1 Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Apartado de Correos 73, Burjassot, 46100 Valencia, Spain
2 Departamento de Medicina Preventiva y Salud Pública, Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universitat de València, Avenida Vicente Andrés Estellés s/n, 46100-Burjassot, Valencia, Spain
Correspondence
Andrew P. MacCabe
andrew{at}iata.csic.es
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ABSTRACT |
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INTRODUCTION |
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Apart from being a principal source of carbon and energy, glucose exerts important regulatory effects on cellular metabolism, and previous studies have identified several genetic loci implicated in the regulation of carbon catabolite repression (CCR) of metabolic processes in A. nidulans (Felenbok & Kelly, 1996). The physiology and genetics of glucose uptake is however largely uncharacterized in this hyphal fungus. Earlier studies provided evidence for acetate- and pyruvate-repressible (Romano & Kornberg, 1968
, 1969
; Desai & Modi, 1977
), energy-dependent D-glucose uptake by A. nidulans mycelia distinct from the bacterial phosphoenolpyruvate sugar phosphotransferase system in that hexose uptake is not dependent on phosphorylation of the transported sugar (Brown & Romano, 1969
; Mark & Romano, 1971
). Since then little direct work on sugar transport in this micro-organism has been conducted.
As part of an effort to investigate sugar transport in A. nidulans we have initiated studies on glucose uptake. We report here an initial characterization of the kinetics of glucose uptake by germinating conidia. Evidence is presented for the existence of at least two kinetically distinct glucose uptake systems and roles for the products of the creA and sorA genes.
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METHODS |
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Obtention of germinating conidia.
Defined numbers of conidia were inoculated into 300 ml SGM containing 0·1 % (w/v) yeast extract and 1 % (w/v) carbon source (added from filter-sterilized stocks as specified in Results) in 1 l Erlenmeyer flasks. Incubation was carried out for 4 h at 37 °C on an orbital shaker at 200 r.p.m. Germinating conidia (prior to emergence of the germ tube) were recovered by low-speed centrifugation (3200 g) for 6 min and washed serially (five or six times) with ice-cold SGM lacking a carbon source to eliminate traces of the growth substrate. Washed conidia were then resuspended by gentle vortexing in ice-cold SGM until a homogeneous suspension was obtained. Aliquots (250 µl) containing a known number of conidia (within the range 5x1065x107 depending on the strain analysed) were dispensed into 2 ml screw-capped Eppendorf tubes. Aliquots were maintained on ice until required.
Glucose uptake measurements.
Glucose uptake rates were measured by assaying the incorporation of D-[u-14c]glucose (11·655 gbq mmol-1; american radiolabeled chemicals) into conidia at various D-glucose concentrations in the range 0·1 µM to 10 mM. Aliquots of germinating conidia (250 µl) were pre-incubated at 37 °C for 10 min in a shaking thermomixer (Eppendorf) prior to addition of an equal volume of warmed (37 °C) substrate solution containing a twofold concentration of radiolabelled glucose of known specific activity. Agitation and incubation were continued for periods of 5, 30, 60 and 90 s and uptake subsequently quenched by rapid addition of 1·5 ml ice-cold 200 mM unlabelled glucose in SGM and filtration over nitrocellulose filters (HAWP02500; Millipore) mounted in a vacuum manifold, followed immediately by two consecutive washes of 1·5 ml with the same ice-cold solution. Filters were subsequently lightly blotted on filter paper to remove excess moisture and immersed in 3 ml OptiPhase Hisafe 3 (Wallac) for liquid scintillation counting. The amount of glucose (nmol) retained on the filters was calculated from the specific activities of the substrate solutions and plotted vs period of uptake. The rate of glucose uptake was determined from the gradient of the slope. Data analysis was done using SigmaPlot v 4.01.
To investigate the energy requirement of glucose uptake 4-nitrophenol was added to conidial aliquots immediately prior to the start of the 10 min pre-incubation period.
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RESULTS |
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Measurement of glucose uptake (see Methods for details) by wild-type conidia germinating in the presence of glucose showed typical MichaelisMenten saturation kinetics (Fig. 1). The Km (
1·3 mM) and Vmax (
0·5 nmol glucose s-1 per 5x107 conidia) values for overall glucose uptake were obtained directly by non-linear regression analysis. Apart from the direct plot (v0 vs [S]), the data were transformed to the EadieHofstee representation (v0 vs v0 [S]-1). This transformation yielded a biphasic plot indicative of the presence of at least two kinetically distinct modes of uptake (Fig. 1
inset). The form of the plot reveals a major contribution to overall glucose transport by a low-affinity uptake system and a minor contribution by a system of much greater glucose affinity. Given that extrapolation of Km and Vmax values from EadieHofstee plots is both relatively error-prone and limited to cases of monophasic kinetics (Leatherbarrow, 1990
; Fuhrmann & Völker, 1993
), non-linear regression to two-component MichaelisMenten kinetics (V={Vmax1[S]/(Km1+[S])}+{Vmax2[S]/(Km2+[S])}) was used to obtain estimates of Km and Vmax values for the low- and high-affinity systems. A high-affinity glucose uptake system was deduced operating at micromolar (Km
16 µM) concentrations of glucose whilst a low-affinity component operates in the millimolar (Km
1·4 mM) concentration range. The Vmax (0·6 fmol min-1 per conidium) of the low-affinity component represents more than 95 % of the Vmax of overall glucose uptake.
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Overall glucose uptake kinetics by sorA3 mutant conidia germinated in glucose medium were found to be similar to those of wild-type (Fig. 7) except that EadieHofstee representation is indicative of monophasic kinetics. Parallel analyses of sorA3 conidia germinating in the presence of glucose or glycerol indicate that uptake is effected by distinct monophasic kinetic systems (Fig. 7
inset) depending on the carbon source used for germination. The estimated Km (
1·3 mM) for uptake by glucose-germinated conidia corresponds to the low-affinity component present in wild-type conidia. However, in glycerol-germinated sorA3 conidia uptake is not effected by a system with a Km as low as that observed for the high-affinity component in glycerol-germinated wild-type conidia. Non-linear regression analysis yields a Km of
400 µM for this component. The absence of the high-affinity uptake system in the glycerol-germinated sorA3 mutant indicates that the sorA gene encodes a function required for high-affinity glucose uptake.
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DISCUSSION |
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The monophasic high-affinity kinetics of glucose uptake by glycerol-germinating wild-type conidia implies that low-affinity uptake is induced by the presence of glucose. Induction of this system is not however apparent in creAd30 mutant conidia germinating in the presence of glucose. Several speculative explanations for this observation are possible: (i) repression of the low-affinity uptake system by some property/component involved in the high-affinity uptake system; (ii) indirect or direct activation effects of CreA, i.e. via repression of a repressor of the low-affinity uptake system or an unidentified direct activation function of CreA exerted on the low-affinity system, respectively; or (iii) CreA involvement in glucose sensing either as a component of the sensing machinery or by regulation of the expression of a sensor such that in creAd mutants elevated levels of glucose fail to be detected. In the context of dual repressor/activator functions, the S. cerevisiae transcription factor Rgt1 has been shown to function as both repressor and activator of hexose transporters in response to glucose concentration (Özcan et al., 1996).
Unlike both wild-type and creAd mutants, germinating conidia of the L-sorbose resistant sorA3 mutant fail to show expression of the high-affinity glucose uptake system even under derepressing conditions. In contrast, creAd mutants take up glucose exclusively by high-affinity uptake and exhibit enhanced susceptibility to L-sorbose toxicity. These observations suggest that the high-affinity glucose uptake system is involved in L-sorbose uptake, which could explain the greatly reduced uptake of L-sorbose and slightly reduced D-glucose uptake previously observed for sorA mutants compared to wild-type (Elorza & Arst, 1971). Of particular interest in this regard are earlier studies in the filamentous fungus Neurospora crassa that showed L-sorbose uptake (Km
4 mM) to be effected by an energy-requiring high-affinity glucose transporter (Km
10 µM) which is expressed under derepressing conditions (Scarborough, 1970
; Schneider & Wiley, 1971
). By analogy to the situation in N. crassa, the sorA gene is likely to be implicated in the high-affinity glucose uptake component of A. nidulans, possibly encoding either a glucose permease or a regulator of its expression. Under derepressing conditions sorA3 mutant conidia display two phenotypic variations compared to wild-type: (i) the absence of the high-affinity system, and (ii) the appearance of a glucose uptake activity of intermediate affinity (Km
400 µM). These two phenomena may be accounted for by a mutation in a structural gene encoding the high-affinity transporter which affects (increases) the Km of glucose (and L-sorbose) uptake. Hence, under derepressing conditions glucose uptake would be effected by a mutant high-affinity transporter having about tenfold less affinity for glucose. However, an alternative possibility of a role for the sorA gene product in the regulation of glucose uptake cannot be discounted. It is noteworthy in the latter context that the A. nidulans malA gene, previously identified in mutants affected in maltose utilization (Roberts, 1963
), was ultimately revealed to encode a zinc finger transcription factor AmyR rather than an enzymic or permease activity (Tani et al., 2001
).
Recognition of the multiplicity of hexose transporters and sensors resulting from the molecular cloning of genes involved in sugar transport in S. cerevisiae and the characterization of their protein products has greatly assisted in clarifying the data from earlier kinetic analyses of hexose uptake carried out in that micro-organism (Özcan & Johnston, 1999, and references therein). The suggestion that the variations observed in the kinetics of uptake could be brought about by alteration of the properties of a single transporter (Walsh et al., 1994
) has been superseded by the concept of regulated expression of a range of transporters of differing kinetic characteristics. Evidence for multiplicity of sugar transport related proteins in A. nidulans (accession numbers AJ251561, AJ278285 and other unpublished data) suggests that Km values may be useful in identifying individual uptake systems. In that regard, the work reported here identifies three glucose uptake components. High- and low-affinity systems have been clearly identified from the analyses of wild-type and creAd germinating conidia. A third, intermediate-affinity system is detected in sorA3 conidia under derepressing conditions. However, the possibility that this system arises from alteration of another uptake activity cannot be excluded (see above).
The current study reveals certain differences between the system of hexose transport in A. nidulans and that of S. cerevisiae: whereas uptake in this yeast is by facilitated diffusion, the process in A. nidulans is energy requiring; Km values reveal the existence of glucose uptake systems of much greater affinity in A. nidulans than those observed in S. cerevisiae. These differences appear to reflect the differences in the niches occupied by these two micro-organisms the relatively sugar-rich environments favoured by S. cerevisiae and the wide variety of substrates which can be utilized by A. nidulans including those having little or no immediately available monosaccharidic carbon source. Further kinetic studies on mutants affected in sugar metabolism coupled with the identification, cloning and characterization of genes involved in sugar uptake will yield considerable insight into the nature and regulation of the process of uptake of basic nutrients by the model organism A. nidulans.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arst, H. N., Jr & Cove, D. J. (1973). Nitrogen metabolite repression in Aspergillus nidulans. Mol Gen Genet 126, 111141.[Medline]
Arst, H. N., Jr & Scazzocchio, C. (1985). Formal genetics and molecular biology of the control of gene expression in Aspergillus nidulans. In Gene Manipulations in Fungi, pp. 309343. Edited by J. W. Bennet & L. L. Lasure. New York: Academic Press.
Arst, H. N., Jr, Tollervey, D., Dowzer, C. E. & Kelly, J. M. (1990). An inversion truncating the creA gene of Aspergillus nidulans results in carbon catabolite derepression. Mol Microbiol 4, 851854.[Medline]
Bailey, C. & Arst, H. N., Jr (1975). Carbon catabolite repression in Aspergillus nidulans. Eur J Biochem 51, 573577.[Medline]
Boles, E. & Hollenberg, C. P. (1997). The molecular genetics of hexose transport in yeasts. FEMS Microbiol Rev 21, 85111.[CrossRef][Medline]
Brown, C. E. & Romano, A. H. (1969). Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans. J Bacteriol 100, 11981203.[Medline]
Ciriacy, M. & Reifenberger, E. (1997). Hexose transport. In Yeast Sugar Metabolism, pp. 4565. Edited by F. K. Zimmermann & K. D. Entian. Lancaster: Technomic Publishing Co.
Clutterbuck, A. J. (1973). Aspergillus nidulans. In Handbook of Genetics, pp. 447510. Edited by R. C. King. New York: Plenum Press.
Coons, D. M., Boulton, R. B. & Bisson, L. F. (1995). Computer-assisted nonlinear regression analysis of the multicomponent glucose uptake kinetics of Saccharomyces cerevisiae. J Bacteriol 177, 32513258.[Abstract]
Cove, D. J. (1966). The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim Biophys Acta 113, 5156.[Medline]
d'Enfert, C. (1997). Fungal spore germination: insights from the molecular genetics of Aspergillus nidulans and Neurospora crassa. Fungal Genet Biol 21, 163172.[CrossRef]
Desai, J. D. & Modi, V. V. (1977). Regulation of glucose transport in Aspergillus nidulans. Experientia 33, 726727.[Medline]
Diallinas, G., Gorfinkel, L., Arst, H. N., Jr, Cecchetto, G. & Scazzocchio, C. (1995). Genetic and molecular characterization of a gene encoding a wide specificity purine permease of A. nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes. J Biol Chem 270, 86108622.
Dowzer, C. E. & Kelly, J. M. (1989). Cloning of the creA gene from Aspergillus nidulans: a gene involved in carbon catabolite repression. Curr Genet 15, 457459.[Medline]
Dowzer, C. E. & Kelly, J. M. (1991). Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol 11, 57015709.[Medline]
Elorza, M. V. & Arst, H. N., Jr (1971). Sorbose resistant mutants of Aspergillus nidulans. Mol Gen Genet 111, 185193.[Medline]
Felenbok, B. & Kelly, J. M. (1996). Regulation of carbon metabolism in mycelial fungi. In The Mycota. III: Biochemistry and Molecular Biology, pp. 369380. Edited by R. Brambl & G. Marzluf. Berlin: Springer.
Fuhrmann, G. F. & Völker, B. (1993). Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots. Biochim Biophys Acta 1145, 180182.[Medline]
Hynes, M. J. & Kelly, J. M. (1977). Pleiotropic mutants of Aspergillus nidulans altered in carbon metabolism. Mol Gen Genet 150, 193204.[Medline]
Johnston, M. (1999). Feasting, fasting and fermenting: glucose sensing in yeast and other cells. Trends Genet 15, 2933.[CrossRef][Medline]
Kruckeberg, A. L. (1996). The hexose transporter family of Saccharomyces cerevisiae. Arch Microbiol 166, 283292.[CrossRef][Medline]
Kruckeberg, A. L., Walsh, M. C. & van Dam, K. (1998). How do yeast cells sense glucose? BioEssays 20, 972976.[CrossRef][Medline]
Kulmburg, P., Mathieu, M., Dowzer, C., Kelly, J. & Felenbok, B. (1993). Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans. Mol Microbiol 7, 847857.[Medline]
Leatherbarrow, R. J. (1990). Using linear and non-linear regression to fit biochemical data. Trends Biochem Sci 15, 455458.[CrossRef][Medline]
MacCabe, A. P., Orejas, M. & Ramón, D. (2001). Aspergillus nidulans as a model organism for the study of the expression of genes encoding enzymes of relevance in the food industry. In Applied Mycology and Biotechnology, vol. I, Agriculture and Food Production, pp. 239265. Edited by G. G. Khachatourians & D. K. Arora. Amsterdam: Elsevier.
Mark, C. G. & Romano, A. H. (1971). Properties of the hexose transport system of Aspergillus nidulans. Biochim Biophys Acta 249, 216226.[Medline]
Özcan, S. & Johnston, M. (1999). Function and regulation of yeast hexose transporters. Microbiol Mol Biol Rev 63, 554569.
Özcan, S., Leong, T. & Johnston, M. (1996). Rgt1p of Saccharomyces cerevisiae, a key regulator of glucose-induced genes, is both an activator and a repressor of transcription. Mol Cell Biol 16, 64196426.[Abstract]
Peñalva, M. A. (2001). A fungal perspective on human inborn errors of metabolism: alkaptonuria and beyond. Fungal Genet Biol 34, 110.[CrossRef][Medline]
Peñalva, M. A. & Arst, H. N., Jr (2002). Regulation of gene expression by ambient pH in filamentous fungi and yeasts. Microbiol Mol Biol Rev 66, 426446.
Roberts, C. F. (1963). The genetic analysis of carbohydrate utilization in Aspergillus nidulans. J Gen Microbiol 31, 4558.[Medline]
Rolland, F., Winderickx, J. & Thevelein, J. M. (2002). Glucose-sensing and -signalling mechanisms in yeast. FEMS Yeast Res 2, 183201.[CrossRef][Medline]
Romano, A. H. & Kornberg, H. L. (1968). Regulation of sugar utilization by Aspergillus nidulans. Biochim Biophys Acta 158, 491493.[Medline]
Romano, A. H. & Kornberg, H. L. (1969). Regulation of sugar uptake by Aspergillus nidulans. Proc Roy Soc B 173, 475490.
Scarborough, G. A. (1970). Sugar transport in Neurospora crassa. II. A second glucose transport system. J Biol Chem 245, 39853987.
Schleissner, C., Olivera, E. R., Fernandez-Valverde, M. & Luengo, J. M. (1994). Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate. J Bacteriol 176, 76677676.[Abstract]
Schneider, R. P. & Wiley, W. R. (1971). Kinetic characteristics of the two glucose transport systems in Neurospora crassa. J Bacteriol 106, 479486.[Medline]
Shroff, R. A., O'Connor, S. M., Hynes, M. J., Lockington, R. A. & Kelly, J. M. (1997). Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans. Fungal Genet Biol 22, 2838.[CrossRef][Medline]
Tani, S., Katsuyama, Y., Hayashi, T., Suzuki, H., Kato, M., Gomi, K., Kobayashi, T. & Tsukagoshi, N. (2001). Characterization of the amyR gene encoding a transcriptional activator for the amylase genes in Aspergillus nidulans. Curr Genet 39, 1015.[CrossRef][Medline]
Tazebay, U. H., Sophianopoulou, V., Cubero, B., Scazzocchio, C. & Diallinas, G. (1995). Post-transcriptional control and kinetic characterization of proline transport in germinating conidiospores of Aspergillus nidulans. FEMS Microbiol Lett 132, 2737.[CrossRef][Medline]
Torres, N. V., Riol-Cimas, J. M., Wolschek, M. & Kubicek, C. P. (1996). Glucose transport by Aspergillus niger: the low-affinity carrier is only formed during growth on high glucose concentrations. Appl Microbiol Biotechnol 44, 790794.[CrossRef]
Walsh, M. C., Smits, H. P., Scholte, M. & van Dam, K. (1994). Affinity of glucose transport in Saccharomyces cerevisiae is modulated during growth on glucose. J Bacteriol 176, 953958.[Abstract]
Received 13 March 2003;
revised 8 May 2003;
accepted 8 May 2003.
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