Search

The Saccharomyces sp. CID1 isolate (CBS 8614) and several other Saccharomyces sensu stricto yeasts were analysed for their mitochondrial and nuclear genes. The data show that Saccharomyces sp. CID1, found so far only in one location in Europe, is a natural hybrid between three different Saccharomyces yeast species. Two of them, Saccharomyces cerevisiae-like and Saccharomyces bayanus-like, are ubiquitous and contributed parts of the nuclear genome; the third, Saccharomyces sp. IFO 1802-like, which has been found only in Japan, contributed the mitochondrial DNA molecule. These data suggest that the yeast cell is able to accommodate, express and propagate genetic material that originates from different species, and the very existence of the resulting natural hybrids indicates that such hybrids are well adapted to their habitats.

Genetic variation among 35 strains representing the four currently recognized species of Saccharomyces sensu stricto (Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus/carlsbergensis and Saccharomyces paradoxus) was estimated by analysing the electrophoretic mobilities of nonspecific esterases, acid phosphatase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes. Twenty-two electrophoretic types were identified, a result in agreement with the phenotypic and genetic polymorphisms reported for this group of yeasts. However, the four species were clearly distinguishable based on the patterns obtained using three of the enzymes assayed, the resolving power not being improved by the introduction of data correspondent to lactate dehydrogenase. The overall diversity was higher among S. cerevisiae isolates, in contrast with S. paradoxus which showed only two patterns, one of which was common to four of the five strains studied. Concordant results from the application of the method and DNA hybridization experiments demonstrate its value for identification purposes.

An improved pulsed-field electrophoresis program was developed to study differently sized chromosomes within the genus Saccharomyces. The number of chromosomes in the type strains was shown to be nine in Saccharomyces castellii and Saccharomyces dairenensis, 12 in Saccharomyces servazzii and Saccharomyces unisporus, 16 in Saccharomyces exiguus and seven in Saccharomyces kluyveri. The sizes of individual chromosomes were resolved and the approximate genome sizes were determined by the addition of individual chromosomes of the karyotypes. Apparently, the genome of S. exiguus, which is the only Saccharomyces sensu lato yeast to contain small chromosomes, is larger than that of Saccharomyces cerevisiae. On the other hand, other species exhibited genome sizes that were 10-25% smaller than that of S. cerevisiae. Well-defined karyotypes represent the basis for future genome mapping and sequencing projects, as well as studies of the origin of the modern genomes.

A phylogenetic investigation of the ascomycetous yeast genus Saccharomyces was performed by using 18S rRNA gene sequence analysis. Comparative sequence analysis showed that the genus is phylogenetically very heterogeneous. Saccharomyces species were found to be phylogenetically interdispersed with members of other ascomycetous genera (e.g., the genera Kluyveromyces, Torulaspora, and Zygosaccharomyces). The four species of the Saccharomyces sensu stricto complex (viz., Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus) were found to be phylogenetically closely related to one another, displaying exceptionally high levels of sequence similarity (> or = 99.9%). These four species formed a natural group that was quite separate from the other Saccharomyces and non-Saccharomyces species examined. Saccharomyces exiguus and its anamorph, Candida holmii, were found to be genealogically almost identical and, along with Saccharomyces barnettii, formed a stable group closely related to, but nevertheless distinct from, Kluyveromyces africanus, Kluyveromyces lodderae, Saccharomyces rosinii, Saccharomyces spencerorum, and Saccharomyces sp. strain CBS 7662T (T = type strain). Saccharomyces spencerorum and Kluyveromyces lodderae displayed a particularly close genealogical affinity with each other, as did Saccharomyces castellii and Saccharomyces dairensis. Similarly, Saccharomyces servazzii, Saccharomyces unisporus, and Saccharomyces sp. strain CBS 6904 were found to be genotypically highly related and to form a phylogenetically distinct lineage. The recently reinstated species Saccharomyces transvaalensis was found to form a distinct lineage and displayed no specific association with any other Saccharomyces or non-Saccharomyces species. Saccharomyces kluyveri formed a very loose association with a group which included Kluyveromyces thermotolerans, Kluyveromyces waltii, Zygosaccharomyces cidri, and Zygosaccharomyces fermentati. Saccharomyces spp. strain CBS 6334T, on the other hand, displayed no specific association with any of the other Saccharomyces spp. studied, although a neighbor-joining analysis did reveal that this strain exhibited a loose phylogenetic affinity with Kluyveromyces polysporus and Kluyveromyces yarrowii. On the basis of the phylogenetic findings, two new Saccharomyces species, Saccharomyces kunashirensis (with type strain CBS 7662) and Saccharomyces martiniae (with type strain CBS 6334), are described.

Allozyme electrophoresis was used to characterize 39 isolates belonging to the wild yeast species Saccharomyces paradoxus for variation at nine enzyme loci. The data revealed significant genetic differentiation between isolates from two geographically distinct regions, one including continental Europe and the other including the Russian Far East and Japan. The results are consistent with previous observations indicating that there is partial reproductive isolation between isolates collected from these regions, and they suggest the possibility that these two populations represent an early stage in speciation.

The type strains and other strains of the phenotypically similar taxa Saccharomyces castelli Capriotti, Saccharomyces dairensis Naganishi, and Pachytichospora transvaalensis van der Walt were studied by comparing the ascospore morphologies of these organisms by examining ultrathin sections by transmission electron microscopy. The results of this study and another investigation of DNA base sequence homology demonstrated that the monotypic genus Pachytichospora van der Walt is invalid. We propose that the species Saccharomyces transvaalensis van der Walt should be reinstated.

A study of DNA reassociation kinetics and electrophoretic karyotypes of several strains classified as Saccharomyces castellii, Saccharomyces dairensis, and Pachytichospora transvaalensis strains revealed that the group included members of at least five distinct species. Two of the four strains classified as P. transvaalensis exhibited very high levels of DNA sequence homology with the type strain of S. castellii, while the type strain and another strain demonstrated intermediate levels of relatedness to the type strains of the unrelated taxa S. castellii and S. dairensis. Five strains that were classified as S. dairensis strains were not related to the type strains of the taxa studied. Two of these strains exhibited intermediate relatedness (65% base sequence homology), while another pair exhibited a level of DNA reassociation of 97%. The remaining strain was distinct from all of the other strains studied on the basis of both DNA base sequences and electrophoretic karyotype data.

Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.

An analytical method in which we used the selective adsorption of several fluorophores by yeast cells is described. The suitability of using binary mixtures of 1-pyrene butyric acid, 3,6-dimethylamino acridine, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, and rhodamine B isothiocyanate for the characterization and identification of microorganisms was tested with 98 yeast strains belonging to the genera Candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces. The application of multivariate statistical methods and pattern recognition methods to the allocation of the yeast strains into genus-species-strain structures and to a comparison of fluorescence data sets for differentiation and identification purposes showed the usefulness of the method.