Protein abundance changes of Zygosaccharomyces rouxii in different sugar concentrations.

Zygosaccharomyces rouxii is a yeast which can cause spoilage in the concentrated juice industries. It exhibits resistance to high sugar concentrations but genome- and proteome-wide studies on Z. rouxii in response to high sugar concentrations have been poorly investigated. Herein, by using a 2-D electrophoresis based workflow, the proteome of a wild strain of Z. rouxii under different sugar concentrations has been analyzed. Proteins were extracted, quantified, and subjected to 2-DE analysis in the pH range 4-7. Differences in growth (lag phase), protein content (13.97-19.23mg/g cell dry weight) and number of resolved spots (196-296) were found between sugar concentrations. ANOVA test showed that 168 spots were different, and 47 spots, corresponding to 40 unique gene products have been identified. These protein species are involved in carbohydrate and energy metabolism, amino acid metabolism, response to stimulus, protein transport and vesicle organization, cell morphogenesis regulation, transcription and translation, nucleotide metabolism, amino-sugar nucleotide-sugar pathways, oxidoreductases balancing, and ribosome biogenesis. The present study provides important information about how Z. rouxii acts to cope with high sugar concentration at molecular levels, which might enhance our global understanding of Z. rouxii's high sugar-tolerance trait.

Early detection of Zygosaccharomyces rouxii--spawned spoilage in apple juice by electronic nose combined with chemometrics.

Spoilage spawned by Zygosaccharomyces rouxii can cause sensory defect in apple juice, which could hardly be perceived in the early stage and therefore would lead to the serious economic loss. Thus, it is essential to detect the contamination in early stage to avoid costly waste of products or recalls. In this work the performance of an electronic nose (e-nose) coupled with chemometric analysis was evaluated for diagnosis of the contamination in apple juice, using test panel evaluation as reference. The feasibility of using e-nose responses to predict the spoilage level of apple juice was also evaluated. Coupled with linear discriminant analysis (LDA), detection of the contamination was achieved after 12h, corresponding to the cell concentration of less than 2.0 log 10 CFU/mL, the level at which the test panelists could not yet identify the contamination, indicating that the signals of e-nose could be utilized as early indicators for the onset of contamination. Loading analysis indicated that sensors 2, 6, 7 and 8 were the most important in the detection of Z. rouxii-contaminated apple juice. Moreover, Z. rouxii counts in unknown samples could be well predicted by the established models using partial least squares (PLS) algorithm with high correlation coefficient (R) of 0.98 (Z. rouxii strain ATCC 2623 and ATCC 8383) and 0.97 (Z. rouxii strain B-WHX-12-53). Based on these results, e-nose appears to be promising for rapid analysis of the odor in apple juice during processing or on the shelf to realize the early detection of potential contamination caused by Z. rouxii strains.

The quaternary structure of the eukaryotic DNA replication proteins Sld7 and Sld3.

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomes. Yeast Sld3 and its metazoan counterpart treslin are the hub proteins mediating protein associations critical for formation of the helicase. The Sld7 protein interacts with Sld3, and the complex formed is thought to regulate the function of Sld3. Although Sld7 is a non-essential DNA replication protein that is found in only a limited range of yeasts, its depletion slowed the growth of cells and caused a delay in the S phase. Recently, the Mdm2-binding protein was found to bind to treslin in humans, and its depletion causes defects in cells similar to the depletion of Sld7 in yeast, suggesting their functional relatedness and importance during the initiation step of DNA replication. Here, the crystal structure of Sld7 in complex with Sld3 is presented. Sld7 comprises two structural domains. The N-terminal domain of Sld7 binds to Sld3, and the C-terminal domains connect two Sld7 molecules in an antiparallel manner. The quaternary structure of the Sld3-Sld7 complex shown from the crystal structures appears to be suitable to activate two helicase molecules loaded onto replication origins in a head-to-head manner.

Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice.

Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR.

A hydrophobic filter confers the cation selectivity of Zygosaccharomyces rouxii plasma-membrane Na+/H+ antiporter.

Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group.

Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain.

Methylation of the N6 position of selected internal adenines (m(6)A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m(6)A residue in the center. The m(6)A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m(6)A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines.

Enhancement of dibenzothiophene desulfurization by Gordonia alkanivorans strain 1B using sugar beet molasses as alternative carbon source.

There are several problems limiting an industrial application of fossil fuel biodesulfurization, and one of them is the cost of culture media used to grow the microorganisms involved in the process. In this context, the utilization of alternative carbon sources resulting from agro-industrial by-products could be a strategy to reduce the investment in the operating expenses of a future industrial application. Recently, Gordonia alkanivorans 1B was described as a fructophilic desulfurizing bacterium, and this characteristic opens a new interest in alternative carbon sources rich in fructose. Thus, the goal of this study was to evaluate the utilization of sugar beet molasses (SBM) in the dibenzothiophene (DBT) desulfurization process using strain 1B. SBM firstly treated with 0.25% BaCl2 (w/v) was used after sucrose acidic hydrolysis or in a simultaneous saccharification and fermentation process with a Zygosaccharomyces bailii Talf1 invertase (1%), showing promising results. In optimal conditions, strain 1B presented a µ max of 0.0795 h(-1), and all DBT was converted to 2-hydroxybiphenyl (250 µM) within 48 h with a maximum production rate of 7.78 µM h(-1). Our results showed the high potential of SBM to be used in a future industrial fossil fuel biodesulfurization process using strain 1B.

Effects of pH and sugar concentration in Zygosaccharomyces rouxii growth and time for spoilage in concentrated grape juice at isothermal and non-isothermal conditions.

The effect of pH (1.7-3.2) and sugar concentration (64-68 °Brix) on the growth of Zygosaccharomyces rouxii MC9 using response surface methodology was studied. Experiments were carried out in concentrated grape juice inoculated with Z. rouxii at isothermal conditions (23 °C) for 60 days. pH was the variable with the highest effect on growth parameters (potential maximum growth rate and lag phase duration), although the effect of sugar concentration were also significant. In a second experiment, the time for spoilage by this microorganism in concentrated grape juice was evaluated at isothermal (23 °C) and non-isothermal conditions, in an effort to reproduce standard storage and overseas shipping temperature conditions, respectively. Results show that pH was again the environmental factor with the highest impact on delaying the spoilage of the product. Thereby, a pH value below 2.0 was enough to increase the shelf life of the product for more than 60 days in both isothermal and non-isothermal conditions. The information obtained in the present work could be used by producers and buyers to predict the growth and time for spoilage of Z. rouxii in concentrated grape juice.

Raman spectroscopy and chemometrics for identification and strain discrimination of the wine spoilage yeasts Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Brettanomyces bruxellensis.

The yeasts Zygosaccharomyces bailii, Dekkera bruxellensis (anamorph, Brettanomyces bruxellensis), and Saccharomyces cerevisiae are the major spoilage agents of finished wine. A novel method using Raman spectroscopy in combination with a chemometric classification tool has been developed for the identification of these yeast species and for strain discrimination of these yeasts. Raman spectra were collected for six strains of each of the yeasts Z. bailii, B. bruxellensis, and S. cerevisiae. The yeasts were classified with high sensitivity at the species level: 93.8% for Z. bailii, 92.3% for B. bruxellensis, and 98.6% for S. cerevisiae. Furthermore, we have demonstrated that it is possible to discriminate between strains of these species. These yeasts were classified at the strain level with an overall accuracy of 81.8%.

Screening of different stress factors and development of growth/no growth models for Zygosaccharomyces rouxii in modified Sabouraud medium, mimicking intermediate moisture foods (IMF).

The microbial stability of intermediate moisture foods (IMF) is linked with the possible growth of osmophilic yeast and xerophilic moulds. As most of these products have a long shelf life the assessment of the microbial stability is often an important hurdle in product innovation. In this study a screening of several Zygosaccharomyces rouxii strains towards individual stress factors was performed and growth/no growth models were developed, incorporating a(w), pH, acetic acid and ethanol concentrations. These stress factors are important for sweet IMF such as chocolate fillings, ganache, marzipan, etc. A comparison was made between a logistic regression model with and without the incorporation of time as an explanatory variable. Next to the model development, a screening of the effect of chemical preservatives (sorbate and benzoate) was performed, in combination with relevant stress factors within the experimental design of the model. The results of the study showed that the influence of the investigated environmental stress factors on the growth/no growth boundary of Z. rouxii is the most significant in the first 30-40 days of incubation. Incorporating time as an explanatory variable in the model had the advantage that the growth/no growth boundary could be predicted at each time between 0 and 60 days of incubation at 22 °C. However, the growth/no growth boundary enlarged significantly leading to a less accurate prediction on the growth probability of Z. rouxii. The developed models can be a useful tool for product developers of sweet IMF. Screening with chemical preservatives revealed that benzoic acid was much less active towards Z. rouxii than sorbic acid or a mixture of both acids.

Cloning of the Zygosaccharomyces bailii GAS1 homologue and effect of cell wall engineering on protein secretory phenotype.

BACKGROUND: Zygosaccharomyces bailii is a diploid budding yeast still poorly characterized, but widely recognised as tolerant to several stresses, most of which related to industrial processes of production. Because of that, it would be very interesting to develop its ability as a cell factory. Gas1p is a beta-1,3-glucanosyltransglycosylase which plays an important role in cell wall construction and in determining its permeability. Cell wall defective mutants of Saccharomyces cerevisiae and Pichia pastoris, deleted in the GAS1 gene, were reported as super-secretive. The aim of this study was the cloning and deletion of the GAS1 homologue of Z. bailii and the evaluation of its deletion on recombinant protein secretion. RESULTS: The GAS1 homologue of Z. bailii was cloned by PCR, and when expressed in a S. cerevisiae GAS1 null mutant was able to restore the parental phenotype. The respective Z. bailii Deltagas1 deleted strain was obtained by targeted deletion of both alleles of the ZbGAS1 gene with deletion cassettes having flanking regions of approximately 400 bp. The morphological and physiological characterization of the Z. bailii null mutant resulted very similar to that of the corresponding S. cerevisiae mutant. As for S. cerevisiae, in the Z. bailii Deltagas1 the total amount of protein released in the medium was significantly higher. Moreover, three different heterologous proteins were expressed and secreted in said mutant. The amount of enzymatic activity found in the medium was almost doubled in the case of the Candida rugosa lipase CRL1 and of the Yarrowia lipolytica protease XPR2, while for human IL-1beta secretion disruption had no relevant effect. CONCLUSIONS: The data presented confirm that the engineering of the cell wall is an effective way to improve protein secretion in yeast. They also confirmed that Z. bailii is an interesting candidate, despite the knowledge of its genome and the tools for its manipulation still need to be improved. However, as already widely reported in literature, our data confirmed that an "always working" solution to the problems related to recombinant protein production can be hardly, if never, found; instead, manipulations have to be finely tuned for each specific product and/or combination of host cell and product.

Arsenic removal from groundwater by pretreated waste tea fungal biomass.

Arsenic contamination in ground water poses a serious threat on human health. The tea fungus, a waste produced during black tea fermentation has been examined for its capacity to sequester the metal ions from ground water samples. Autoclaved tea fungal mat and autoclaving followed by FeCl3 pretreated tea fungal mat were exploited for removal of As(III), As(V) and Fe(II) from ground water sample collected from Kolkata, West Bengal, India. The biosorption rate tends to increase with the increase in contact time and adsorbent dosage. FeCl3 pretreated and autoclaved fungal mats removed 100% of As(III) and Fe(II) after 30 min contact time and 77% of As(V) after 90 min contact time. The optimum adsorbent dosage was 1.0 g/50 mL of water sample. The results revealed that the FeCl3 pretreated fungal mat could be used as an effective biosorbent for As(III) and As(V); autoclaved fungal mat for Fe(II) removal from ground water sample.

Zygocin, a secreted antifungal toxin of the yeast Zygosaccharomyces bailii, and its effect on sensitive fungal cells.

Zygocin, a protein toxin produced and secreted by a killer virus-infected strain of the osmotolerant yeast Zygosaccharomyces bailii, kills a great variety of human and phytopathogenic yeasts and filamentous fungi. Toxicity of the viral toxin is envisaged in a two-step receptor-mediated process in which the toxin interacts with cell surface receptors at the level of the cell wall and the plasma membrane. Zygocin receptors were isolated and partially purified from the yeast cell wall mannoprotein fraction and could be successfully used as biospecific ligand for efficient one-step purification of the 10-kDa protein toxin by receptor-mediated affinity chromatography. Evidence is presented that zygocin-treated yeast cells are rapidly killed by the toxin, and intensive propidium iodide staining of zygocin-treated cells indicated that the toxin is affecting cytoplasmic membrane function, most probably by lethal ion channel formation. The presented findings suggest that zygocin has potential as a novel antimycotic in combating fungal infections.

Cloning, sequencing and characterization of a gene encoding dihydroxyacetone kinase from Zygosaccharomyces rouxii NRRL2547.

The dihydroxyacetone pathway, an alternative pathway for the dissimilation of glycerol via reduction by glycerol dehydrogenase and subsequent phosphorylation by dihydroxyacetone (DHA) kinase, is activated in the yeasts Saccharomyces cerevisiae and Zygosaccharomyces rouxii during osmotic stress. In experiments aimed at investigating the physiological function of the DHA pathway in Z. rouxii, a typical osmotolerant yeast, we cloned and characterized a DAK gene encoding dihydroxyacetone kinase from Z. rouxii NRRL 2547. Sequence analysis revealed a 1761 bp open reading frame, encoding a peptide composed of 587 deduced amino acids with the predicted molecular weight of 61 664 Da. As the amino acid sequence was most closely homologous (68% identity) to the S. cerevisiae Dak1p, we named the gene and protein ZrDAK1 and ZrDak1p, respectively. A putative ATP binding site was also found but no consensus element associated with osmoregulation was found in the upstream region of the ZrDAK1 gene. The ZrDAK1 gene complemented a S. cerevisiae W303-1A dak1delta dak2 delta strain by improving the growth of the mutant on 50 mmol/l dihydroxyacetone and by increasing the tolerance to dihydroxyacetone in a medium containing 5% sodium chloride, suggesting that it is a functional homologue of the S. cerevisiae DAK1. However, expression of the ZrDAK1 gene in the S. cerevisiae dak1delta dak2 delta strain had no significant effect on glycerol levels during osmotic stress. The ZrDAK1 sequence has been deposited in the public data bases under Accession No. AJ294719; regions upstream and downstream of ZrDAK1are deposited as Accession Nos AJ294739 and AJ294720, respectively.

Organization of specific genomic regions of Zygosaccharomyces rouxii and Pichia sorbitophila: comparison with Saccharomyces cerevisiae.

The genomes of Zygosaccharomyces rouxii and Pichia sorbitophila were partially explored. The genome of Z. rouxii CBS 732 consists of seven chromosomes with an approximate size of 1.0-2.75 Mb, 12.8 Mb in total. Five of the chromosomes were labelled with specific probes. Three Z. rouxii genomic DNA fragments were sequenced; all 10 ORFs found were without introns and they have homologues in S. cerevisiae. Gene order comparison revealed that the organization is partially conserved in both species. The genome of P. sorbitophila CBS 7064 consists of seven chromosomes with an approximate size of 1.0-2.9 Mb, 13.9 Mb in total. Three of the chromosomes were labelled with specific probes. The sequencing of a 5.2 kb genomic DNA fragment revealed three ORFs, but no conservation of their organization was found, although all of them have their respective homologues in S. cerevisiae. According to our results, the presence of two overlapping ORFs in S. cerevisiae (YJL107c-YJL108c) could be interpreted as the result of a frameshift mutation.