these strains, the ura4+ cassette replaces –85 to +1527, –68 to +1543, and –141 to +1422 (relative to the A of the initiation codon) of the amt1, amt2, and amt3 genes, respectively. amt strains, in which the ura4+ cassette had been popped out, were obtained from the amt::ura4+ strains by selecting colonies on EMM+Ura medium containing 1 mg/mL 5-fluoroorotic acid. Double and triple deletion strains were constructed by genetic crosses between amt::ura4+ and amt strains. The deletion allele ura4-D18 (Grimm et al. 1988) was converted to the wild-type allele through transformation with a ura4+ fragment that was PCR-amplified from the genomic DNA of FY7507. In order to facilitate homologous recombination, PCR primers used for the amt disruption or the ura4 conversion were designed so that both the 5' terminal nucleotide and its 5' adjacent nucleotide are T, allowing the termini of the amplified fragment to remain homologous to its target sequences even if 3'-A overhangs were added by the terminal transferase activity of Taq DNA polymerase. Primers used in this study are listed in Supplementary Table S1.
Genetic cross and random spore analysis
To construct double and triple amt mutants, crosses between h90 strains were carried out as described (Gutz et al. 1974). All the crosses were performed with Ura+ and Ura– strains by mixing an excess amount of the Ura– cells with the Ura+ cells on ME medium. After 3–5 days at 27 °C, the mating mixture was suspended in 0.5%
ß-glucuronidase (Sigma) and incubated at 30 °C overnight. The resulting spores were washed and resuspended in water, spotted on EMM medium, and streaked for single colonies, whose amt genotypes were then determined by PCR.
Test for growth at various ammonium concentrations or in the presence of methylammonium
Cells of a set of amt strains grown on YE medium were suspended in water, counted, and diluted to 1 x 107 or 5 x 107 cells/mL, and 3-µL aliquots of the suspensions were spotted onto plates to be tested.
Ammonium removal assay
The ammonium concentration in the culture medium was determined essentially as described (Marini et al. 1994; Lorenz & Heitman 1998a). Cells grown overnight in minimal proline medium were harvested and inoculated into the same medium at an OD600 of 1. After incubation at 30 °C for 90 min, NH4Cl was added to a final concentration of 1 mM. At intervals, an aliquot of the culture was taken and filtered, and the supernatant was used for assaying ammonium by the glutamate dehydrogenase-based method (Bergmeyer & Beutler 1985), in which the reduction of NADH was determined by measuring A340. In these assays, samples were diluted so that a A340 of 1 corresponded to 1 mM NH4+.
Invasive growth assay
Cells grown on YE medium were suspended in water, counted, and diluted to 2 x 107 cells/mL, and 5-µL aliquots of the suspensions were spotted onto plates to be tested. After incubation at 30 °C for the indicated periods, cells on the surface of the agar were washed off by rubbing gently with a finger under a stream of running water, which revealed the cells that had penetrated the agar.
Microscopy
The morphology of colonies within the agar was observed under an Olympus microscope with a 4x or 10x objective by placing plates on the microscope stage.
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* Correspondence: E-mail: mitsuzawa.hiroshi{at}nihon-u.ac.jp
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Received: 31 May 2006
Accepted: 14 July 2006
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