The novel function of the Saccharomyces cerevisiaeCBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasiiLSU intron in vivo

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiaeCBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiaeCBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron. Received: 1 October 1997 / Accepted: 18 November 1997     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

Divergence of the mitochondrial leucyl tRNA synthetase genes in two closely related yeasts Saccharomyces cerevisiae and Saccharomyces douglasii: a paradigm of incipient evolution

Summary We studied the NAM2 genes of Saccharomyces douglasii and Saccharomyces cerevisiae, and showed that they are interchangeable for all the known functions of these genes, both mitochondrial protein synthesis and mitochondrial mRNA splicing. This confirms the prediction that the S. douglasii NAM2D gene encodes the mitochondrial leucyl tRNA synthetase (EC 6.1.1.4). The observation that these enzymes are interchangeable for their mRNA splicing functions, even though there are significant differences in the intron/exon structure of their mitochondrial genome, suggests that they may have a general role in yeast mitochondrial RNA splicing. A short open reading frame (ORF) precedes the synthetase-encoding ORF, and we showed that at least in S. cerevisiae this is not essential for the expression of the gene; however, it may be involved in a more subtle type of regulation. Sequence comparisons of S. douglasii and S. cerevisiae revealed a particularly interesting situation from the evolutionary point of view. It appears that the two yeasts have diverged relatively recently: there is remarkable nucleotide sequence conservation, with no deletions or insertions, but numerous (albeit non-saturating) silent substitutions resulting from transitions. This applies not only to the NAM2 coding regions, but also to two other ORFs flanking the NAM2 ORF. The regions between the ORFs (believed to be intergenic regions) are much less conserved, with several deletions and insertions. Thus S. douglasii and S. cerevisiae provide an ideal system for the study of molecular evolution, being two yeasts caught in the act of speciation.     

Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae

We have determined the physical and genetic map of the 73,000 base-pair mitochondrial genome of a novel yeast species Saccharomyces douglasii. Most of the protein and RNA-coding genes known to be present in the mitochondrial DNA of Saccharomyces cerevisiae have been identified and located on the S. douglasii mitochondrial genome. The nuclear genomes of the two species are thought to have diverged some 50 to 80 million years ago and their nucleo-mitochondrial hybrids are viable but respiratorily deficient. The mitochondrial genome of S. douglasii displays many interesting features in comparison with that of S. cerevisiae. The three mosaic genes present in both genomes are quite different with regard to their structure. The S. douglasii COXI gene has two new introns and is missing the five introns of the S. cerevisiae gene. The S. douglasii cytochrome b gene has one new intron and lacks two introns of the S. cerevisiae gene. Finally, the L-rRNA gene of S. douglasii, like that of S. cerevisiae, has one intron of which the structure is different. Another salient feature of the S. douglasii mitochondrial genome reported here is that the gene order is different in comparison with S. cerevisiae mitochondrial DNA. In particular, a segment of approximately 15,000 base-pairs including the genes coding for COXIII and S-rRNA has been translocated to a position between the genes coding for varl and L-rRNA.     

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.     

New Hybrids between Saccharomyces Sensu Stricto Yeast Species Found among Wine and Cider Production Strains

Two yeast isolates, a wine-making yeast first identified as a Mel⁺ strain (ex. S. uvarum) and a cider-making yeast, were characterized for their nuclear and mitochondrial genomes. Electrophoretic karyotyping analyses, restriction fragment length polymorphism maps of PCR-amplified MET2 gene fragments, and the sequence analysis of a part of the two MET2 gene alleles found support the notion that these two strains constitute hybrids between Saccharomyces cerevisiae and Saccharomyces bayanus. The two hybrid strains had completely different restriction patterns of mitochondrial DNA as well as different sequences of the OLI1 gene. The sequence of the OLI1 gene from the wine hybrid strain appeared to be the same as that of the S. cerevisiae gene, whereas the OLI1 gene of the cider hybrid strain is equally divergent from both putative parents, S. bayanus and S. cerevisiae. Some fermentative properties were also examined, and one phenotype was found to reflect the hybrid nature of these two strains. The origin and nature of such hybridization events are discussed.     

Chimeric Genomes of Natural Hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.The genus Saccharomyces consists of seven biological species: S. arboricolus, S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus (29, 59) and the partially allotetraploid species S. pastorianus (46, 58).The hybrid species S. pastorianus, restricted to lager brewing environments, arose from two or more natural hybridization events between S. cerevisiae and a S. bayanus-like yeast (7, 16, 28, 46). Recent studies of S. bayanus have also revealed the hybrid nature of certain strains of this species, which has subsequently been subdivided into two groups, S. bayanus var. bayanus, containing a variety of hybrid strains, and S. bayanus var. uvarum, also referred to as S. uvarum, that contains nonhybrid strains (45, 46).New hybrids of other species from the genus Saccharomyces have recently been described. Hybrid yeasts of S. cerevisiae and S. kudriavzevii have been characterized among wine (6, 20, 33) and brewing yeasts (21); even triple hybrids of S. cerevisiae, S. bayanus, and S. kudriavzevii have been identified (20, 41).The first natural Saccharomyces interspecific hybrid identified, the lager brewing yeast S. pastorianus (S. carlsbergensis) (42, 57), has become one of the most investigated types of yeast hybrids. The genome structure of these hybrids has been examined by competitive array comparative genome hybridization (aCGH) (5, 16, 28), complete genome sequencing (28), and PCR-restriction fragment length polymorphism (RFLP) analysis of 48 genes and partial sequences of 16 genes (46). The aCGH analyses of several S. pastorianus strains with S. cerevisiae-only DNA arrays (5, 28) revealed the presence of aneuploidies due to deletions of entire regions of the S. cerevisiae fraction of the hybrid genomes. A recent aCGH analysis of S. pastorianus strains with S. cerevisiae and S. bayanus DNA arrays (16) showed two groups of strains according to their genome structure and composition. These groups arose from two independent hybridization events, and each one is characterized by a reduction and an amplification of the S. cerevisiae genome fraction, respectively.The genetic characterization of the wine S. cerevisiae and S. kudriavzevii hybrids by restriction analysis of five nuclear genes located in different chromosomes, 5.8S-ITS rDNA region and the mitochondrial COX2 gene, revealed the presence of three types of hybrids in Swiss wines, thus indicating the presence of different hybrid genomes (20). In a recent study (21), we identified six new types of S. cerevisiae and S. kudriavzevii hybrids among brewing strains, which were compared to wine hybrids by a genetic characterization based on RFLP analysis of 35 protein-encoding genes. This analysis confirmed the presence of three different genome types among wine hybrids that contain putative chimeric chromosomes, probably generated by a recombination between homeologous chromosomes of different parental origins.The aim of the present study is to investigate the genome composition and structure of wine hybrids of S. cerevisiae and S. kudriavzevii. This has been achieved by a combined approach based on the RFLP analysis of 35 gene regions from our previous study, comparative genome hybridizations using S. cerevisiae DNA macroarrays, a ploidy analysis by flow cytometry, and gene dose determinations by quantitative real-time PCR. This multiple approach allowed us to confirm the presence of chimeric chromosomes and define the mechanisms involved in their origins.     

Pure and Mixed Genetic Lines of Saccharomyces bayanus and Saccharomyces pastorianus and Their Contribution to the Lager Brewing Strain Genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified “pure” strains containing a single type of genome and “hybrid” strains that contained portions of the genomes from the “pure” lines, as well as alleles termed “Lager” that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho⁰) or those with deleted mitochondrial DNA (rho^–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho^– and rho⁰ cells were ~2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho^– and rho⁰) led to decreased intracellular levels of ROS. We also demonstrate that in rho⁰ cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.     

Incipient mitochondrial evolution in yeasts. II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution.

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.     

Population structure of mitochondrial genomes in Saccharomyces cerevisiae

Background

Rigorous study of mitochondrial functions and cell biology in the budding yeast, Saccharomyces cerevisiae has advanced our understanding of mitochondrial genetics. This yeast is now a powerful model for population genetics, owing to large genetic diversity and highly structured populations among wild isolates. Comparative mitochondrial genomic analyses between yeast species have revealed broad evolutionary changes in genome organization and architecture. A fine-scale view of recent evolutionary changes within S. cerevisiae has not been possible due to low numbers of complete mitochondrial sequences.

Results

To address challenges of sequencing AT-rich and repetitive mitochondrial DNAs (mtDNAs), we sequenced two divergent S. cerevisiae mtDNAs using a single-molecule sequencing platform (PacBio RS). Using de novo assemblies, we generated highly accurate complete mtDNA sequences. These mtDNA sequences were compared with 98 additional mtDNA sequences gathered from various published collections. Phylogenies based on mitochondrial coding sequences and intron profiles revealed that intraspecific diversity in mitochondrial genomes generally recapitulated the population structure of nuclear genomes. Analysis of intergenic sequence indicated a recent expansion of mobile elements in certain populations. Additionally, our analyses revealed that certain populations lacked introns previously believed conserved throughout the species, as well as the presence of introns never before reported in S. cerevisiae.

Conclusions

Our results revealed that the extensive variation in S. cerevisiae mtDNAs is often population specific, thus offering a window into the recent evolutionary processes shaping these genomes. In addition, we offer an effective strategy for sequencing these challenging AT-rich mitochondrial genomes for small scale projects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1664-4) contains supplementary material, which is available to authorized users.     

The NAM8 gene in Saccharomyces cerevisiae encodes a protein with putative RNA binding motifs and acts as a suppressor of mitochondrial splicing deficiencies when overexpressed.

Summary We have characterized the nuclear geneNAM8 inSaccharomyces cerevisiae. It acts as a suppressor of mitochondrial splicing deficiencies when present on a multicopy plasmid. The suppressed mutations affect RNA folding and are located in both group I and group II introns. The gene is weakly transcribed in wildtype strains, its overexpression is a prerequisite for the suppressor action. Inactivation of theNAM8 gene does not affect cell viability, mitochondrial function or mitochondrial genome stability. TheNAM8 gene encodes a protein of 523 amino acids which includes two conserved (RNP) motifs common to RNA-binding proteins from widely different organisms. This homology with RNA-binding proteins, together with the intronic location of the suppressed mitochondrial mutations, suggests that the NAM8 protein could be a non-essential component of the mitochondrial splicing machinery and, when present in increased amounts, it could convert a deficient intron RNA folding pattern into a productive one.     

Redox Interactions between Saccharomyces cerevisiae and Saccharomyces uvarum in Mixed Culture under Enological Conditions

Wine yeast starters that contain a mixture of different industrial yeasts with various properties may soon be introduced to the market. The mechanisms underlying the interactions between the different strains in the starter during alcoholic fermentation have never been investigated. We identified and investigated some of these interactions in a mixed culture containing two yeast strains grown under enological conditions. The inoculum contained the same amount (each) of a strain of Saccharomyces cerevisiae and a natural hybrid strain of S. cerevisiae and Saccharomyces uvarum. We identified interactions that affected biomass, by-product formation, and fermentation kinetics, and compared the redox ratios of monocultures of each strain with that of the mixed culture. The redox status of the mixed culture differed from that of the two monocultures, showing that the interactions between the yeast strains involved the diffusion of metabolite(s) within the mixed culture. Since acetaldehyde is a potential effector of fermentation, we investigated the kinetics of acetaldehyde production by the different cultures. The S. cerevisiae-S. uvarum hybrid strain produced large amounts of acetaldehyde for which the S. cerevisiae strain acted as a receiving strain in the mixed culture. Since yeast response to acetaldehyde involves the same mechanisms that participate in the response to other forms of stress, the acetaldehyde exchange between the two strains could play an important role in inhibiting some yeast strains and allowing the growth of others. Such interactions could be of particular importance in understanding the ecology of the colonization of complex fermentation media by S. cerevisiae.     

Sequence rearrangements between mitochondrial DNAs of Torulopsis glabrata and Kloeckera africana identified by hybridization with six polypeptide encoding regions from Saccharomyces cerevisiae mitochondrial DNA

Sequences hybridizing to six mitochondrial DNA encoded polypeptide genes of Saccharomyces cerevisiae have been mapped in the 18·9 and 27·1 kbp² circular mitochondrial DNAs from Torulopsis glabrata and Kloeckera africana. With the possible exception of cytochrome oxidase subunit 1 and ATPase subunit 6 genes, no two hybridizable sequences share the same order in the two mtDNAs nor is there any topographical similarity to S. cerevisiae mtDNA apart from the grouping mentioned above. Because sequence rearrangements are prevalent in yeast mitochondrial DNAs we infer that order is not critical for mitochondrial gene expression and that prokaryotic-like operons do not exist. In contrast to S. cerevisiae, the cytochrome b region in T. glabrata and K. africana is confined to 1·46 or 1·58 kbp, respectively, which suggests that intervening sequences in this gene are either small or absent. On the other hand, hybridizable sequences to a 5·2 kbp portion of the S. cerevisiae cytochrome oxidase subunit 1 gene, retaining exons 3 to 7 or 8, span 3 to 4 kbp in the two mtDNAs. In addition an 0·8 to 0·9 kbp intervening sequence is present in each case, which does not hybridize to either exon or intron regions of the S. cerevisiae probe. These results imply that the cytochrome oxidase subunit 1 gene in both mtDNAs has a mosaic organization of coding and noncoding sequences.     

Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

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.     

Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces

There have been an increasing number of reports of yeast systemic infection involving Saccharomyces cerevisiae strains. The development of a rapid and reliable diagnostic tool is therefore warranted in order to explore the distribution of S. cerevisiae as an opportunistic pathogen in humans. In this study, we designed and validated five primer sets targeting the 26S rRNA gene of S. cerevisiae and the S. sensu stricto complex using 26 yeast strains. Among them, two sets of primers specifically amplified the 26S rRNA gene and the ITS region of S. cerevisiae strains, and three sets were specific for amplifying the same genes in the S. sensu stricto complex. After determining the optimal conditions of two primer pairs for quantitative real time PCR, human fecal samples were analyzed to examine the distribution of S. cerevisiae and the S. sensu stricto complex. It was possible to detect a single cell of S. cerevisiae in environmental sample. Qualitative PCR revealed that out of eleven fecal samples tested, one sample contained S. cerevisiae and four samples contained the S. sensu stricto complex. Quantitative real time PCR revealed that the target gene copy numbers of S. cerevisiae and the S. sensu stricto complex were 0.84 and 2.44 respectively, in 1 ng of DNA from the bulk fecal community.     

Brewing up a storm: The genomes of lager yeasts and how they evolved

Yeasts used in the production of lager beers belong to the species Saccharomyces pastorianus, an interspecies hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus. The hybridisation event happened approximately 500–600 years ago and therefore S. pastorianus may be considered as a newly evolving species. The happenstance of the hybridisation event created a novel species, with unique genetic characteristics, ideal for the fermentation of sugars to produce flavoursome beer. Lager yeast strains retain the chromosomes of both parental species and also have sets of novel hybrid chromosomes that arose by recombination between the homeologous parental chromosomes. The lager yeasts are subdivided into two groups (I and II) based on the S. cerevisiae: S. eubayanus gene content and the types and numbers of hybrid chromosomes. Recently, whole genome sequences for several Group I and II lager yeasts and for many S. cerevisiae and S. eubayanus isolates have become available. Here we review the available genome data and discuss the likely origins of the parental species that gave rise to S. pastorianus. We review the compiled data on the composition of the lager yeast genomes and consider several evolutionary models to account for the emergence of the two distinct types of lager yeasts.