Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-242, 04510 Mexico City, Mexico1
Author for correspondence: Alicia González. Tel: +52 56 22 56 31. Fax: +52 56 22 56 30. e-mail: amanjarr{at}ifisiol.unam.mx
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ABSTRACT |
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Keywords: glutamate biosynthesis, glutamate synthase, glutamate dehydrogenase
Abbreviations: GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthetase
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INTRODUCTION |
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Mutants affected in either NADP-GDH or GOGAT have been obtained in a number of fungal species. In the yeast Saccharomyces cerevisiae, besides the GS-GOGAT pathway, there are two NADP-GDH isozymes, NADP-GDH1 and NADP-GDH3, encoded by GDH1 and GDH3, respectively (Avendaño et al., 1997 ). In this yeast, only the triple mutant lacking GOGAT, GDH1 and GDH3 shows a tight glutamate auxotrophy (Avendaño et al., 1997
). In Neurospora crassa and Aspergillus nidulans, the simultaneous lack of GOGAT and NADP-GDH leads to full auxotrophy, indicating that in this species, only two pathways for glutamate biosynthesis are functional (Romero & Dávila, 1986
; Macheda et al., 1999
). Thus, S. cerevisiae is so far the only example of a micro-organism possessing three pathways for glutamate biosynthesis.
In Kluyveromyces lactis, both NADP-GDH and GOGAT activities have been detected (Valenzuela et al., 1995 ). Mutants devoid of NADP-GDH showed a wild-type phenotype, suggesting that either the GS-GOGAT pathway plays a major role in glutamate biosynthesis or that, as found for S. cerevisiae gdh1 mutants, glutamate biosynthesis could be maintained by the combined action of the GS-GOGAT pathway and a third biosynthetic route. To analyse this matter, we isolated a K. lactis single mutant impaired in GOGAT activity and a double mutant lacking both GOGAT and NADP-GDH. The first mutant strain did not display any growth defect phenotype whilst the double mutant was a full glutamate auxotroph. This implies that, as in N. crassa and A. nidulans, K. lactis has only two pathways for glutamate biosynthesis.
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METHODS |
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Determination of GOGAT and NADP-GDH activities.
Soluble extracts for enzyme assays were prepared as previously described (Valenzuela et al., 1995 ). NADP-GDH and GOGAT were assayed by the methods of Doherty (1970)
and Roon et al. (1974)
, respectively. Protein was measured by the Lowry method with bovine serum albumin as a standard.
Purification of GOGAT from K. lactis.
K. lactis MD2/1 was grown in 12 l MM, harvested at an OD600 of 0·5 and stored at -70 °C until used. All purification steps were carried out at 4 °C.
Step 1: crude extract.
Cells were thawed and resuspended in 2 ml buffer A [0·1 M potassium phosphate (pH 7·5), 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 50 µg N-p-tosyl-L-lysine chloromethyl ketone (TLCK) ml-1 and 100 µg N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) ml-1] (g cells)-1. Crude extracts were obtained after mechanical disruption of cells with a Bead-Beater (10 cycles of 1 min), using 0·5 g 710180 µm diameter glass beads (ml cell suspension)-1 and centrifugation at 35000 g for 30 min.
Step 2: ammonium sulphate precipitation.
Crude extract was treated with a saturated solution of ammonium sulphate (pH 7·5). Proteins precipitating at 30% saturation were discarded and the supernatant was dialysed four times (30 min each) against 2 l buffer A but lacking DTT and protease inhibitors (buffer B).
Step 3: affinity chromatography.
The dialysed fraction was applied to a 40 ml blue Sepharose column (23x1·7 cm) previously equilibrated with buffer A plus 0·2 mM 2-oxoglutarate and 5% glycerol (buffer C). After sample application, the column was washed with 20 column volumes of buffer C and GOGAT activity was eluted with buffer C that also contained 0·1 mM NADH. Fractions with GOGAT activity were pooled and dialysed three times against buffer B for 30 min; protease inhibitors (0·5 mM DTT, 25 µg ml-1 TLCK, 50 µg ml-1 TPCK and 0·05 mM PMSF) were added and protein was concentrated by ultrafiltration using an Amicon YM30 membrane.
Molecular mass determination.
The apparent molecular mass of the GOGAT polypeptide was determined by SDS-PAGE (Schägger et al., 1986 ). Molecular mass standards were obtained from Sigma.
Western blot analysis.
Immunoblotting was carried out as described by Towbin et al. (1979) , with the modifications of González-Halphen et al. (1988)
. S. cerevisiae GOGAT anti-serum, obtained as described by Cogoni et al. (1995)
, was diluted 1:50000 and goat anti-rabbit immunoglobulin Galkaline phosphatase conjugate was used as the secondary antibody.
Northern analysis.
Northern analysis was carried out as previously described (González et al., 1992 ). Total RNA of the MD2/1 wild-type strain was prepared from 50 ml of overnight cultures in MM, as described by Struhl & Davis (1981)
. Prehybridization was carried out at 65 °C for 60 min and hybridization was carried out at 65 °C for 12 h. Filters were first washed with a tenfold dilution of 20xSSC (Maniatis et al., 1982
) containing 0·10% SDS at 65 °C for 30 min and then with a 130-fold dilution of 20xSSC containing 0·10% SDS at 65 °C for 30 min.
Southern analysis.
Southern analysis was carried out as described by Nasmyth & Reed (1980) . Total K. lactis DNA was prepared from 50 ml of overnight cultures of the MD2/1 wild-type strain or of the WM37 glt1 mutant in MM. When a heterologous probe was used, prehybridization was carried out at 45 °C for 45 min and hybridization was carried out at 45 °C for 24 h. Filters were washed twice with a tenfold dilution of 20xSSC containing 0·10% SDS at 45 °C for 35 min. When an homologous probe was used, prehybridization and hybridization conditions were those described for the Northern analysis.
PCR amplification.
K. lactis GLT1 (GOGAT) PCR amplification was conducted using the deoxyoligonucleotides and the programme described by Cogoni et al. (1995) . Total DNA from K. lactis strain WM37 was used as template for amplification by PCR in a Stratagene Robocycler 40. The amplification product (300 bp as judged on agarose gel) was cloned in the EcoRI restriction site of the Stratagene SK plasmid and sequenced with an ABI PRISM Genetic Analyzer, using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit from Applied Biosystems-Perkin Elmer. To obtain a PCR product suitable for gene disruption, the PCR-based ORF replacement protocol described by Wach et al. (1994)
was used with kanMX as a marker. Two deoxyoligonucleotides were designed based on the nucleotide sequence of the K. lactis GOGAT fragment obtained by PCR and on the sequence of the multiple cloning site present in the pFA6a vector (Wach et al., 1994
). The deoxyoligonucleotide S1 (5'-GAG ACT CAT CAA ACA TTA GTT TTG AAT GAT TTG AGA GGA AAC GTC CGT ACG CTG CAG GTC GAC-3') comprises 45 bp from the most 5' end of the K. lactis PCR GOGAT fragment and 18 bp (indicated above in bold lettering) of the pFA6a multiple cloning site. The deoxyoligonucleotide S2 (5'-GAA TTT GTC TCT CAA AAC GGG GTC TTG AGT GGC AAT ACC AAC TGC ATC GAT GAA TTC GAG CTC G-3') contains 45 bp from the 3' end of the PCR GOGAT fragment and 19 bp (bold) from the pFA6a multiple cloning site. Qiagen purified pFA6a DNA was used as template for amplification by PCR, carried out in a Stratagene Robocycler 40 with the following programme: one denaturing cycle for 30 s at 94 °C, followed by 26 cycles of 30 s denaturation at 94 °C, 1 min annealing at 50 °C and 3 min extension at 72 °C. A 1500 bp PCR product obtained was gel-purified and used to transform strain WM37.
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RESULTS AND DISCUSSION |
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The apparent molecular mass of the pure polypeptide was 199 kDa, as estimated by SDS-PAGE. This value is comparable to those of S. cerevisiae, N. crassa and plant NADH-GOGAT (Hummelt & Mora, 1980 ; Benny & Bolland, 1977
; Chen & Cullimore, 1988
: Hayakawa et al., 1992
; Anderson et al., 1989
; Cogoni et al., 1995
).
Western blot analysis was carried out with antibodies raised against the S. cerevisiae GOGAT protein (Cogoni et al., 1995 ). These antibodies cross-reacted with crude extracts (data not shown) and pure GOGAT protein from K. lactis (Fig. 1b
), suggesting that these two proteins bear similar antigenic epitopes. The same antibodies did not react with Escherichia coli NADPH-GOGAT (Cogoni et al., 1995
).
Since the immunoblotting results suggested similarity between the S. cerevisiae and K. lactis GOGAT enzymes, we decided to use an internal EcoRIEcoRI 4·3 kb fragment of the S. cerevisiae GOGAT gene (Cogoni et al., 1995 ) as a hybridization probe against total DNA from K. lactis strain MD2/1. Using low-stringency hybridizing conditions we were able to detect an EcoRIEcoRI 4·3 kb band in total DNA of K. lactis digested with EcoRI (Fig. 2a
). This result suggested that the K. lactis genome contained a sequence homologous to the S. cerevisiae GOGAT probe used.
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To determine the size of the mRNA that encodes the K. lactis GOGAT, the 300 bp PCR product was hybridized against total RNA extracted from the K. lactis wild-type strain (MD2/1). As shown in Fig. 2(b), a major band of around 7 or 8 kb was identified. These results indicate that the GOGAT enzyme from K. lactis should be constituted by a high-molecular-mass polypeptide. In order to analyse whether the 199 kDa polypeptide was the only GOGAT in K. lactis we decided to disrupt the KlGLT1 gene that encodes this protein.
To obtain a null GOGAT mutant, the PCR product flanked by GLT1 sequences, obtained as described in Methods, was used to transform strain WM37, following the lithium acetate protocol described by Gietz & Woods (1994) . After transformation, cells were allowed to grow for 3 h in liquid YPD (see Methods). Transformation mix was then plated on YPD plates containing 200 mg G418 l -1. After 3 d incubation, four colonies appeared on the transformation plates. These were streaked out on YPD/G418 plates and three of them (WM37a, WM37b and WM37c) grew out to form single colonies. Chromosomal DNA was isolated from the three transformants and digested with BglII. Southern analysis was carried out with the 300 bp PCR fragment as the probe. The pattern obtained from the transformants clearly indicated the insertion of the construct in the wild-type genomic sequence of KlGLT1 (Fig. 3
). The null mutants were completely devoid of GOGAT activity (Table 2
).
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Table 2 shows that when grown on glutamate, the double mutant C4A showed neither NADP-GDH nor GOGAT activity. As can be seen in Fig. 4
, single GOGAT null mutants grew as well as the wild-type strain on ammonium as sole nitrogen source. However the double mutant (C4A) was completely unable to grow in the absence of glutamate. These results show that in K. lactis two pathways exist for glutamate biosynthesis and that both of them have to be knocked out in order to obtain a glutamate auxotroph.
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K. lactis mutants devoid of NADP-GDH, showed no growth defect phenotype. Since these mutant strains have increased GS and GOGAT activities, it has been suggested that in this yeast the GS-GOGAT pathway could constitute either the major pathway for ammonium assimilation or a compensatory mechanism for the lack of NADP-GDH (Valenzuela et al., 1995 ). The isolation of K. lactis null GOGAT mutants has allowed us to show that, similar to the situation occurring in E. coli, neither null NADP-GDH mutants nor GOGAT null mutants exhibit a growth defect phenotype. Further experiments will have to be conducted in order to determine whether there are particular physiological conditions in which each of the two pathways present in K. lactis plays a major biosynthetic role. As has been pointed out previously, Schizosaccharomyces pombe mutants devoid of GOGAT activity grow slowly and show a fivefold increase in their NADP-GDH levels, implying that the GS-GOGAT pathway is the primary route of glutamate biosynthesis (Barel & MacDonald, 1993
). Thus, the primary route for glutamate biosynthesis is not the same in all yeast species, although so far all the species studied have both GDH and GOGAT enzymes.
As mentioned earlier, besides GOGAT, S. cerevisiae has two non-allelic genes encoding two different NADP-GDH isozymes, one of which most probably evolved as the result of duplication of a primary gene. In this regard, it has been proposed (Wolfe & Shields, 1997 ), that S. cerevisiae is a degenerate tetraploid resulting from whole-genome duplication, and that Kluyveromyces and Saccharomyces diverged before this proposed genome duplication of S. cerevisiae occurred. Accordingly, it has been found that Kluyveromyces homologues of many S. cerevisiae genes are only found in single copy. This study shows that K. lactis has a single copy of the NADP-GDH-encoding genes, thus constituting another example of genes that were duplicated sometime during the evolutionary history of S. cerevisiae, but which evolved as a single allele in K. lactis. In addition, it has been proposed, that S. cerevisiae genome duplication may have played a role in the evolutionary adaptation of S. cerevisiae to fermentative growth (Wolfe & Shields, 1997
). In this regard, it could be speculated that the presence of two NADP-GDH isozymes in S. cerevisiae provides a mechanism to equilibrate utilization of 2-oxoglutarate and energy production under fermentative or respiratory conditions. Since K. lactis is a strict aerobe, only able to grow under respiratory conditions, it could be proposed that in this yeast, a single NADP-GDH enzyme allows glutamate biosynthesis and thus 2-oxoglutarate utilization without compromising energy production.
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ACKNOWLEDGEMENTS |
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This work was supported in part by the Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (IN212898).
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Received 29 June 1999;
revised 13 September 1999;
accepted 4 October 1999.