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M Côrte-Real

Publications and source records attributed to M Côrte-Real.

16 recordsLinked to original sources

Isolation and sequence analysis of the gene encoding triose phosphate isomerase from Zygosaccharomyces bailii.

The ZbTPI1 gene encoding triose phosphate isomerase (TIM) was cloned from a Zygosaccharomyces bailii genomic library by complementation of the Saccharomyces cerevisiae tpi1 mutant strain. The nucleotide sequence of a 1.5 kb fragment showed an open reading frame (ORF) of 746 bp, encoding a protein of 248 amino acid residues. The deduced amino acid sequence shares a high degree of homology with TIMs from other yeast species, including some highly conserved regions. The analysis of the promoter sequence of the ZbTPI1 revealed the presence of putative motifs known to have regulatory functions in S. cerevisiae. The GenBank Accession No. of ZbTPI1 is AF325852.

Amino Acid Sequence↗

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry.

In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential (Delta Psi m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure Delta Psi m with only little dependence on cell 'volume' and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the Delta Psi m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of Delta Psi m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.

Flow Cytometry↗

Construction of a genomic library of the food spoilage yeast Zygosaccharomyces bailii and isolation of the beta-isopropylmalate dehydrogenase gene (ZbLEU2).

A genomic library of the yeast Zygosaccharomyces bailii ISA 1307 was constructed in pRS316, a shuttle vector for Saccharomyces cerevisiae and Escherichia coli. The library has an average insert size of 6 kb and covers the genome more than 20 times assuming a genome size similar to that of S. cerevisiae. This new tool has been successfully used, by us and others, to isolate Z. bailii genes. One example is the beta-isopropylmalate dehydrogenase gene (ZbLEU2) of Z. bailii, which was cloned by complementation of a leu2 mutation in S. cerevisiae. An open reading frame encoding a protein with a molecular mass of 38.7 kDa was found. The nucleotide sequence of ZbLEU2 and the deduced amino acid sequence showed a significant degree of identity to those of beta-isopropylmalate dehydrogenases from several other yeast species. The sequence of ZbLEU2 has been deposited in the EMBL data library under accession number AJ292544.

3-Isopropylmalate Dehydrogenase↗

Oxygen requirements of the food spoilage yeast Zygosaccharomyces bailii in synthetic and complex media.

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.

Aerobiosis↗

Red fluorescent protein (DsRed) as a reporter in Saccharomyces cerevisiae.

We describe the utilization of a red fluorescent protein (DsRed) as an in vivo marker for Saccharomyces cerevisiae. Clones expressing red and/or green fluorescent proteins with both cytoplasmic and nuclear localization were obtained. A series of vectors are now available which can be used to create amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) fusions with the DsRed protein.

Genes, Reporter↗

Rapid detection of efflux pumps and their relation with drug resistance in yeast cells.

BACKGROUND: Cell drug resistance can be due to the presence of active efflux pumps (AEP). Identification of yeast cells with a resistance phenotype is important either from a clinical, agricultural or biotechnological point of view. Rapid and reliable methods to detect AEP can be therefore very useful. METHODS: Some yeast cells change their staining by calcein-AM, BCECF-AM, rhodamine 123 and DiOC(5), when pretreated with verapamil, CCCP or ATP depletion, or when pretreated with specific antimicrobial agents. This fact may be interpreted as an indication of the presence/absence of AEP. Six yeast species were tested with a flow cytometric method (FCM) and an epifluorescence microscopic method (EFM), and ten other species were evaluated only by EFM. The minimum inhibitory concentration (MIC) of penconazol, benomyl and cycloheximide for Saccharomyces cerevisiae and Kluyveromyces marxianus, were determined by growth inhibition on solid medium and were compared to the staining changes detected by FCM. RESULTS: The FCM and the EFM allowed the detection of AEP in all the yeast species tested. High MIC values for a drug were related with the presence of at least one AEP indicated by the cytometric data. CONCLUSIONS: The FCM revealed to be a robust assay whereas the EFM can be used as a preliminary test. It is possible to identify resistance/sensitivity patterns in yeast cells through cytometric detection methods of different efflux pumping systems.

ATP-Binding Cassette Transporters↗

A differential medium for the enumeration of the spoilage yeast Zygosaccharomyces bailii in wine.

A collection of yeasts, isolated mostly from spoiled wines, was used in order to develop a differential medium for Zygosaccharomyces bailii. The 118 selected strains of 21 species differed in their origin and resistance to preservatives and belonged to the genera Pichia, Torulaspora, Dekkera, Debaryomyces, Saccharomycodes, Issatchenkia, Kluyveromyces, Kloeckera, Lodderomyces, Schizosaccharomyces, Rhodotorula, Saccharomyces, and Zygosaccharomyces. The design of the culture medium was based on the different ability of the various yeast species to grow in a mineral medium with glucose and formic acid (mixed-substrate medium) as the only carbon and energy sources and supplemented with an acid-base indicator. By manipulating the concentration of the acid and the sugar it was possible to select conditions where only Z. bailii strains gave rise to alkalinization, associated with a color change of the medium (positive response). The final composition of the mixed medium was adjusted as a compromise between the percentage of recovery and selectivity for Z. bailii. This was accomplished by the use of pure or mixed cultures of the yeast strains and applying the membrane filtration methodology. The microbiological analysis of two samples of contaminated Vinho Verde showed that the new medium can be considered as a differential medium to distinguish Z. bailii from other contaminating yeasts, having potential application in the microbiological control of wines and probably other beverages and foods.

Colony Count, Microbial↗

Flow cytometric assessment of cell structural and functional changes induced by acetic acid in the yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae.

Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.

Acetic Acid↗

Glucose respiration and fermentation in Zygosaccharomyces bailii and Saccharomyces cerevisiae express different sensitivity patterns to ethanol and acetic acid.

In the yeast Zygosaccharomyces bailii ISA 1307, respiration and fermentation of glucose were exponentially inhibited by ethanol, both processes displaying similar sensitivity to the alcohol. Moreover, the degree of inhibition on fermentation was of the same magnitude as that reported for Saccharomyces cerevisiae. Acetic acid also inhibited these two metabolic processes in Z. bailii, with the kinetics of inhibition again being exponential. However, inhibition of fermentation was much less pronounced than in S. cerevisiae. The values estimated with Z. bailii for the minimum inhibitory concentration of acetic acid ranged from 100 to 240 mmol 1(-1) total acetic acid compared with values of near zero reported for S. cerevisiae. The inhibitory effects of acetic acid on Z. bailii were not significantly potentiated by ethanol.

Acetic Acid↗

Biochemical characterization of a mutant of the yeast Pichia anomala derepressed for malic acid utilization in the presence of glucose.

The mutant IGC 40 x 1001 of the yeast Pichia anomala IGC 4380, which displays inverse diauxic growth in a medium with glucose and malic acid, was studied to elucidate the biochemical mechanisms underlying that behavior. Time course changes of enzyme activities during growth of the mutant in that mixture of substrates indicated that the gluconeogenic enzymes remained active during the first phase of diauxic growth, while glycolytic enzyme activities were significantly reduced. This reduction was essentially due to an alteration in the maximum velocity and not in substrate affinity. Malate, citrate, and adenosine triphosphate did not affect significantly the activities of the glucose phosphorylating enzymes in cell extracts of either the mutant or the wild strain. In P. anomala, unlike Saccharomyces cerevisiae, the fructose/glucose phosphorylating ratio was not associated with repression/derepression conditions.

Enzyme Repression↗

Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments.

Cells of Zygosaccharomyces bailii ISA 1307 grown in a medium with acetic acid, ethanol, or glycerol as the sole carbon and energy source transported acetic acid by a saturable transport system. This system accepted propionic and formic acids but not lactic, sorbic, and benzoic acids. When the carbon source was glucose or fructose, the cells displayed activity of a mediated transport system specific for acetic acid, apparently not being able to recognize other monocarboxylic acids. In both types of cells, ethanol inhibited the transport of labelled acetic acid. The inhibition was noncompetitive, and the dependence of the maximum transport rate on the ethanol concentration was found to be exponential. These results reinforced the belief that, under the referenced growth conditions, the acid entered the cells mainly through a transporter protein. The simple diffusion of the undissociated acid appeared to contribute, with a relatively low weight, to the overall acid uptake. It was concluded that in Z. bailii, ethanol plays a protective role against the possible negative effects of acetic acid by inhibiting its transport and accumulation. Thus, the intracellular concentration of the acid could be maintained at levels lower than those expected if the acid entered the cells only by simple diffusion.

Acetates↗

Quantitative analysis of proton movements associated with the uptake of weak carboxylic acids. The yeast Candida utilis as a model.

A quantitative analysis of the proton movements associated with the initial uptake rate of weak short-chain carboxylic acids was developed in order to estimate proton/carboxylate symports stoichiometries. The yeast Candida utilis was used as a biological model and the deduced equations were applied on the elucidation of the proton/carboxylate symports stoichiometries of lactate, succinate and citrate in a strain of that yeast species at different pH values. At pH 5.0, the proton/lactate and the proton/succinate symport stoichiometry was 1:1. In the cases of the proton/lactate and proton/citrate symports it appears that the stoichiometry ratio increased with increasing extracellular pH.

Biological Transport↗

Inverse Diauxy in the Yeast Hansenula anomala: Mutants Derepressed for Malic Acid Utilization in the Presence of Glucose.

Utilization of l-malic acid by yeast strain Hansenula anomala IGC 4380 is subject to glucose repression. Derepressed mutants were obtained with UV light by use of the nonmetabolizable glucose analog 2-deoxyglucose as a selective agent. Three mutant strains degraded l-malic acid in the presence of up to 30% (wt/vol) glucose and are of potential interest for the biological deacidification of grape must. The mutant strains, as compared with the parent strain, displayed inverse diauxy in glucose-malate medium, glucose being metabolized only after malate consumption had been completed.

Journal Article↗

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.

Biological Transport, Active↗