Zygosaccharomyces Species Research Articles

Molecular evidence for the existence of natural hybrids in the genus

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.

Molecular monitoring of spoilage yeasts during the production of candied fruit nougats to determine food contamination sources

In the present work, we have analysed the yeast microbiota present in a manufacturing plant of candied fruits and nougats. Four yeasts species ( Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Sporobolomyces roseus, and Debaryomyces hansenii) and a filamentous fungi ( Nectria mauriiticola) were identified according to restriction analysis of 5.8S-ITS rDNA. These identifications were subsequently confirmed by sequencing the D1/D2 domain of the 26S rRNA gene. Z. rouxii and Z. bailii were isolated at high frequency along the whole manufacturing process. Since food alteration by Z. bailii and Z. rouxii is the cause of important economic losses for the food industry, there is a need for differentiating yeasts at the strain level as an essential part of quality control programs in this industry. For this purpose, we have tested the performance of three molecular techniques (RFLP mtDNA, RAPD-PCR, and microsatellite with (GAC) 5 and (GTG) 5 primers) to differentiate strains belonging to these two Zygosaccharomyces species. Those techniques with the best discriminatory power were applied to differentiate Zygosaccharomyces species isolates. The results of this analysis indicate that one strain of Z. bailii and two strains of Z. rouxii were involved in the spoilage of candied fruits. Moreover, the Z. bailii strain was also present in the spoiled nougat, hence being responsible of this alteration.

Isoenzyme Patterns: A Valuable Molecular Tool for the Differentiation of Zygosaccharomyces Species and Detection of Misidentified Isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii.

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.

Zymological indicators: a new concept applied to the detection of potential spoilage yeast species associated with fruit pulps and concentrates

In a survey of the microbial quality of raw materials used in fruit juice processing, yeast counts in fruit concentrates and pulps were found to range from <1 to 2·9×103cfu g−1. Ascomycetous yeasts were represented by 76% of the isolates while 24% were basidiomycetes. The identification of strains isolated by the simplified identification system (SIM) revealed 19 yeast species representing 12 genera. The most frequently isolated yeasts belonged to the genera Saccharomyces, Pichia, Cryptococcus, Kluyveromyces andCandida .Fatty acid yeast composition allowed the separation of contaminating yeasts into one of three major groups. Group I included yeasts without linoleic (C 18:2) and linolenic (C 18:3) fatty acids such as Saccharomyces cerevisiae. Group II comprised yeasts without C 18:3 fatty acid like Zygosaccharomyces rouxii and Torulaspora delbrueckii, and group III included yeasts with C 18:2 and C 18:3 acids that belong, among others, to one of the following yeast genera:Pichia, Candida, Kluyveromyces or Cryptococcus.Species-specific PCR primers were used for the rapid detection and identification of the most dangerous species affecting fruit concentrate stability. The simplified protocol used consisted of PCR-amplification of conserved tracts in the ITS region of the rDNA unit, thus enabling the detection of potentially dangerous flora such as Zygosaccharomyces species and T. delbrueckii in contaminated fruit concentrates. Results from PCR-typing were in full agreement with the fatty acid compositions of these species.The grouping of contaminant yeasts into three main groups showed that fatty acid composition may be used to differentiate yeasts according to their technological significance. Yeasts isolated in this work as being most dangerous to product stability belong to either group II (Z. rouxii and T. delbrueckii) or group I (Saccharomyces spp.). Group III was comprised of several species regarded as indicators of deficiencies in ‘good manufacturing practices’. Thus, each of the groups delineated may be considered to be a zymological indicator of technological significance. The conjugation of fatty acid profiles with PCR-typing methods may be used as a rapid detection system for contaminant yeasts. The fatty acid profiles provide a preliminary identification of yeasts potentially dangerous to product stability present within 48 h of isolation. Whereas the PCR-typing method is mainly used to confirm isolate identity, when required, after the initial diagnosis has been performed, over a period of 4 h.

Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker.

Unusual growth characteristics of a spoilage yeast, originally isolated from spoiled whole-orange drink and previously identified as Zygosaccharomyces bailii, prompted careful re-examination of its taxonomic position. Small-subunit rRNA gene sequences were determined for this strain and for four other strains also originally described as Z. bailii but which, in contrast to other strains of this species, grew poorly or not at all under aerobic conditions with agitation, failed to grow in the presence of 1% acetic acid and failed to grow at 30 degrees C. Comparative sequence analysis revealed that these strains represented a phylogenetically distinct taxon closely related to, but distinct from, Z. bailii and Zygosaccharomyces bisporus. Furthermore, sequence analysis of the internal transcribed spacer (ITS) region showed that, while all five strains had identical ITS2 sequences, they could be subdivided into two groups based on ITS1 sequences. Despite such minor inter-strain sequence variation, these yeasts could readily be distinguished from all other currently described Zygosaccharomyces species by using ITS sequences. On the basis of the phylogenetic results presented, a new species comprising the five strains, Zygosaccharomyces lentus sp. nov., is described and supporting physiological data are discussed, including a demonstration that growth of this species is particularly sensitive to the presence of oxygen. The type strain of Z. lentus is NCYC D2627T.

Use of an rRNA internal transcribed spacer region to distinguish phylogenetically closely related species of the genera Zygosaccharomyces and Torulaspora.

Analyses of the sequences of the small-subunit (18S) rRNA gene and two internal transcribed spacers (ITSs), ITS1 and ITS2, revealed that members of the yeast genera Torulaspora and Zygosaccharomyces are phylogenetically intermixed. Despite some minor differences in 18S rRNA-, ITS1-, and ITS2-derived trees, in general the patterns of the relationships inferred from the three chronometers were in good agreement. The ITS sequences of Torulaspora and Zygosaccharomyces species exhibited far greater interspecies differences than the 18S rRNA sequences and were better than 18S rRNA sequences for measuring close genealogical relationships. Despite the existence of interstrain ITS sequence variation in some species, it is possible to identify conserved regions in both ITSs that are useful in species differentiation.

Differetiation of the spoilage yeast Zygosaccharomyces bailii from other Zygosaccharomyces species using 18S rDNA as target for a non-radioactive ligase detection reaction

A non-radioactive PCR coupled ligase detection reaction was developed to discriminate the food spoilage yeasts Zygosaccharomyces bailii and Z. bisporus from each other and from other members of the genus. A short region of the 18S rRNA gene was amplified from boiled cell lysates and polymerase chain reaction (PCR) products used as target in the template directed ligation of two adjacent oligonucleotides. Ligated products were captured using biotin-streptavidin chemistry and detected using digoxigenin immuno-chemiluminescence. The ligase detection reaction was able to discriminate to the species level, targeting a single base deletion. The specificity of the reaction was assessed using seven species of the genus Zygosaccharomyces. Only strains of Z. bailii and Z. bisporus gave positive results with their respective primer sets. The lower detection limit of the strategy was 10pg (3 x 10(7) targets) of amplified product.

Characterization of MEL genes in the genus Zygosaccharomyces

We cloned and sequenced a Zygosaccharomyces cidri MEL gene with a view to investigating the structure and regulation of yeast MEL genes. The amino acid sequence deduced from the nucleotide sequence showed 78.6% and 78.2% similarity to Saccharomyces cerevisiae and Saccharomyces pastorianus alpha-galactosidases, respectively. The expression of the MEL gene in several Zygosaccharomyces strains was induced by galactose. An electrophoretic karyotype of several Zygosaccharomyces species was obtained using contour-clamped electric field gel electrophoresis. The minimum number of chromosomes was five for Z. cidri, six for Z. fermentati, three for Z. florentinus, and four for Z. microellipsoides. The sizes of the chromosomes were generally larger than those of S. cerevisiae, the smallest containing approximately 0.4 megabase. The MEL gene was located, using the Z. cidri MEL gene as a probe, on the largest chromosome of the Z. cidri strains. In addition, a smaller chromosome (600 kb) in Z. cidri strain CBS4575 showed hybridization to the homologous MEL probe. This chromosome was absent in Z. cidri strain CBS5666. The probe hybridized to the largest chromosome of Mel+ Z. fermentati strains but failed to hybridize to any chromosome of Mel+ Z. mrakii or Z. florentinus strains. These results suggest the existence of a polymorphic MEL gene family in the yeast Zygosaccharomyces.

Use of electrophoretic karyotyping and DNA-DNA hybridization in yeast identification

Contour-clamped homogenous electric field (CHEF) gel electrophoresis was used to separate intact, chromosome-size DNA of different species of Saccharomyces and Zygosaccharomyces. Strains of the same Saccharomyces species had similar electrophoretic karyotypes. However, reproducible differences between individual bands indicated that strain-specific chromosome length polymorphism (CLP) is widely spread in these organisms. Strains of Zygosaccharomyces spp. showed karyotype differences beyond chromosome length polymorphism. A new, DNA-DNA hybridization technique was developed to test conspecificity, using individual chromosomes as templates to prepare randomly primed, radioactive probes. Under our conditions of stringency, species and chromosome-specific hybridization reactions were achieved with these probes. Using isolated chromosomes of Sacch. cerevisiae, Sacch. bayanus, and Zygosacch. rouxii as templates for probe preparation, conspecificity of strains was established, and closely related yeast species were distinguished with high reproducibility. This method is an efficient tool for studying genetic diversity of a given yeast species. Also, it can assist in yeast species identification and taxonomy.

The phylogenetic relationships of the q6-equipped generatorulaspora lindner and zygosaccharomyces barker (saccharomycetaceae) based on the partial sequences of 18s and 26s ribosomal ribonucleic acids.

Eleven strains of Torulaspora and Zygosaccharomyces species were examined for the partial base sequences of 18S and 26S rRNAs. In the partial base sequences in positions 493-622 (130 bases) of 26S rRNA, there were 88-91% and 70-92% maximum homologies within the genera Torulaspora and Zygosaccharomyces, respectively. The maximum homologies were 70-90% between the two genera. The species of the two genera had 73-88% maximum homologies with Saccharomyces cerevisiae. In the partial base sequences in positions 1611-1835 (225 bases) of 26S rRNA, there were 3-0 and 16-1 base differences within the two genera, respectively. The base differences were 15-0 between the two genera. The two genera had 17-8 base differences with S. cerevisiae. In the partial base sequences in positions 1451-1618 (168 bases) of 18S rRNA, there were 0 and 3-0 base differences within the two genera, respectively. The base differences were 2-0 between the two genera. The two genera had 3-1 base differences with S. cerevisiae. Zygosaccharomyces cidri and Z. fermentati occupied a unique situation (base differences, 3-2). The data obtained were discussed with regard to the phylogenetic relationships and the taxonomic positions of Torulaspora, Zygosaccharomyces, and Saccharomyces.

DNA relatedness among species of the genus Zygosaccharomyces

The extent of nuclear DNA complementarity was determined for members of the genus Zygosaccharomyces. From these comparisons, nine species have been identified: Z. baillii, Z. bisporus, Z. cidri, Z. fermentati, Z. florentinus, Z. mellis, Z. microellipsoides, Z. mrakii and Z. rouxii. Candida mogii, the proposed anamorph of Z. rouxii, showed low relatedness to all nine species. The recently described Saccharomyces astigiensis and S. albasitensis were conspecific with Z. fermentati, and S. placentae showed high relatedness with Z. rouxii. Growth tests were defined that allow recognition of all Zygosaccharomyces species.

THE TYPES OF OSMOPHILIC YEASTS FOUND IN NORMAL HONEY AND THEIR RELATION TO FERMENTATION

A study of the predominant sugar-tolerant yeasts infecting 163 samples of normal Canadian honey led to the recognition of eight different species, comprising the genera Zygosaccharomyces, Schizosaccharomyces and Torula. The frequency of their occurrence varied greatly, one type, Z. richteri, being by far the most commonly encountered. The yeast predominating originally is not necessarily the most abundant type after fermentation. Analysis of samples fermenting within 14 months showed species of Zygosaccharomyces only to be most abundant, while Z. richteri, in addition to being the predominant type infecting a large majority of samples, was able, even in certain cases where it was originally outnumbered, to develop and apparently assume the leading role in fermentation.