Isolation and characterization of the HO gene from the yeast Saccharomyces paradoxus

A DNA fragment homologous to the homothallism (HO) gene of Saccharomyces cerevisiae was isolated from Saccharomyces paradoxus and was found to contain an open reading frame that was 90.9% identical to the coding sequence of the S. cerevisiae HO gene. The putative HO gene was shown to induce diploidization in a heterothallic haploid strain from S. cerevisiae. Phylogenetic analysis revealed that the coding and 5'-upstream regulatory regions from five Saccharomyces sensu stricto HO genes have coevolved, and that S. paradoxus is phylogenetically closer to S. cerevisiae than to S. bayanus. Finally, heterothallic haploid strains were isolated from the original homothallic type strain of S. paradoxus by disrupting the S. paradoxus HO gene with the S. cerevisiae URA3 gene.     

PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers

The aim of the present study was to design species-specific primers capable of distinguishing between Saccharomyces cerevisiae, Saccharomyces bayanus/Saccharomyces pastorianus. The 5'-specific primers were designed from the ITS-1 region (between positions 150 and 182 from the 3'-SSU end) and the 3'-specific primers were located in the LSU gene (positions 560-590 from the 5'-end of this gene). These primers were tested with different collections and wild strains of these species and the results showed that the primers were capable of distinguishing between S. cerevisiae strains and S. bayanus/S. pastorianus. Not enough sequence differences were found between S. bayanus and S. pastorianus to design specific primers for these species using this region. This method offers an effective tool for a quick differentiation of the Saccharomyces strains of the most common species involved in industrial processes.     

Evolutionary relationships between the former species Saccharomyces uvarum and the hybrids Saccharomyces bayanus and Saccharomyces pastorianus; reinstatement of Saccharomyces uvarum (Beijerinck) as a distinct species

Analysis of the nucleotide sequence of the GDH1 homologues from Saccharomyces bayanus strain CBS 380T and S. pastorianus strains showed that they share an almost identical sequence, SuGDH1*, which is a diverged form of the SuGDH1 from the type strain of the former species S. uvarum, considered as synonym of S. bayanus. SuGDH1* is close to but differs from SuGDH1 by the accumulation of a high number of neutral substitutions designated as Multiple Neutral Mutations Accumulation (MNMA). Further analysis carried out with three other markers, BAP2, HO and MET2 showed that they have also diverged from their S. uvarum counterparts by MNMA. S. bayanus CBS 380T is placed between S. uvarum and S. pastorianus sharing MET2, CDC91 sequences with the former and BAP2, GDH1, HO sequences with the latter. S. bayanus CBS 380T has been proposed to be a S. uvarum/S. cerevisiae hybrid and this proposal is confirmed by the presence in its genome a S. cerevisiae SUC4 gene. Strain S. bayanus CBS 380T, with a composite genome, is genetically isolated from strains of the former S. uvarum species, thus justifying the reinstatement of S. uvarum as a distinct species.     

Heterothallism in Saccharomyces cerevisiae isolates from nature: effect of HO locus on the mode of reproduction

Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae , we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother–daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother–daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother–daughter mating might not be as significant as was considered.     

Divergence of glyceraldehyde-3-phosphate dehydrogenase isozymes in Saccharomyces cerevisiae complex

Although sake yeasts are placed in Saccharomyces cerevisiae, we have been interested in their difference from the other subgroups of the species, and examined their proteins. When SDS-PAGE patterns of their soluble proteins were compared, specific differences between subgroups were found in their 36,000 Da regions. Proteins isolated therefrom were found to be subunits of three isomers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from their N-terminal amino acid sequences and identified with anti-GAPDH serum. Therefore, comparison of zymogram was carried out by a modified method: denatured monomers were observed and the enzyme activity of their oligomers was not considered. SDS-PAGE patterns of all the sake yeasts differed from those of the other strains of S. cerevisiae. Strains of Saccharomyces bayanus showed uniform patterns which are different from the above two groups. Saccharomyces pastorianus strains resembled S. bayanus and were partly similar to S. cerevisiae in their patterns, in agreement with the hypothesis that S. pastorianus is a hybrid between these two species. Patterns of S. paradoxus appeared to be rather similar to those of sake yeasts. Results on the other species of the genus and on the preliminary experiments on PAGE of native isozymes are also described.     

Variability of HXT2 at the protein and gene level among the Saccharomyces sensu stricto group

Variability of HXT2 at the protein and gene level was investigated among Saccharomyces sensu stricto and other yeast species. Results showed that the HXT2 gene is probably present in yeast genera other than Saccharomyces, suggesting that this gene is widely distributed in the yeast world. Chromosomal analyses indicated the stable location of HXT2 on the same chromosome and with the same copy number throughout the entire sensu stricto group. Results of the immunoblotting assay demonstrated that all strains tested (with the exception of S. cerevisiae DBVPG 6042) exhibited a lower level of Hxt2p expression than that shown by laboratory wild-type. Moreover, Hxt2p expression seems to reinforce the taxonomical differences between the two pairs of species (S. cerevisiae and S. paradoxus vs. S. pastorianus and S. bayanus) within the sensu stricto group of the genus of Saccharomyces that also reflect their different ecological niche.     

Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species

Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.     

Deciphering the hybridisation history leading to the Lager lineage based on the mosaic genomes of Saccharomyces bayanus strains NBRC1948 and CBS380

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380(T) and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380(T) harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380(T) and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380(T) or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Rapid identification and differentiation of Saccharomyces cerevisiae, Saccharomyces bayanus and their hybrids by multiplex PCR

AIMS: To develop a multiplex PCR assay for the specific identification and differentiation of Saccharomyces cerevisiae, S. bayanus and their hybrids. METHODS AND RESULTS: Two sets of primers with sequences complementary to the region YBR033w were used. A single amplicon of 1710 bp or 329 bp was obtained with species S. cerevisiae and S. bayanus, respectively, while the presence of both bands was observed in S. pastorianus because of its hybrid nature. Both amplification products were also obtained after amplification from DNA of several laboratory S. cerevisiae x S. bayanus hybrid strains. CONCLUSIONS: Multiplex PCR was optimized for the rapid and reliable identification of S. cerevisiae, S. bayanus and their hybrids. SIGNIFICANCE AND IMPACT OF THE STUDY: The procedure may be used for routine detection of the most common Saccharomyces sensu stricto yeasts involved in industrial fermentation processes, overcoming the problems of conventional techniques.     

Molecular analysis of alpha-galactosidase MEL genes from Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the alpha-galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1-99.3%), comparable with interspecific homology (94.8-100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae (MELj) was 76.2-81.7%, suggesting certain species specificity. On this evidence, the alpha-galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

An Opportunity to distingush species of Saccharomyces Sensu stricto by electrophoretic separation of the larger Chromosomes

J. TORNAI-LEHOCZKI AND D. DLAUCHY. 1996. Intact chromosomal DNA of the type strains of Saccharomyces sensu stricto species and some of their synonyms and 38 Hungarian wine and 14 brewing yeast strains were separated by rotating field gel electrophoresis (RFE). The applied electrophoretic sdnditions enabled us to distinguish strains of S. bayanus, S. pastorianus and S. cerevisiae from each other, for strains of the three species showed clear differences in their electrophoretic patterns at heavy chromosomes (> 1300 kb).     

SED1 polymorphism within the genus Saccharomyces

The SED1 gene is characterised by abundant length and sequence polymorphisms within the species Saccharomyces cerevisiae, due to the expansion and contraction of minisatellite-like sequences located within the ORF. A survey of the SED1 ORFs of 26 yeasts ascribed to the species S. cerevisiae, S. bayanus, S. pastorianus, S. paradoxus, S. cariocanus, S. kudriavzevii and S. mikatae revealed SED1 gene length and sequence variations between the species of the genus. Moreover, results obtained by Neighbour-Joining analysis of a dataset comprising the partial predicted amino acid sequences of SED1 ORFs agreed with the phylogenetic relationships of the seven species. Thus, the SED1 gene may represent a further molecular target for the identification of Saccharomyces isolates.     

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.     

A multiplex set of species-specific primers for rapid identification of members of the genus Saccharomyces

The Saccharomyces genus (previously Saccharomyces sensu stricto) formally comprises Saccharomyces arboricola, Saccharomyces bayanus, Saccharomyces cariocanus, Saccharomyces cerevisiae, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces paradoxus and Saccharomyces pastorianus. Species-specific primer pairs that produce a single band of known and different product size have been developed for each member of the clade with the exception of S. pastorianus, which is a polyphyletic allopolyploid hybrid only found in lager breweries, and for which signature sequences could not be reliably created. Saccharomyces cariocanus is now regarded as an American variant of S. paradoxus, and accordingly a single primer pair that recognizes both species was developed. A different orthologous and essential housekeeping gene was used to detect each species, potentially avoiding competition between PCR primers and overlap between amplicons. In multiplex format, two or more different species could be identified in a single reaction; double and triple hybrids could not always be correctly identified. Forty-two unidentified yeasts from sugar cane juice fermentations were correctly identified as S. cerevisiae. A colony PCR method was developed that is rapid, robust, inexpensive and capable of automation, requires no mycological expertise on the part of the user and is thus useful for large-scale preliminary screens.     

The Genetic System Controlling Homothallism in Saccharomyces Yeasts

There are four types of life cycles in Saccharomyces cerevisiae and its related species. A perfect homothallic life cycle (the Ho type) is observed in the classic D strain. Two other types show semi-homothallism; one of them shows a 2-homothallic diploid:2alpha heterothallic haploid segregation (the Hp type) and another, a 2-homothallic:2a segregation (the Hq type). In the segregants from these Ho, Hp, and Hq diploids, each homothallic segregant shows the same segregation pattern as its parental diploid. The fourth type has a heterothallic life cycle showing a 2a:2alpha segregation and the diploids are produced by the fusion of two haploid cells of opposite mating types. The diploids prepared by the crosses of alpha Hp (an alpha haploid segregant from the Hp diploid) to a Hq (an a haploid from the Hq diploid) segregated two types (Type I and II) of the Ho type homothallic clone among their meiotic segregants. Genetic analyses were performed to investigate this phenomenon and the genotypes of the Ho type homothallic clones of Type I and Type II. Results of these genetic analyses have been most adequately explained by postulating three kinds of homothallic genes, each consisting of a single pair of alleles, HO/ho, HMalpha/hmalpha, and HMa/hma, respectively. One of them, the HMalpha locus, was proved to be loosely linked (64 stranes) to the mating-type locus. A spore having the HO hmalpha hma genotype gives rise to an Ho type homothallic diploid (Type I), the same as in the case of the D strain which has the HO HMalpha HMa genotype (Type II). A spore having the a HO hmalpha HMa or alpha HO HMalpha hma genotype will produce an Hp or Hq type homothallic diploid culture, respectively. The other genotypes, a HO HMalpha hma, alpha HO hmalpha HMa, and the genotypes combined with the ho allele give a heterothallic character to the spore culture. A possible molecular hypothesis for the mating-type differentiation with the controlling elements produced by the HMalpha and HMa genes is proposed.     

Karyotyping of Saccharomyces strains with different temperature profiles

This study examined the karyotype, the fermentation performance and the optimum growth temperature (Topt) of 28 yeast strains all identified as species belonging to Saccharomyces sensu stricto . The strains were isolated from fermented musts, which had not been inoculated, at two temperature ranges: 20–40 °C and approximately 0–6 °C. The results demonstrated a correlation between the Topt and the chromosome organization. In particular, strains with Topt of less than 30 °C showed only two bands in the region between 365 and 225 kb, while those with a Topt greater than 30 °C had three bands in this size range. From a taxonomic viewpoint, the Topt is a better indicator for the Saccharomyces sp. than the ceiling temperature of 37 °C currently used to differentiate cryotolerant Saccharomyces bayanus and S. pastorianus from non-cryotolerant S. cerevisiae and S. paradoxus strains.     

Duplicated Genes Producing Transposable Controlling Elements for the Mating-Type Differentiation in SACCHAROMYCES CEREVISIAE

Mutation of the two homothallic genes, HML alpha/HMLa and HMRa/HMR alpha, in homothallic strains of Saccharomyces cerevisiae was studied. Of 11 mutants of the HML alpha gene, eight were due to a phenotypic mutation from HML alpha to HMLa, i.e., a mutation causing a change in function of the original HML allele to that of the other HML allele (functional mutation), and three were due to a defective mutation at the HML alpha gene, i.e., a mutation causing a nonfunctional allele (nonfunctional mutation). All 14 mutants of the HMRa gene, on the other hand, were due to a phenotypic mutation from HMRa to HMR alpha i.e., a functional mutation. Phenotypic reverse mutations, i.e., HMLa to HML alpha and HMR alpha to HMRa, were also observed in the cultivation of EMS (ethyl methanesulfonate) treated spores having the HO HMR alpha HMLa genotype. Mutation from heterothallic cells to homothallism was observed in a nonfunctional mutant of the HML alpha gene, by mutagenesis with EMS, but not in the functional mutants of the HML alpha and HMRa genes or in the authentic strains having the alpha HO HMR alpha HML alpha (alpha Hp) and a HO HMRa HMLa (a Hq) genotypes. These observations suggest that the functional mutation is not caused by the direct mutation from a homothallic allele to the opposite, but by replacement of a transposable genic element produced from a homothallic locus with a region of a different homothallic locus. These observations also support the controlling-element model and the cassette model, which have been proposed to explain the mating-type differentiation by the homothallic genes.     

Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.     

PCR–DGGE differentiation of strains of Saccharomyces sensu stricto

A quick molecular biology method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was developed for distinguishing strains belonging to the Saccharomyces sensu stricto group. Differentiation was obtained between S. cerevisiae, S. paradoxus and S. bayanus / S. pastorianus although no distinction was possible between S. bayanus and S. pastorianus using the amplification of the ITS regions. The ability to distinguish between different strains of the Saccharomyces sensu stricto group could allow for a better understanding of the ecology of these species on grapes as well as in musts and wines and the method developed can be useful for the quick identification of Saccharomyces sensu stricto strains from numerous isolates.     

A murine monoclonal antibody directed against a yeast cell wall glycoprotein antigen of the yeast genus Saccharomyces

Abstract Murine monoclonal antibodies (mAbs) were selected against a cell wall glycoprotein of Saccharomyces cerevisiae . One of the mAbs (92-276/018) specifically identified S. cerevisiae and the sibling species S. paradoxus, S. pastorianus and S. bayanus in immunofluorescence studies and immunoblot analyses, while no other yeast genera except Saccharomyces were recognized. Further analysis indicated that the mAb 92-276/018 reacts with an epitope in the carbohydrate chain of the cell wall glycoproteins.