Draft genome sequence of Wickerhamomyces anomalus LBCM1105, isolated from cachaça fermentation.

Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

Monitoring yeast intracellular Ca2+ levels using an in vivo bioluminescence assay.

This protocol describes the use of the jellyfish Aequorea victoria aequorin protein to measure Ca(2+) levels in living yeast cells. All yeast strains to be analyzed must express the A. victoria apoprotein of the aequorin calcium biosensor, to be reconstituted into fully active aequorin by association with its cofactor, coelenterazine, which cannot be synthesized by yeast itself. The simplest way to achieve reconstitution is to transform yeast cells with a vector driving apoaequorin expression, and then supply commercially available coelenterazine cofactor in the medium. Coelenterazine is a hydrophobic molecule and is able to permeate yeast cells.

Total cellular Ca2+ measurements in yeast using flame photometry.

A photoelectric flame photometer is a device used in inorganic chemical analysis for determining the concentrations of certain metals in solution. It does this by measuring the intensity of the light emitted by the metal when the solution is sprayed under controlled conditions into a nonluminous flame. This protocol describes how to measure total cellular calcium (maximal emission at 622 nm, orange flame) in yeast using this technique.

Measurement of calcium uptake in yeast using 45Ca.

This protocol describes how to measure calcium uptake in yeast by (45)Ca radioactive isotope incorporation.

Glucose-induced calcium influx in budding yeast involves a novel calcium transport system and can activate calcineurin.

Glucose addition to glucose-starved Saccharomyces cerevisiae cells triggers a quick and transient influx of calcium from the extracellular environment. In yeast at least two different carrier systems were identified: a high affinity system, requiring Cch1 channel, and a low affinity system. Here we report that another calcium transport system exists in yeast, not yet identified, that can substitute the two known systems when they are inactivated. This system was called GIC (for Glucose Induced Calcium) system and it is a high affinity calcium transport system, magnesium-sensitive but nickel-resistant. Moreover, GIC transport is sensitive to gadolinium and nifedipine, but it is not sensitive to inhibition by verapamil, which conversely behaves as an agonist on glucose response. GIC transport is fully functional in conditions when calcineurin is active, a serine/threonine specificity phosphatase involved in the regulation of calcium homeostasis and in many other cellular phenomena such as tolerance to high concentrations of Na(+) and Li(+), response to pheromones and gene transcription regulation. Here it is reported for the first time that calcineurin can also be activated by nutrients: the activation of Crz1 transcription factor by calcineurin was observed in derepressed cells after addition of glucose in the presence of extracellular calcium.

Protective effect of ions against cell death induced by acid stress in Saccharomyces.

Saccharomyces boulardii is a probiotic used to prevent or treat antibiotic-induced gastrointestinal disorders and acute enteritis. For probiotics to be effective they must first be able to survive the harsh gastrointestinal environment. In this work, we show that S. boulardii displayed the greatest tolerance to simulated gastric environments compared with several Saccharomyces cerevisiae strains tested. Under these conditions, a pH 2.0 was the main factor responsible for decreased cell viability. Importantly, the addition of low concentrations of sodium chloride (NaCl) protected cells in acidic conditions more effectively than other salts. In the absence of S. boulardii mutants, the protective effects of Na(+) in yeast viability in acidic conditions was tested using S. cerevisiae Na(+)-ATPases (ena1-4), Na(+)/H(+) antiporter (nha1Delta) and Na(+)/H(+) antiporter prevacuolar (nhx1Delta) null mutants, respectively. Moreover, we provide evidence suggesting that this protection is determined by the plasma membrane potential, once altered by low pH and low NaCl concentrations. Additionally, the absence or low expression/activity of Ena proteins seems to be closely related to the basal membrane potential of the cells.

Carbonyl cyanide m-chlorophenylhydrazone induced calcium signaling and activation of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae.

The plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is an enzyme that plays a very important role in the yeast physiology. The addition of protonophores, such as 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), also triggers a clear in vivo activation of this enzyme. Here, we demonstrate that CCCP-induced activation of the plasma membrane H(+)-ATPase shares some similarities with the sugar-induced activation of the enzyme. Phospholipase C and protein kinase C activities are essential for this activation process while Gpa2p, a G protein involved in the glucose-induced activation of the ATPase, is not required. CCCP also induces a phospholipase C-dependent increase in intracellular calcium. Moreover, we show that the availability of extracellular calcium is required for CCCP stimulation of H(+)-ATPase, suggesting a possible connection between calcium signaling and activation of ATPase.

Calcium signaling and sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells.

In this work, we show that glucose-induced activation of plasma membrane H(+)-ATPase from Saccharomyces cerevisiae is strongly dependent on calcium metabolism and that the glucose sensor Snf3p works in a parallel way with the G protein Gpa2p in the control of the pathway. The role of Snf3p is played by the Snf3p C-terminal tail, since in a strain with the deletion of the SNF3 gene, but also expressing a chimera protein formed by Hxt1p (a glucose transporter) and the Snf3p C-terminal tail, a normal glucose-activation process can be observed. We present evidences indicating that Snf3p would be the sensor for the internal signal (phosphorylated sugars) of this pathway that would connect calcium signaling and activation of the plasma membrane ATPase. We also show that Snf3p could be involved in the control of Pmc1p activity that would regulate the calcium availability in the cytosol.

Deficiency of Pkc1 activity affects glycerol metabolism in Saccharomyces cerevisiae.

Protein kinase C is apparently involved in the control of many cellular systems: the cell wall integrity pathway, the synthesis of ribosomes, the appropriated reallocation of transcription factors under specific stress conditions and also the regulation of N-glycosylation activity. All these observations suggest the existence of additional targets not yet identified. In the context of the control of carbon metabolism, previous data had demonstrated that Pkc1p might play a central role in the control of cellular growth and metabolism in yeast. In particular, it has been suggested that it might be involved in the derepression of genes under glucose-repression by driving an appropriated subcellular localization of transcriptional factors, such as Mig1p. In this work, we show that a pkc1Delta mutant is unable to grow on glycerol because it cannot perform the derepression of the GUT1 gene that encodes glycerol kinase. Additionally, active transport is also partially affected. Using this phenotype, we were able to isolate a new pkc1Delta revertant. We also isolated two transformants identified as the nuclear exportin Msn5 and the histone deacetylase Hos2 extragenic suppressors of this mutation. Based on these results, we postulate that Pkc1p may be involved in the control of the cellular localization and/or regulation of the activity of nuclear proteins implicated in gene expression.

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii.

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.

Regulation of N-acetyl-beta-D-glucosaminidase produced by Trichoderma harzianum: evidence that cAMP controls its expression.

Trichoderma harzianum is a filamentous fungus reported to be a producer of extracellular N-acetyl-beta-D-glucosaminidase (NAGase) when grown in chitin-containing medium. An approximately 64-kDa protein with NAGase activity was purified by gel filtration and ion exchange chromatography. The involvement of cyclic AMP (cAMP) in the synthesis of NAGase from T. harzianum in chitin-containing medium was also investigated. Molecules that increase the intracellular levels of cAMP, including caffeine, aluminium tetrafluoride and dinitrophenol, were used. Western blot analysis showed that NAGase synthesis was repressed by increasing the levels of intracellular cAMP. Using specific nag primers in a reverse transcription-polymerase chain reaction-based approach, NAGase synthesis was shown to be regulated at the level of gene transcription.

Regulation of Sugar Transport Systems in Fusarium oxysporum var. lini.

Fusarium oxysporum var. lini (ATCC 10960) formed a facilitated diffusion system for glucose (K(s), about 10 mM) when grown under repressed conditions. Under conditions of derepression, the same system was present together with a high-affinity (K(s), about 40 muM) active system. The maximum velocity of the latter was about 5% of that of the facilitated diffusion system. The high-affinity system was under the control of glucose repression and glucose inactivation. When lactose was the only carbon source in the medium, a facilitated diffusion system for lactose was found (K(s), about 30 mM).