1 Dep. Botânica e Engenharia Biológica, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal
2 Centro de Recursos Microbiológicos, SABT, FCT/UNL, 2825-516 Caparica, Portugal
Correspondence
Maria C. Loureiro-Dias
mcdias{at}isa.utl.pt
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ABSTRACT |
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INTRODUCTION |
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Zygosaccharomyces bailii is a food-spoilage yeast that has evolved the ability to grow under rather inhospitable conditions, such as those present in preserved food and beverages: low water activity, low pH and the presence of weak acid preservatives, in particular (Fleet, 1992). In contrast with most yeasts, and in particular with S. cerevisiae, Z. bailii consumes fructose faster than glucose, deserving the designation of a fructophilic yeast. This behaviour was explained by Sousa-Dias et al. (1996)
by taking into account the kinetics of hexose uptake observed in this yeast. Fructose is taken up by a high-capacity, low-affinity transporter, specific for fructose (no other sugar inhibits fructose uptake); a second transporter takes up glucose, fructose and 2-deoxyglucose in a low-capacity and high-affinity manner. Both involve facilitated diffusion mechanisms. Moreover, fructose promotes the inactivation of the glucose transporter, preventing the utilization of this sugar when fructose is also available (Sousa-Dias et al., 1996
).
This work deals with the cloning and characterization of the fructose-specific transporter, Ffz1, of Z. bailii. Our strategy involved functional complementation of a strain of S. cerevisiae incapable of growth on hexoses.
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METHODS |
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Screening of the library.
The genomic library of Z. bailii ISA1307 constructed by Rodrigues et al. (2001) was used in this work. For library plasmid DNA isolation, 45 000 clones, representing four times the total number of independent clones of the genomic library, were grown on LB medium supplemented with ampicillin. Strain EBY.VW4000 was transformed with library plasmid DNA (about 4 µg DNA yielded approximately 2x106 transformants), using the lithium acetate method (Agatep et al., 1998
). The transformation mixture was first plated onto solid YNB medium with 20 g maltose l1 as the sole carbon and energy source, supplemented with leucine, tryptophan and histidine. After 7 days at 28 °C, colonies were replica plated onto the same medium containing 10 g l1 of either fructose or glucose, instead of maltose. The growth phenotype on both media was evaluated after 7 days at 28 °C. The transformants were cured of the library plasmids by selection in 5-fluoroorotic acid (5-FOA)-containing medium, according to the procedure described by Boeke et al. (1987)
.
Initial uptake measurements.
Cells were harvested at an OD640 of 0·50·8 by centrifugation (5000 g for 5 min), washed twice with cold distilled water and resuspended in distilled water to a final concentration of approximately 50 mg dry weight ml1. The cell suspension (20 µl) was mixed with 20 µl 100 mM Tris/citrate buffer, pH 5·0, in 10 ml conical centrifuge tubes. After 2 min incubation in a water bath at 22 °C, uptake was initiated by the addition of D-[u-14c]fructose (283 mci mmol1; 10·5 gbq mmol1) or D-[U-14C]glucose (310 mCi mmol1; 11·5 GBq mmol1) at the appropriate concentrations. After 5 s, the incorporation was stopped by the addition of 10 ml cold distilled water. Cells were filtered through wet glass-fibre filters (Whatman CF/C) at reduced pressure, and washed with 10 ml cold distilled water. All determinations were performed in triplicate. For blanks, cold distilled water was added before the labelled sugar. The values obtained for the blank samples were subtracted from the measurements.
For competition assays, 250 mM of either glucose, 2-deoxyglucose, mannose or sorbose, or 2·5 mM uranyl nitrate, in the same buffer, were added to the D-[U-14C]fructose solutions. In this case, the incorporation was started by the addition of the cell suspension.
The filters were placed in scintillation liquid (OptiPhase HiSafe II; Amersham). The radioactivity remaining on the filters was measured with a Beckman LS6000LL scintillation counter.
DNA manipulations.
The procedure followed for plasmid rescue from yeast transformants was as described by Hoffman & Winston (1987). DNA manipulations were performed essentially as described by Sambrook et al. (1989)
. The complete DNA sequence of both strands of the FFZ1-coding region was obtained by primer walking, using an ABI PRISM 310 Genetic Analyser (Perkin-Elmer).
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RESULTS |
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Kinetics of sugar transport
Transformant TF1 grew in liquid YNB medium containing 1 % fructose as sole carbon and energy source with a duplication time of 3 h. TF1 was able to take up D-[U-14C]fructose, but no D-[U-14C]glucose uptake could be measured (Fig. 2). The kinetic parameters calculated for fructose uptake were Vmax=3·3 mmol h1 g1 and Km=80·4 mM, at 22 °C and pH 5. The specificity of the carrier was determined by testing the effect of other sugars as possible competitive inhibitors of initial [14C]fructose uptake. The uptake of fructose was not affected by the presence of 100 mM glucose, mannose, sorbose or 2-deoxyglucose (results not shown). Uranyl (1 mM), which functions as a competitive inhibitor of glucose uptake in several biological systems (Fuhrmann et al., 1992
), also did not interfere with fructose uptake.
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The FFZ1 gene
Strain EBY.VW4000 transformed with plasmid pTF1 displayed fructose-uptake characteristics that resembled those found for the specific fructose facilitator of Z. bailii, indicating that pTF1 encodes this transporter.
The plasmid was found to carry a Z. bailii genomic DNA insert of approximately 4·5 kb. The insert was sequenced in its entire length, revealing the presence of an ORF of 616 amino acids that exhibited the characteristics of an integral membrane protein with 12 membrane-spanning regions (as predicted by the HMMTOP server, version 1.1). The gene was named FFZ1 (fructose facilitator of Zygosaccharomyces) (see footnote for EMBL accession number). A similarity search in public databases, using the predicted amino-acid sequence of Ffz1p, disclosed a low degree of homology with fungal membrane proteins, among which were sugar transporters and multidrug resistance proteins (Fig. 3). The closest relative of Ffz1p appeared to be a hitherto uncharacterized membrane transporter from Schizosaccharomyces pombe (Yao5p).
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DISCUSSION |
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Two closely related genes encoding specific H+-fructose symporters were previously cloned from yeasts: FSY1 from S. pastorianus (Gonçalves et al., 2000) and FRT1 from K. lactis (Diezemann & Boles, 2003
). Ffz1p, a facilitated diffusion system, does not resemble these proteins (Fig. 3
), suggesting that fructose specificity does not have a common phylogenetic background in these cases. The homology with other facilitated diffusion systems that transport glucose and fructose, like the Hxt family from S. cerevisiae, is also poor. The putative protein was found to be most similar to Yao5p (probable membrane transporter) of Schizosaccharomyces pombe (28 % identity).
The values previously reported for the kinetic parameters of fructose uptake in Z. bailii are quite similar to those measured in S. cerevisiae transformant TF1 carrying the FFZ1 gene in a centromeric plasmid (low copy number). The Km values measured in the original strain and in transformant TF1 were, respectively, 65 mM and 80 mM. Transport via Ffz1p can, therefore, be classified as low affinity. The H+-fructose symporters displayed a much higher affinity (Km values of 0·16 mM in both cases) (Gonçalves et al., 2000; Diezemann & Boles, 2003
). In general, the H+-glucose symporters that have been characterized so far in yeasts also present higher affinities than facilitated diffusion systems (Spencer-Martins & van Uden, 1985
; Loureiro-Dias, 1987
; Peinado et al., 1989
). We can speculate that yeasts invest proton motive force in sugar transport only when the sugar is present in low concentrations in the environment; energy is then necessary to provide an adequate intracellular concentration of the sugar in order for metabolism to proceed. In environments where sugars are present at high levels, the concentration gradient across the plasma membrane is enough to maintain an active catabolism; in this situation it is favourable that sugars cross the membrane by facilitated diffusion without energy dissipation. The presence of a low-affinity, fructose-specific carrier in Z. bailii is, therefore, in keeping with the fact that this yeast is frequently isolated from high-sugar-content foods and beverages.
The maximum velocity of fructose uptake measured in the transformant expressing only Ffz1p (TF1) was 3·3 mmol h1 g1. This value is comparable to that previously reported for the original strain of Z. bailii (5 mmol h1 g1) (Sousa-Dias et al., 1996), and is much higher than the values found for the H+-fructose symporters of S. pastorianus and K. lactis, which were of the order of 0·1 mmol h1 g1(Cason et al., 1986
; Diezemann & Boles, 2003
). Also, H+-glucose symporters characterized in yeasts display, in general, much lower maximum velocities than facilitated diffusion systems (Spencer-Martins & van Uden, 1985
; Loureiro-Dias, 1987
; Peinado et al., 1989
).
Many questions remain to be answered. An intriguing issue concerns the specificity of the novel permease. What are the key structural differences between Ffz1p and the many multi-substrate facilitated-diffusion hexose carriers, like those belonging to the Hxt family? A first clue may reside in the observation that the absence of the 67 amino acids at the C-terminus clearly affects Ffz1p function, causing not only reduced growth of the respective S. cerevisiae transformant (TFG1) on fructose, but also affecting the specificity of the transporter, since growth of TFG1 on glucose could be detected (Fig. 1). This suggests that the C-terminus has a prominent structural/functional role.
Although the fructophilic behaviour of Z. bailii is not yet fully elucidated, a first step has been accomplished: since Ffz1p is specific for fructose, when this sugar is present in high concentrations in the environment it crosses the plasma membrane at a higher rate than glucose and, consequently, can be metabolized faster.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 10 December 2003;
revised 24 March 2004;
accepted 7 April 2004.
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