Genotype-dependent sulphite tolerance of Australian Dekkera (Brettanomyces) bruxellensis wine isolates

Aims: The aim of this study was to determine sulphite tolerance for a large number of Dekkera bruxellensis isolates and evaluate the relationship between this phenotype and previously assigned genotype markers. Methods and Results: A published microplate‐based method for evaluation of yeast growth in the presence of sulphite was benchmarked against culturability following sulphite treatment, for the D. bruxellensis type strain (CBS 74) and a reference wine isolate (AWRI 1499). This method was used to estimate maximal sulphite tolerance for 41 D. bruxellensis isolates, which was found to vary over a fivefold range. Significant differences in sulphite tolerance were observed when isolates were grouped according to previously assigned genotypes and ribotypes. Conclusions: Variable sulphite tolerance for the wine spoilage yeast D. bruxellensis can be linked to genotype markers. Significance and Impact of the Study: Strategies to minimize risk of wine spoilage by D. bruxellensis must take into account at least a threefold range in effective sulphite concentration that is dependent upon the genotype group(s) present. The isolates characterized in this study will be a useful resource for establishing the mechanisms conferring sulphite tolerance for this industrially important yeast species.     

Isolation,Functional Characterization,and Expression Pattern of a Vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter Gene <Emphasis Type="Italic">TrNHX1</Emphasis> from <Emphasis Type="Italic">Trifolium repens</Emphasis> L.

Plant vacuolar Na⁺/H⁺ antiporter plays an important role in salt tolerance. A vacuolar Na⁺/H⁺ antiporter gene TrNHX1 was cloned from Trifolium repens L., a forage legume, by RT-PCR and RACE methods using degenerate oligonucleotide primers. The TrNHX1 sequence contains 2,394 nucleotides and an open-reading frame of 1,626 nucleotides that encodes a protein of 541 amino acids with a deduced molecular mass of 59.5 kDa. The deduced amino acid sequence of TrNHX1 is 78% identical to that of a vacuolar Na⁺/H⁺ antiporter of Arabidopsis thaliana, AtNHX1, and contains the consensus amiloride-binding domain. TrNHX1 could partially complement the NaCl-sensitive phenotypes of yeast mutants Δnhx1 and Δena1-4Δnhx1, and a similar complementation was also observed in the presence of LiCl and KCl. In addition, it was found that TrNHX1 suppressed the hygromycin-sensitive phenotype of yeast mutant Δena1-4Δnhx1. The expression of TrNHX1 in T. repens increased in the presence of 150 mM NaCl, and this result accords with that of Na⁺ contents determination under the same treatment. These results suggest that TrNHX1 functions as a vacuolar Na⁺/H⁺ antiporter and plays an important role in salt tolerance and ion homeostasis in T. repens.     

Intracellular transport of a heterologous membrane protein,the human transferrin receptor,in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae. The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the plasma membrane (PM). The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar compartment. Degradation is further dependent on favorable transport conditions to this compartment. The main bottleneck of transport seems to be the transition from the ER to the PM. The chaperone Cne1p, which is involved in quality control in the ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein. This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins. Electronic Publication     

Vacuolar morphology of Saccharomyces cerevisiae during the process of wine making and Japanese sake brewing

Although ethanol and osmotic stress affect the vacuolar morphology of Saccharomyces cerevisiae, little information is available about changes in vacuolar morphology during the processes of wine making and Japanese sake (rice wine) brewing. Here, we elucidated changes in the morphology of yeast vacuoles using Zrc1p-GFP, a vacuolar membrane protein, so as to better understand yeast physiology during the brewing process. Wine yeast cells (OC-2 and EC1118) contained highly fragmented vacuoles in the sake mash (moromi) as well as in the grape must. Although sake yeast cells (Kyokai no. 9 and no. 10) also contained highly fragmented vacuoles during the wine-making process, they showed quite a distinct vacuolar morphology during sake brewing. Since the environment surrounding sake yeast cells in the sake mash did not differ much from that surrounding wine yeast cells, the difference in vacuolar morphology during sake brewing between wine yeast and sake yeast was likely caused by innate characters.     

Genome-wide identification of genes involved in tolerance to various environmental stresses inSaccharomyces cerevisiae

During fermentation, yeast cells are exposed to a number of stresses — such as high alcohol concentration, high osmotic pressure, and temperature fluctuation — so some overlap of mechanisms involved in the response to these stresses has been suggested. To identify the genes required for tolerance to alcohol (ethanol, methanol, and 1-propanol), heat, osmotic stress, and oxidative stress, we performed genome-wide screening by using 4828 yeast deletion mutants. Our screens identified 95, 54, 125, 178, 42, and 30 deletion mutants sensitive to ethanol, methanol, 1-propanol, heat, NaCl, and H₂O₂, respectively. These deleted genes were then classified based on their cellular functions, and cross-sensitivities between stresses were determined. A large number of genes involved in vacuolar H⁺-ATPase (V-ATPase) function, cytoskeleton biogenesis, and cell wall integrity, were required for tolerance to alcohol, suggesting their protective role against alcohol stress. Our results revealed a partial overlap between genes required for alcohol tolerance and those required for thermotolerance. Genes involved in cell wall integrity and the actin cytoskeleton are required for both alcohol tolerance and thermotolerance, whereas the RNA polymerase II mediator complex seems to be specific to heat tolerance. However, no significant overlap of genes required for osmotic stress and oxidative stress with those required for other stresses was observed. Interestingly, although mitochondrial function is likely involved in tolerance to several stresses, it was found to be less important for thermotolerance. The genes identified in this study should be helpful for future research into the molecular mechanisms of stress response.     

Subunit composition,biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase

The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H⁺-ATPase) that closely resembles the vacuolar H⁺-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H⁺-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H⁺-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of thevma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

Over-expression of <Emphasis Type="Italic">pcvA</Emphasis> involved in vesicle–vacuolar fusion affects the conidiation and penicillin production in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Rab GTPase is required for vesicle–vacuolar fusion during the vacuolar biogenesis in fungi. Rab GTPase-encoding gene, pcvA, was cloned from Penicillium chrysogenum: it contained five introns and its predicted protein contained the conserved Rab GTPase domain involved in GTP-binding and hydrolysis. Over-expression of pcvA significantly stimulated the vesicle–vacuolar fusion but repressed the conidiation and decreased conidial tolerance against thermal stress. Penicillin production was decreased in the pcvA over-expressed strain suggesting that pcvA is involved in vesicle–vacuolar fusion participates in the penicillin biosynthesis in P. chrysogenum.     

An important step forward for the future development of an easy and fast procedure for identifying the most dangerous wine spoilage yeast,Dekkera bruxellensis,in wine environment

Dekkera bruxellensis is the main reason for spoilage in the wine industry. It renders the products unacceptable leading to large economic losses. Fluorescence In Situ Hybridization (FISH) technique has the potential for allowing its specific detection. Nevertheless, some experimental difficulties can be encountered when FISH technique is applied in the wine environment (e.g. matrix and cells’ autofluorescence, fluorophore inadequate selection and probes’ low specificity to the target organisms). An easy and fast in-suspension RNA-FISH procedure was applied for the first time for identifying D. bruxellensis in wine. A previously designed RNA-FISH probe to detect D. bruxellensis (26S D. brux.5.1) was used, and the matrix and cells’ fluorescence interferences, the influence of three fluorophores in FISH performance and the probe specificity were evaluated. The results revealed that to apply RNA-FISH technique in the wine environment, a red-emitting fluorophore should be used. Good probe performance and specificity were achieved with 25% of formamide. The resulting RNA-FISH protocol was applied in wine samples artificially inoculated with D. bruxellensis. This spoilage microorganism was detected in wine at cell densities lower than those associated with phenolic off-flavours. Thus, the RNA-FISH procedure described in this work represents an advancement to facilitate early detection of the most dangerous wine spoilage yeast and, consequently, to reduce the economic losses caused by this yeast to the wine industry.     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

Construction and evaluation of self‐cloning bottom‐fermenting yeast with high SSU1 expression

Aim: To construct a self‐cloning brewer’s yeast that can minimize the unfavourable flavours caused by oxidation and certain kinds of sulfur compounds. Methods and Results: DNA fragments of a high‐expression promoter from the TDH3 gene originating from Saccharomyces cerevisiae were integrated into the promoter regions of the S. cerevisiae‐type and Saccharomyces bayanus‐type SSU1 genes of bottom‐fermenting brewer’s yeast. PCR and sequencing confirmed the TDH3 promoter was correctly introduced into the SSU1 regions of the constructed yeasts, and no foreign DNA sequences were found. Using the constructed yeasts, the concentration of sulfite in fermenting wort was higher when compared with the parent strain. In addition, the concentrations of hydrogen sulfide, 3‐methyl‐2‐buten‐1‐thiol (MBT) and 2‐mercapto‐3‐methyl‐1‐butanol (2M3MB) were lower when compared with the parent strain. Conclusion: We successfully constructed a self‐cloning brewer’s yeast with high SSU1 expression that enhanced the sulfite‐excreting ability and diminished the production ability of hydrogen sulfide, MBT and 2M3MB. Significance and Impact of the Study: The self‐cloning brewer’s yeast with high SSU1 expression would contribute to the production of superior quality beer with a high concentration of sulfite and low concentrations of hydrogen sulfide, MBT and 2M3MB.     

Root bacterial endophytes confer drought resistance and enhance expression and activity of a vacuolar H+-pumping pyrophosphatase in pepper plants

It has been previously shown that the transgenic overexpression of the plant root vacuolar proton pumps H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase) confer tolerance to drought. Since plant-root endophytic bacteria can also promote drought tolerance, we hypothesize that such promotion can be associated to the enhancement of the host vacuolar proton pumps expression and activity. To test this hypothesis, we selected two endophytic bacteria endowed with an array of in vitro plant growth promoting traits. Their genome sequences confirmed the presence of traits previously shown to confer drought resistance to plants, such as the synthesis of nitric oxide and of organic volatile organic compounds. We used the two strains on pepper (Capsicuum annuum L.) because of its high sensitivity to drought. Under drought conditions, both strains stimulated a larger root system and enhanced the leaves' photosynthetic activity. By testing the expression and activity of the vacuolar proton pumps, H⁺-ATPase (V-ATPase) and H⁺-PPase (V-PPase), we found that bacterial colonization enhanced V-PPase only. We conclude that the enhanced expression and activity of V-PPase can be favoured by the colonization of drought-tolerance-inducing bacterial endophytes.     

Expression of tobacco cDNA encoding phytochelatin synthase promotes tolerance to and accumulation of Cd and As inSaccharomyces cerevisiae

The first tobacco cDNA encoding phytochelatin synthase (NtPCS1) has been cloned by complementing the YCF1 (vacuolar ABC type transporter)-depleting yeast mutant DTY167 with an expression library fromNicotiana tabacum. When NtPCSI was over-expressed in DTY165 (WT) and DTY167 (mutant), tolerance to and the accumulation of cadmium (Cd) were enhanced. Interestingly, its expression promoted these responses as well to arsenic (As), but only in DTY167. We conclude thatNtPCS1 plays a role in tolerance to and the accumulation of both toxic metals inSaccharomyces cerevisiae. These authors contributed equally to the work.     

Aspects of physiology of Histoplasma capsulatum (A review)

Yeast and mycelial forms of several strains of Histoplasma capsulatum have been analysed with respect to their ability to grow on a defined medium with or without the amino acid supplement. It appeared that whereas mycelial cells of all strains tested were prototrophic, the yeast cells of most strains stringently required L-cysteine for growth. This was due to the absence from these cells of an active form of an enzyme, sulfite reductase, normally needed for cysteine biosynthesis. We have found that the yeast cells of two strains (Downs and G 184 B) can grow without cysteine supplement if L-serine is added to the medium. These cells have an active sulfite reductase but the enzyme disappears when cysteine is added. Thus, the regulation of sulfite reductase is different in mycelium and yeast — the enzyme is constitutive or repressible, respectively.Examination of RNA synthetic components of H. capsulatum revealed that the major proportion of RNA polymerase of the yeast form is sensitive to inhibition by -amanitin. The sensitivity to the toxin disappears completely upon conversion to mycelial phase. The yeast cells possess an unusual enzyme capable of synthesizing oligonucleotides without the aid of a DNA template. The enzyme stimulates DNA synthesis in the reaction catalyzed by DNA polymerase from H. capsulatum or Escherichia coli. The above data are discussed in terms of regulatory mechanisms involved in the process of morphological conversion. It is proposed that efforts be directed toward the identification and isolation of specific gene products so that qualitative and quantitative analysis of the conversion could be carried out.presented, in part, at the 1st International Histoplasmosis Conference, held on April 10–12, 1978 in Atlanta, Georgia, U.S.A.     

Inactivation of wine spoilage yeasts Dekkera bruxellensis using low electric current treatment (LEC)

Aims: The objective of this study was to investigate the inactivation of a selected yeast Dekkera bruxellensis strain 4481 in red wine by application of low electric current treatment (LEC). Methods and Results: LEC (200 mA) was applied for 60 days to a red wine, Montepulciano d’Abruzzo, in an alternative strategy to the SO₂ addition during wine storage. The LEC effect on both cell activity and microflora viability was assessed. LEC decreased significantly the survival viable cells and increased the death rate of D. bruxellensis strain 4481 yeast. A final comparison was made of the main physico‐chemical parameters of the wine after the different treatments. The study suggests the importance of an appropriate LEC treatment which limits wine deterioration in terms of off‐flavours synthesis. Conclusions: The results demonstrate that the growth of undesirable Dekkera can be inhibited by low voltage treatment; LEC was shown to be useful to prevent wine spoilage and has the potential of being a concrete alternative method for controlling wine spoilage. Significance and Impact of the Study: Wine spoilage can be avoided by preventing the growth of undesirable Dekkera yeasts, through the effective use of LEC in the winemaking process.