M. Teresa Fernández-Espinar

RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces : a fast method for species identification and the differentiation of flor yeasts

The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus × S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.

A simplified procedure to analyse mitochondrial DNA from industrial yeasts.

A rapid method based on mtDNA restriction analysis is described for yeast strain identification. The method is an adaptation of that devised by Querol et a]. [Syst. Appl. Microbiol. 15 (1992) 439] for Saccharomyces cerevisiae wine strains, and consists of the standard miniprep isolation of yeast total DNA, and the use of restriction endonucleases that recognise a large number of sites in yeast nuclear DNA, but few sites in the mitochondrial DNA. In the adapted method, the propagation of yeast cells and restriction analysis were the steps mainly affected: cell growth was reduced to 36 h by using microfuge tubes, and the restriction analysis was carried out in just 33 min using a microwave oven for DNA digestion, and minigels for restriction fragment separation. The DNA extraction procedure was performed in the same way as in the original protocol. but slightly reducing the duration of each step and scaling down the volumes of the different solutions. enzymes and reagents used. As result, a large time reduction (52.5 h) was obtained compared to the original method. The DNA obtained can be directly digested with endonucleases displaying clear restriction patterns useful for S. cerevisiae yeast strain differentiation. In addition, strains belonging to other foodborne yeast species, including spoilage yeast species, can also be identified.

Identification of species of the genus Candida by analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers

The PCR amplification and subsequent restriction analysis of the ribosomal region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene is applied to the identification of yeasts belonging to the genus Candida. This methodology has previously been used for the identification of some species of this genus, but in the present work this application has been applied to the identification and characterisation of a greater number of species of the genus Candida, with a special survey of species of clinical and biotechnological interest. Among the species of the genus Candida, the high variability observed, both in the length of the amplified region (ranging between 390 and 900 bp) and in their restriction patterns, allows the unequivocal identification to the species level, with the exception of the group of species that comprises C. membranifaciens, C. conglobata, C. atlantica, C. atmosphaerica, and C. oleophila, that required the sequencing of the D1/D2 domain of the 26S rRNA gene or the 5.8S–ITS region for their proper differentiation. The 5.8S–ITS restriction analysis also failed in the differentiation of species within the pairs C.aaseri/C.butyri,C.fructus/C.musae,C.santamariae var. santamariae/C. beechii and C. zeylanoides/C. krissii. In this case, the high sequence similarities obtained for their 26S D1/D2 domain and the 5.8S–ITS region indicate that each pair of species should be considered as a single species. The main purpose of this work is to generate a database for a high number of yeast species, of both biotechnological and clinical interest, and to facilitate their easy, fast, and reliable identification. The present work improves the database available online at the IATA web page (http://motor.edinfo.es/iata/) with the patterns of 75 species belonging to the genus Candida.

A new PCR-based method for monitoring inoculated wine fermentations.

A new PCR-based method has been developed to monitor inoculated wine fermentations. The method is based on the variation in the number and position of introns in the mitochondrial gene COX1. Oligonucleotide primers homologous to the regions flanking the Saccharomyces cerevisiae COX1 introns have been designed and tested for S. cerevisiae wine yeast strain differentiation. Four primers were selected for their subsequent use in a multiplex PCR reaction and have proved to be very effective in uncovering polymorphism in natural and commercial yeast strains. An important point is that the speed and simplicity of the technique, which does not require the isolation of DNA, allows early detection of the starter yeast strain throughout the fermentation process. The main advantage for the wineries is that the must sample can be used directly for the PCR reaction obtaining very fast results (in approximately 8 h). This allows the wine industries to intervene quickly if necessary.

Analysis of the genetic variability in the species of the Saccharomyces sensu stricto complex

Random amplified polymorphic DNA–polymerase chain reaction (RAPD–PCR) analysis was applied to differentiate the sibling species Saccharomyces bayanus, S. cerevisiae, S. paradoxus and S. pastorianus, which constitute the most common strains of the Saccharomyces sensu stricto complex. Six decamer primers of arbitrary sequences were used to amplify the DNA of 58 strains. Species‐specific (diagnostic) bands were obtained for each species. Two phylogenetic trees constructed by the neighbour‐joining and maximum parsimony methods clearly showed that the delimitation of these related yeast species is possible by using RAPD analysis. Four groups of strains, corresponding to the species S. bayanus, S. cerevisiae, S. paradoxus and S. pastorianus, were obtained. Within the S. bayanus taxon, two groups of strains were observed. One includes the former type strain of S. uvarum, CECT1969T, and closely related wine strains (S. bayanus var. uvarum), whilst the other contains S. bayanus type strain CECT1941T and strains CECT1991 and 10513 (S. bayanus var. bayanus). The heterogeneous S. paradoxus group was divided into three lineages, corresponding to different geographic origin, American, Japanese and European populations. In addition, due to the multilocus nature of the RAPD–PCR marker, this method is both useful and appropriate for the identification of the hybrid origin of S. pastorianus. The hybrid nature was deduced from the analysis of the fraction of bands shared by each hybrid strain and the parental species. Among the 58 strains analysed, six S. pastorianus strains were hybrids, although the fraction of genome coming from each parent varied depending on the strain. Copyright

A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors

Saccharomyces cerevisiae is the most widely used yeast in industrial/commercial food and beverage production and is even consumed as a nutritional supplement. Various cases of fungemia caused by this yeast species in severely debilitated traumatized or immune-deficient patients have been reported in recent years, suggesting that this species could be an opportunistic pathogen in such patients. To determine whether the industrial S. cerevisiae strains can be included in this virulent group of strains, we carried out a comparative study between clinical and industrial yeasts based on the various phenotypic traits associated with pathogenicity in two other yeast species (Candida albicans and Cryptococcus neoformans). The majority of the clinical isolates were found to secrete higher levels of protease and phospholipase, grow better at 42°C and show strong pseudohyphal growth relative to industrial yeasts. However three industrial yeast strains, one commercial wine strain, baker’s yeast and one commercial strain of S. cerevisiae (var. boulardii), were exceptions and based on their physiological traits these yeasts would appear to be related to clinical strains.

Authentication and identification of Saccharomyces cerevisiae‘flor’ yeast races involved in sherry ageing

Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate ‘flor’ and non-‘flor’S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that ‘flor’ yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.

Molecular Characterization of Clinical Saccharomyces cerevisiae Isolates and their Association with Non-Clinical Strains

We assessed the molecular characterization of 96 clinical isolates of S. cerevisiae from a Spanish medical institution and we compared them with 6 non-clinical strains isolated from wine, beer and bread and 1 S. boulardii strain collected from a commercial preparation. The strains were subjected to HinfI mtDNA restriction analysis and PCR amplification of delta sequences. Although both techniques are appropriate for routine clinical analysis, that based on PCR turned out to be the most discriminating. This study, apart from providing tools for clinical application, deals with the relationships between clinical and non-clinical strains. The two bakers yeasts analysed shared mtDNA and PCR patterns with a group of 31 clinical isolates. An exogenous entry of a strain was also reflected in the case of 19 clinical isolates and the therapeutic strain S. boulardii. Both bakers yeasts and S. boulardii were identified respectively among 32.3% and 19.8% of the clinical isolates and there seemed to be a connection between their ability to colonize humans and their ability to cause vaginal infection. The rest of food isolates were not grouped with clinical strains.

Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains.

BackgroundIn recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of Saccharomyces cerevisiae. Some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent.ResultsIn this work, we studied the transcriptional profiles of two S. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. This response involves an mRNA levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. We observed that the clinical strains were more resistant to reactive oxygen species in vitro. In addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic Candida albicans strain. Furthermore, a virulent strain mutant in the transcription factor Yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or YAP1 reconstituted strain.ConclusionsOur data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models.

Molecular Identification and Characterization of Wine Yeasts

The transformation of grape must into wine is a complex microbiological process involving the sequential growth of bacteria and yeasts, although only the yeasts are responsible for alcoholic fermentation. In the past, winemaking was purely empirical, but it is now a well-understood, controlled process that has been gradually improved over time. Advances have largely been made possible by the development of molecular techniques to identify and characterize wine yeasts based on analysis of their DNA. These methods are rapid, reproducible, and sensitive, and continue to be used for a variety of purposes, such as analyzing variation in naturally occurring and inoculated yeast populations, monitoring the dynamics of inoculated strains, characterizing wine yeasts, and detecting spoilage yeasts.