Complex Nature of the Genome in a Wine Spoilage Yeast,Dekkera bruxellensis

When the genome organizations of 30 native isolates belonging to a wine spoilage yeast, Dekkera (Brettanomyces) bruxellensis, a distant relative of Saccharomyces cerevisiae, were examined, the numbers of chromosomes varied drastically, from 4 to at least 9. When single gene probes were used in Southern analysis, the corresponding genes usually mapped to at least two chromosomal bands, excluding a simple haploid organization of the genome. When different loci were sequenced, in most cases, several different haplotypes were obtained for each single isolate, and they belonged to two subtypes. Phylogenetic reconstruction using haplotypes revealed that the sequences from different isolates belonging to one subtype were more similar to each other than to the sequences belonging to the other subtype within the isolate. Reanalysis of the genome sequence also confirmed that partially sequenced strain Y879 is not a simple haploid and that its genome contains approximately 1% polymorphic sites. The present situation could be explained by (i) a hybridization event where two similar but different genomes have recently fused together or (ii) an event where the diploid progenitor of all analyzed strains lost a regular sexual cycle, and the genome started to accumulate mutations.Recent achievements in genome sequencing have revealed that gene contents vary among distantly related organisms but are relatively constant among closely related species. For example, among hemiascomycete yeasts, which originated more than 250 million years ago and include well-studied yeasts such as Saccharomyces cerevisiae and Candida albicans (3, 4), an average genome contains approximately 5,000 genes. Approximately one-half of the protein-coding gene families are preserved in all of the yeasts sequenced to date. However, there is a large variation in the gene order and configuration of chromosomes among different species.Chromosome configuration is usually well preserved among populations belonging to the same species. Only rarely do geographically separated populations, for example, Mus musculus (8, 32), differ in the number and form of chromosomes. The mutability of the genome enhances the adaptability of the species, but it also decreases the viability of the new variant. In addition, these changes can preclude successful reproduction and can be a decisive factor in the emergence of new species (2; for a review, see references 6 and 7).Among closely related yeasts belonging to the Saccharomyces sensu stricto clade (including S. cerevisiae), which originated approximately 20 million years ago, the gene contents are relatively similar (13). Their genomes are almost colinear and consist of 16 chromosomes. Some inter- and intraspecific variations are observed predominantly at the chromosome ends (18, 19). Sensu stricto species are semifertile, meaning that they can successfully mate and produce F1 offspring but that the hybrids are largely sterile. It appears that this clade has still not completed the speciation process (7). The relatively low chromosome variability among Saccharomyces sensu stricto yeasts is probably promoted by regular sexual cycles. These yeasts are diploid, but heterozygosity is almost absent because of the homothallic life-style, which enables haploid spores from the same yeast cell to mate. Only for “sterile” hybrids, such as the lager brewing yeast Saccharomyces pastorianus (Saccharomyces carlsbergensis), originating upon the mating of two different species, has a pronounced heterozygosity been observed (14). The parental genomes came from S. cerevisiae and a close relative, Saccharomyces bayanus. A study of allotetraploid hybrids between a diploid S. cerevisiae strain and a diploid S. bayanus strain demonstrated that these hybrids behave essentially as diploids regarding meiosis and sporulation and had 77% spore viability (1, 22). The extent of intra- and interspecific genome variability is not well known for other yeasts, especially among distant relatives of S. cerevisiae. The only well-studied exception is a pathogen, Candida albicans, that is believed to be predominantly asexual. This yeast diverged from the S. cerevisiae lineage prior to the origin of the efficient homothallic life-style (reviewed in reference 25). The genome is diploid and shows a low level of heterozygosity (12), and large variations in the configurations of the chromosomes among different isolates have been reported (reviewed in reference 29).Dekkera bruxellensis is often isolated in wineries and is well known as a major microbial cause of wine spoilage. The lineages of D. bruxellensis and S. cerevisiae separated at approximately the same time as the lineages of S. cerevisiae and C. albicans separated, approximately 200 million years ago (40). However, D. bruxellensis and S. cerevisiae share several characteristics, such as the production of ethanol, the ability to propagate in the absence of oxygen (anaerobic growth), and petite positivity (the ability to produce offspring without mitochondrial DNA mtDNA]), that are rarely found among other yeasts (16, 20). So far, a sexual cycle in D. bruxellensis has not been found.In this paper, we analyzed the genome structures of 30 isolates of D. bruxellensis originating from different geographical localities around the world. We show that these isolates have different numbers and sizes of chromosomes and also that the numbers of copies of several analyzed genes and their sequences vary. In addition, we could detect heterozygosity in the partial genome sequence of strain Y879.