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We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.

Transport of sugars is a fundamental property of all eukaryotic cells. Of particular importance is the uptake of glucose, a preferred carbon and energy source. The rate of glucose utilization in yeast is often dictated by the activity and concentration of glucose transporters in the plasma membrane. Given the importance of transport as a site of control of glycolytic flux, the regulation of glucose transporters is necessarily complex. The molecular analysis of these transporters in Saccharomyces has revealed the existence of a multigene family of sugar carriers. Recent data have raised the question of the actual role of all of these proteins in sugar catabolism, as some appear to be lowly expressed, and point mutations of these genes may confer pleiotropic phenotypes, inconsistent with a simple role as catabolic transporters. The transporters themselves appear to be intimately involved in the process of sensing glucose, a model for which there is growing support.

Uptake of glucose, fructose, and the nonmetabolizable analog 6-deoxyglucose was measured in wild-type Saccharomyces cerevuiae and two mutant strains, one (hxkl hxk2) lacking both hexokinase A (P-I) and B (P-Il) but containing glucokinase (and hence able to grow on glucose but not fructose) and the other (hxkl hxk2 gik) also lacking glucokinase (and not able to grow on glucose either). Uptake of the nonmetabolized substances (i.e., 6-deoxyglucose in all three strains, fructose in the two mutants, and glucose in the triple mutant) reached a plateau at or below the external concentration. The kinetic characteristics of uptake were determined from 5-sec incubations by plotting velocity (V) vs. velocity/substrate concentration (V/S) curves. According to such plots, in the wild-type strain uptake had two components, "high affinity uptake" with Km values of ca. 1 mM for glucose and 6 mM for fructose and "low affinity uptake" with Km values of ca. 20 and 50 mM, respectively. The double kinase mutant showed both components for glucose but only the high Km component for fructose, while the triple kinase mutant showed only high Km uptake for both glucose and fructose. Genetic analysis showed that only in strains lacking both hexokinases (hxkl hxk2) was the low Km system for fructose absent. Low Km uptake was restored to the triple mutant by introduction of the cloned wild-type genes: HXK1 orHXK2, for fructose uptake, and HXKl, HXK2, or GLKl, for glucose uptake. A phosphoglucose isomerase mutant had both low and high Km uptake for glucose. These results indicate the presence of two types of uptake mechanism for glucose and fructose in yeast, the functioning of one of which, the low Km system, is influenced by the cognate kinases.Glucose transport in yeast is not well understood. Studies of specificity have implicated a constitutive membrane carrier for uptake of glucose, fructose, and mannose and their analogs (1-3). The internal concentration of glucose is low during its metabolism, and the problem, as posed by van Steveninck and Rothstein (4), has been whether the low concentration reflects metabolic phosphorylation following entry by facilitated diffusion or a more intimate connection between entry and metabolism-for example, a transient phosphorylation during entry, or even formation of the first metabolic intermediate, hexose-6-phosphate, occurring by an obligatory vectorial phosphorylation (as with the bacterial phosphotransferase system). Several experimental lines of evidence seem to implicate metabolism with transport. (i) Considerations of kinetics do not simply accord with a membrane carrier of fixed properties delivering hexose to the cytoplasm (5, 6). (ii) Metabolic inhibitors, such as iodoacetate, are known to affect apparent affinity for uptake (7) and, in derepressed cells, as measured by fermentation rate, affinity for glucose is decreased by oxygen (8). (iii) Experiments with 2-deoxyglucose have established that its internal free poolin whatever compartment-is preceded by 2-deoxyglucose 6-phosphat...

Wine production is both art and science, a blend of individual creativity and innovative technology. But wine production is also business, with economic factors driving manufacturing practices. To be successful in the modern marketplace, a winemaker must integrate the artistic and economic aspects of wine production, and possess a solid understanding of the intrinsic and extrinsic factors that underlie purchase motivation.