Isabel Spencer-Martins

In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucleotide Probes

ABSTRACT Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3′ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.

Two glucose/xylose transporter genes from the yeast Candida intermedia: first molecular characterization of a yeast xylose–H+ symporter

Candida intermedia PYCC 4715 was previously shown to grow well on xylose and to transport this sugar by two different transport systems: high-capacity and low-affinity facilitated diffusion and a high-affinity xylose-proton symporter, both of which accept glucose as a substrate. Here we report the isolation of genes encoding both transporters, designated GXF1 (glucose/xylose facilitator 1) and GXS1 (glucose/xylose symporter 1) respectively. Although GXF1 was isolated by functional complementation of an HXT-null (where Hxt refers to hexose transporters) Saccharomyces cerevisiae strain, isolation of the GXS1 cDNA required partial purification and micro-sequencing of the transporter, identified by its relative abundance in cells grown on low xylose concentrations. Both genes were expressed in S. cerevisiae and the kinetic parameters of glucose and xylose transport were determined. Gxs1 is the first yeast xylose/glucose-H+ symporter to be characterized at the molecular level. Comparison of its amino acid sequence with available sequence data revealed the existence of a family of putative monosaccharide-H+ symporters encompassing proteins from several yeasts and filamentous fungi.

Estimation and Diversity of Phylloplane Mycobiota on Selected Plants in a Mediterranean–Type Ecosystem in Portugal

Mediterranean ecosystems have not been consistently investigated as natural habitats for microbes in general, and fungi in particular. Here we present the results of a survey of epiphytic mycobiota (filamentous fungi and yeasts) on the phylloplane of selected plants in the Arrábida Natural Park, an ecosystem of Mediterranean characteristics in Portugal, using conventional culture-dependent isolation methods. Leaves from the species Acer monspessulanum and Quercus faginea (deciduous trees) and Cistus albidus, Pistacia lentiscus, and Osyris quadripartita (evergreen shrubs) were collected twice a year for two consecutive years, at two distinct locations of Serra da Arrábida: the more humid northern slope and the drier southern slope. A total of 1029 strains of filamentous fungi and 540 strains of yeasts were isolated, which represented at least 36 and 46 distinct species, respectively. Total counts were higher on the plants from the northern slope and there was a general increase from spring to autumn, notably on the deciduous trees for the yeasts. Plant species that had higher numbers of leaf colonists (A. monspessulanum, C. albidus, and Q. faginea) also yielded a wider range of species. Among the filamentous fungi there was a predominance of species of ascomycetous affinity, whereas basidiomycetous species dominated among yeast isolates. Some of the taxa recovered were common to other phylloplane studies (e.g., ubiquitous molds and yeasts such as Cladosporium spp. and Cryptococcus spp., respectively), but less common species were also found, some of which appeared to represent undescribed taxa. Interestingly, a few species seemed to be associated with a particular plant, notably in the case of the evergreen shrub C. albidus. However, for a considerable number of fungi and yeasts the same taxon was recovered throughout the year from more than one plant and at both sites, suggesting that such species might be genuine phylloplane inhabitants (or at least of aerial plant surfaces) even though they appeared not to display host specificity.

FSY1, a Novel Gene Encoding a Specific Fructose/H+ Symporter in the Type Strain of Saccharomyces carlsbergensis

A novel gene, FSY1, encoding a permease involved in active fructose uptake by a proton symport mechanism in the type strain of Saccharomyces carlsbergensis has been isolated. Fsy1p is only distantly related to the Hxt proteins that mediate facilitated diffusion of glucose and fructose in Saccharomyces cerevisiae and related species.

l‐Arabinose transport and catabolism in yeast

Two yeasts, Candida arabinofermentans PYCC 5603T and Pichia guilliermondii PYCC 3012, which show rapid growth on l‐arabinose and very high rates of l‐arabinose uptake on screening, were selected for characterization of l‐arabinose transport and the first steps of intracellular l‐arabinose metabolism. The kinetics of l‐arabinose uptake revealed at least two transport systems with distinct substrate affinities, specificities, functional mechanisms and regulatory properties. The l‐arabinose catabolic pathway proposed for filamentous fungi also seems to operate in the yeasts studied. The kinetic parameters of the initial l‐arabinose‐metabolizing enzymes were determined. Reductases were found to be mostly NADPH‐dependent, whereas NAD was the preferred cofactor of dehydrogenases. The differences found between the two yeasts agree with the higher efficiency of l‐arabinose metabolism in C. arabinofermentans. This is the first full account of the initial steps of l‐arabinose catabolism in yeast including the biochemical characterization of a specific l‐arabinose transporter.

Maltotriose Utilization by Industrial Saccharomyces Strains: Characterization of a New Member of the α-Glucoside Transporter Family

ABSTRACT Maltotriose utilization by Saccharomyces cerevisiae and closely related yeasts is important to industrial processes based on starch hydrolysates, where the trisaccharide is present in significant concentrations and often is not completely consumed. We undertook an integrated study to better understand maltotriose metabolism in a mixture with glucose and maltose. Physiological data obtained for a particularly fast-growing distillers strain (PYCC 5297) showed that, in contrast to what has been previously reported for other strains, maltotriose is essentially fermented. The respiratory quotient was, however, considerably higher for maltotriose (0.36) than for maltose (0.16) or glucose (0.11). To assess the role of transport in the sequential utilization of maltose and maltotriose, we investigated the presence of genes involved in maltotriose uptake in the type strain of Saccharomyces carlsbergensis (PYCC 4457). To this end, a previously constructed genomic library was used to identify maltotriose transporter genes by functional complementation of a strain devoid of known maltose transporters. One gene, clearly belonging to the MAL transporter family, was repeatedly isolated from the library. Sequence comparison showed that the novel gene (designated MTY1) shares 90% and 54% identity with MAL31 and AGT1, respectively. However, expression of Mty1p restores growth of the S. cerevisiae receptor strain on both maltose and maltotriose, whereas the closely related Mal31p supports growth on maltose only and Agt1p supports growth on a wider range of substrates, including maltose and maltotriose. Interestingly, Mty1p displays higher affinity for maltotriose than for maltose, a new feature among all the α-glucoside transporters described so far.

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae.

Abstract Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)−1] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)−1]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)−1. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)−1 (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)−1. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol.

Efficient Identification of Clinically Relevant Candida Yeast Species by Use of an Assay Combining Panfungal Loop-Mediated Isothermal DNA Amplification with Hybridization to Species-Specific Oligonucleotide Probes

ABSTRACT The occurrence of invasive mycoses has progressively increased in recent years. Yeasts of the genus Candida remain the leading etiologic agents of those infections. Early identification of opportunistic yeasts may contribute significantly to improved disease management and the selection of appropriate antifungal therapy. We developed a rapid and reliable molecular identification system for clinically relevant yeasts that makes use of nonspecific primers to amplify a region of the 26S rRNA gene, followed by reverse hybridization of the digoxigenin-labeled products to a panel of species-specific oligonucleotide probes arranged on a nylon membrane macroarray format. DNA amplification was achieved by the recently developed loop-mediated isothermal DNA amplification technology, a promising option for the development of improved laboratory diagnostic kits. The newly developed method was successful in distinguishing among the major clinically relevant yeasts associated with bloodstream infections by using simple, rapid, and cost-effective procedures and equipment.

Ethanol tolerance of sugar transport, and the rectification of stuck wine fermentations

The incomplete consumption of sugar resulting from stuck wine fermentation is associated with important economic losses. One of the solutions to this serious problem consists of reinoculating the brew with a yeast starter culture that is both alcohol tolerant and a vigorous fructose fermenter. The present work aimed to select yeast strains capable of restarting stuck wine fermentations, and identify key parameters that contribute to the efficiency of the strains. Commercial and non-commercial Saccharomyces wine strains were tested, as well as strains of the fermentative non-Saccharomyces species Zygosaccharomyces bailii and Torulaspora delbrueckii. Although the latter species were shown to be more resistant to a combination of ethanol- and acetic-acid-induced cell death, commercial Saccharomyces cerevisiae strains were the most efficient fructose consumers in medium simulating a stuck fermentation. Stationary-phase S. cerevisiae cells performed better than inocula prepared from exponentially growing cultures, which correlates with the higher resistance to ethanol of non-growing populations. Stationary-phase cells pre-adapted to ethanol did not improve fructose consumption rates; this was in contrast to exponential-phase cells that benefited from prior incubation in ethanol-containing medium. Notably, a correlation was observed between yeast fructose consumption capacity and glucose (or fructose) transport. Our results challenge the current belief that ethanol tolerance, expressed in terms of cell viability, is a reliable criterion for the selection of yeast strains to restart stuck fermentations. Instead, this capacity seems to be based on sugar transport and its resistance to ethanol. In an attempt to further improve cell viability in the presence of high ethanol concentrations, hybrid strains of T. delbrueckii and S. cerevisiae were produced, and they showed high potential as restarter strains. The present work opens perspectives for the application of innovative strategies in the wine-making industry.

In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

ABSTRACT Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.