Yumi Imanishi

A population genomics insight into the Mediterranean origins of wine yeast domestication

The domestication of the wine yeast Saccharomyces cerevisiae is thought to be contemporary with the development and expansion of viticulture along the Mediterranean basin. Until now, the unavailability of wild lineages prevented the identification of the closest wild relatives of wine yeasts. Here, we enlarge the collection of natural lineages and employ whole‐genome data of oak‐associated wild isolates to study a balanced number of anthropic and natural S. cerevisiae strains. We identified industrial variants and new geographically delimited populations, including a novel Mediterranean oak population. This population is the closest relative of the wine lineage as shown by a weak population structure and further supported by genomewide population analyses. A coalescent model considering partial isolation with asymmetrical migration, mostly from the wild group into the Wine group, and population growth, was found to be best supported by the data. Importantly, divergence time estimates between the two populations agree with historical evidence for winemaking. We show that three horizontally transmitted regions, previously described to contain genes relevant to wine fermentation, are present in the Wine group but not in the Mediterranean oak group. This represents a major discontinuity between the two populations and is likely to denote a domestication fingerprint in wine yeasts. Taken together, these results indicate that Mediterranean oaks harbour the wild genetic stock of domesticated wine yeasts.

Method for identifying heat-resistant fungi of the genus Neosartorya.

Species of the genus Neosartorya are heat-resistant fungi that cause the spoilage of heat-processed acidic foods due to the formation of heat-resistant ascospores, and they produce mycotoxins, such as fumitremorgins and gliotoxin. Their anamorphs are phylogenetically and morphologically very close to Aspergillus fumigatus, which has never been reported as a spoilage agent in heat-processed food products. Therefore it is important to discriminate between the species of Neosartorya and A. fumigatus in the food industry. In the present study, we examined β-tubulin and calmodulin genes to identify Neosartorya and A. fumigatus at the species level and found a region for specifically detecting these species. We succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets. Moreover, we developed specific primer sets to identify Neosartorya species, N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex, which are important in food spoilage; these fungi vary in heat resistance and productivity of mycotoxins, depending on the species. PCR using these primer sets did not detect other fungi involved in food spoilage and environmental contamination. These identification methods are rapid and simple with extremely high specificity.

Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans

Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.

Method for Rapid Detection and Identification of Chaetomium and Evaluation of Resistance to Peracetic Acid

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

Difference in FKS1 Gene Sequences between Serotypes A and D of Cryptococcus neoformans

ABSTRACT We compared sequences of the glucan synthase (FKS1) gene in serotypes A and D of Cryptococcus neoformans. Four introns were present in serotype D but not serotype A. PCR with primers that flank these introns permits simple differentiation of serotypes A and D.

Mode of vegetative reproduction of the bipolar budding yeast species Wickerhamomyces pijperi and related strains

To clarify the budding pattern of Wickerhamomyces pijperi, the vegetative cells were observed by scanning electron microscopy. The cells grew by bipolar budding, but cells that budded from the shoulder of a mother cell were occasionally observed. We examined the cell morphology and phylogeny of five strains of Wickerhamomyces sp. isolated in Thailand as well as seven W. pijperi and three Wickerhamomyces sp. strains that were preserved in culture collections. Phylogenetic analysis based on three different nucleotide sequences (D1/D2 domain of 26S rDNA, the actin gene ACT1 and the elongation factor 2 gene EF2) indicated that all the strains belonged to the genus Wickerhamomyces and were neighbours of the type strain W. pijperi NBRC 1290(T). The strains fell into two groups in this analysis. The budding patterns of the strains were carefully observed by staining the bud scars, and these patterns were categorized into three groups: types I-III. Type I included cells that grew by bipolar budding and formed multiple scars, type III included cells that grew by multilateral budding and formed a single scar, and type II included cells that exhibited a mixture of type I and type III patterns. Among the 15 strains, 12 strains, including W. pijperi NBRC 1290(T), mainly exhibited type I or type II budding patterns; these strains belonged to group 1 of the phylogenetic analysis. The remaining three strains, which belonged to group 2, exhibited either type II or type III patterns. Thus the phylogenetic relationship and budding patterns are related. Moreover, some cells also exhibited budding characteristics that were intermediate between bipolar and multilateral budding.

Zygotorulaspora chibaensis sp. nov. and Zygotorulaspora danielsina sp. nov., novel ascomycetous yeast species from tree bark and soil

Multiple isolates belonging to the ascomycetous genus Zygotorulaspora were obtained from forest soils and tree bark in Shiba Prefecture in Japan, and Lake Daniels, Lewis Pass, in New Zealand. Phylogenetic analyses employing combined sequences of the D1/D2 domain and ITS region support the recognition of two new species: Zygotorulaspora chibaensis sp. nov. (type strain PYCC 6970T=CBS 15364T) and Zygotorulaspora danielsina sp. nov. (type strain PYCC 6984T=CBS 15365T). Both species are able to grow on d-xylose and l-arabinose and at 35 °C, unlike Zygotorulaspora florentina and Zygotorulaspora mrakii, the other two species in the genus.