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The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H(+)-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased [(3)H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production.

The biosynthesis of the exopolysaccharide (EPS) cepacian by Burkholderia cepacia complex strains requires the 16.2-kb bce cluster of genes. Two of the clustered genes, bceD and bceF, code for two proteins homologous to phosphotyrosine phosphatases and tyrosine kinases, respectively. We show experimental evidence indicating that BceF is phosphorylated on tyrosine and that the conserved lysine residue present at position 563 in the Walker A ATP-binding motif is required for this autophosphorylation. It was also proved that BceD is capable of dephosphorylating the phosphorylated BceF. Using the artificial substrate p-nitrophenyl phosphate (PNPP), BceD exhibited a V(max) of 8.8 mumol of PNPP min(-1) mg(-1) and a K(m) of 3.7 mM PNPP at 30 degrees C. The disruption of bceF resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain's EPS production yield was still registered for the bceD mutant. The exopolysaccharide produced by the bceD mutant led to less viscous solutions and exhibited the same degree of acetylation as the wild-type cepacian, suggesting a lower molecular mass for this mutant biopolymer. The size of the biofilm produced in vitro by bceD and bceF mutant strains is smaller than the size of the biofilm formed by the parental strain, and this phenotype was confirmed by complementation assays, indicating that BceD and BceF play a role in the establishment of biofilms of maximal size.

The Saccharomyces cerevisiae SPI1 gene encodes a member of the glycosylphosphatidylinositol-anchored cell wall protein family. In this work we show results indicating that SPI1 expression protects the yeast cell from damage caused by weak acids used as food preservatives. This is documented by a less extended period of adaptation to growth in their presence and by a less inhibited specific growth rate for a parental strain compared with a mutant with SPI1 deleted. Maximal protection exerted by Spi1p against equivalent concentrations of the various weak acids tested was registered for the more lipophilic acids (octanoic acid, followed by benzoic acid) and was minimal for acetic acid. Weak-acid adaptation was found to involve the rapid activation of SPI1 transcription, which is dependent on the presence of the Msn2p transcription factor. Activation of SPI1 transcription upon acetic acid stress also requires Haa1p, whereas this recently described transcription factor has a negligible role in the adaptive response to benzoic acid. The expression of SPI1 was found to play a prominent role in the development of yeast resistance to 1,3-beta-glucanase in benzoic acid-stressed cells, while its involvement in acetic acid-induced resistance to the cell wall-lytic enzyme is slighter. The results are consistent with the notion that Spi1p expression upon weak-acid stress leads to cell wall remodeling, especially for the more lipophilic acids, decreasing cell wall porosity. Decreased cell wall porosity, in turn, reduces access to the plasma membrane, reducing membrane damage, intracellular acidification, and viability loss.

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 microM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 microM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).

The possible roles of 13 Msn2p- and Msn4p-regulated genes in the adaptation of Saccharomyces cerevisiae to the herbicide 2,4-D-dichlorophenoxyacetic acid (2,4-D) were examined. Single deletion of genes involved in defense against oxidizing agents (CTT1, GRX1, and GRX2/TTR1) or encoding chaperones of the HSP70 family (SSA1, SSA4, and SSE2) showed a slight effect. A more significant role was observed for the heat shock genes HSP78, HSP26, HSP104, HSP12, and HSP42, most of which encode molecular chaperones. However, the SPI1 gene, encoding a member of the glycosylphosphatidylinositol-anchored cell wall protein family, emerged as the major determinant of 2,4-D resistance. SPI1 expression reduced the loss of viability of an unadapted yeast population suddenly exposed to the herbicide, allowing earlier growth resumption. Significantly, yeast adaptation to 2,4-D involves the rapid and transient Msn2p- and Msn4p-mediated activation (fivefold) of SPI1 transcription. SPI1 mRNA levels were reduced to values slightly above those in unstressed cells when the adapted population started duplication in the presence of 2,4-D. Since SPI1 deletion leads to the higher beta-1,3-glucanase sensitivity of 2,4-D-stressed cells, it was hypothesized that adaptation may involve an Spi1p-mediated increase in the diffusional restriction of the liposoluble acid form of the herbicide across the cell envelope. Such a cell response would avoid a futile cycle due to acid reentry into the cell counteracting the active export of the anionic form, presumably through an inducible plasma membrane transporter(s). Consistent with this concept, the concentration of (14)C-labeled 2,4-D in 2,4-D-energized adapted Deltaspi1 mutant cells and the consequent intracellular acidification are higher than in wild-type cells.

The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway.

The dairy industry produces large quantities of whey as a by-product of cheese production and is increasingly looking for new ways to utilize this waste product. Gellan gum is reliably produced by Sphingomonas paucimobilis in growth media containing lactose, a significant component of cheese whey, as a carbon source. We studied and compared polysaccharide biosynthesis by S. paucimobilis ATCC 31461 in media containing glucose, lactose (5 to 30 g/liter), and sweet cheese whey. We found that altering the growth medium can markedly affect the polysaccharide yield, acyl substitution level, polymer rheological properties, and susceptibility to degradation. Depression of gellan production from lactose compared with gellan production from glucose (approximately 30%) did not appear to occur at the level of synthesis of sugar nucleotides, which are the donors of monomers used for biosynthesis of the repetitive tetrasaccharide unit of gellan. The lactose-derived biopolymer had the highest total acyl content; the glucose- and whey-derived gellans had similar total acyl contents but differed markedly in their acetate and glycerate levels. Rheological studies revealed how the functionality of a gellan polysaccharide is affected by changes in the acyl substitution.

Saccharomyces cerevisiae plasma membrane H(+)-ATPase activity was stimulated during octanoic acid-induced latency, reaching maximal values at the early stages of exponential growth. The time-dependent pattern of ATPase activation correlated with the decrease of cytosolic pH (pHi). The cell population used as inoculum exhibited a significant heterogeneity of pHi, and the fall of pHi correlated with the loss of cell viability as determined by plate counts. When exponential growth started, only a fraction of the initial population was still viable, consistent with the role of the physiology and number of viable cells in the inoculum in the duration of latency under acid stress.

Ethanol stimulated the leakage of amino acids and 260-nm-light-absorbing compounds from cells of Saccharomyces cerevisiae. The efflux followed first-order kinetics over an initial period. In the presence of lethal concentrations of ethanol, the efflux rates at 30 and 36 degrees C were an exponential function of ethanol concentration: k(e) = k(e)e, where k(e) and k(e) are the efflux rate constants, respectively, in the presence of a concentration X of ethanol or the minimal concentration of ethanol, X(m), above which the equation was applicable, coincident with the minimal lethal concentration of ethanol. E is the enhancement constant. At 36 degrees C, as compared with the corresponding values at 30 degrees C, the efflux rates were higher and the minimal concentration of ethanol (X(m)) was lower. The exponential constants for the enhancement of the rate of leakage (E) had similar values at 30 or 36 degrees C and were of the same order of magnitude as the corresponding exponential constants for ethanol-induced death. Under isothermic conditions (30 degrees C) and up to 22% (vol/vol) ethanol, the resistance to ethanol-induced leakage of 260-nm-light-absorbing compounds was found to be closely related with the ethanol tolerance of three strains of yeasts, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Saccharomyces bayanus. The resistance to ethanol-induced leakage indicates the possible adoption of the present method for the rapid screening of ethanol-tolerant strains. The addition to a fermentation medium of the intracellular material obtained by ethanol permeabilization of yeast cells led to improvements in alcohol fermentation by S. cerevisiae and S. bayanus. The action of the intracellular material, by improving yeast ethanol tolerance, and the advantages of partially recycling the fermented medium after distillation were discussed.

The addition of Ca (as CaCl(2)) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with a trace contaminating concentration of Ca (0.025 mM) led to the rapid production of higher concentrations of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation mu(xi) = mu(oi) [1 - (X/X(mi))], where mu(oi) and mu(xi) are, respectively, the specific growth (i = g) and fermentation (i = f) rates in the absence or presence of a concentration (X) of added ethanol, and X(mi) is the maximal concentration of ethanol which allows growth or fermentation. The toxic power is given by n(i). In Ca - supplemented medium (0.75 mM), n(g) = 0.42 for growth and n(f) = 0.43 for fermentation compared with 0.52 and 0.55, respectively, in unsupplemented medium; for both media, X(mg) = 10% (vol/vol) and X(mf) = 13% (vol/vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and incubated with ethanol in medium with an optimal concentration of Ca, maximal for cells grown and incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca on the acidification curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect on growth, fermentation, and viability.

The supplementation of a simple medium with soy flour led to an increase in the specific growth rate and viable cell concentration of Saccharomyces bayanus during fermentation. Increasing the amount of soy flour led to an increase in the maximum number of viable yeast cells and the percentage of glucose fermented. It was possible in 64 h to reach 12.8% (wt/vol) ethanol by adding 4% soy flour (wt/vol) to a simple medium with 300 g of glucose per liter. The aqueous extract from soy flour was nearly as effective as whole-soy flour, whereas the lipidic fraction had no positive effect.

The inhibition of growth by octanoic or decanoic acids, two subproducts of ethanolic fermentation, was evaluated in Saccharomyces cerevisiae and Kluyveromyces marxianus in association with ethanol, the main product of fermentation. In both strains, octanoic and decanoic acids, at concentrations up to 16 and 8 mg/liter, respectively, decreased the maximum specific growth rate and the biomass yield at 30 degrees C as an exponential function of the fatty acid concentration and increased the duration of growth latency. These toxic effects increased with a decrease in pH in the range of 5.4 to 3.0, indicating that the undissociated form is the toxic molecule. Decanoic acid was more toxic than octanoic acid. The concentrations of octanoic and decanoic acids were determined during the ethanolic fermentation (30 degrees C) of two laboratory media (mineral and complex) by S. cerevisiae and of Jerusalem artichoke juice by K. marxianus. Based on the concentrations detected (0.7 to 23 mg/liter) and the kinetics of growth inhibition, the presence of octanoic and decanoic acids cannot be ignored in the evaluation of the overall inhibition of ethanolic fermentation.