Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

Revised karyotyping and gene mapping of the Biomphalaria glabrata embryonic (Bge) cell line.

The fresh water snail Biomphalaria glabrata (2n=36) belongs to the taxonomic class Gastropoda (family Planorbidae) and is integral to the spread of the human parasitic disease schistosomiasis. The importance of this mollusc is such that it has been selected as a model molluscan organism for whole genome sequencing. In order to understand the structure and organisation of the B. glabrata's genome it is important that gene mapping studies are established. Thus, we have studied the genomes of two B. glabrata embryonic (Bge) cell line isolates 1 and 2 grown in separate laboratories, but both derived from Eder L. Hansen's original culture from the 1970s. This cell line continues to be an important tool and model system for schistosomiasis and B. glabrata. Using these cell line isolates, we have investigated the genome content and established a revised karyotype based on chromosome size and centromere position for these cells. Unlike the original karyotype (2n=36) established for the cell line, our investigations now show the existence of extensive aneuploidy in both cell line isolates to the extent that the total complement of chromosomes in both greatly exceeds the original cell line's diploid number of 36 chromosomes. The isolates, designated Bge 1 and 2, had modal chromosome complements of 64 and 67, respectively (calculated from 50 metaphases). We found that the aneuploidy was most pronounced, for both isolates, amongst chromosomes of medium metacentric morphology. We also report, to our knowledge for the first time using Bge cells, the mapping of single-copy genes peroxiredoxin (BgPrx4) and P-element induced wimpy testis (piwi) onto Bge chromosomes. These B. glabrata genes were mapped onto pairs of homologous chromosomes using fluorescence in situ hybridization (FISH). Thus, we have now established a FISH mapping technique that can eventually be utilized for physical mapping of the snail genome.

Chromosomal polymorphism in the yeast species Debaryomyces hansenii.

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and beta-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon.

Chromosomal polymorphism in the yeast species Debaryomyces hansenii.

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and beta-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon.

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera.

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.

Molecular characterisation of Hanseniaspora species.

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6-7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.

Use of conventional taxonomy, electrophoretic karyotyping and DNA-DNA hybridization for the classification of fermentative apiculate yeasts.

A taxonomic study was conducted that considered strains of the genera Hanseniaspora/Kloeckera held in the Industrial Yeasts Collection (DBVPG) of the Dipartimento di Biologia Vegetale of the Università di Perugia, Italy. Standard phenotypic as well as molecular criteria were considered in a effort to revisit the classification of these strains, some of which have been in the collection for about 50 years. Results of salient physiological tests showed that some of the DBVPG and type strains could not be identified by current taxonomic keys. Electrophoretic karyotypes were identical for some species, with the type strains of the seven accepted species showing only five distinct chromosomal patterns. DNA-DNA hybridization analyses, using a non-radioactive dot-blot technique, allowed for the distinction of taxa. The taxonomic implications of these results are discussed.

Description of Saccharomyces turicensis sp. nov., a new species from kefyr.

The new species Saccharomyces turicensis sp. nov. isolated from different kefyr grains is described. Although its morphological properties differ, its physiological characteristics come close to those of Saccharomyces bayanus Saccardo and Saccharomyces pastorianus Reess ex E. C. Hansen. However, electrophoretic karyotyping and restriction fragment length polymorphism of the internal transcribed spacer region yield clear differences. Sequences (270 nucleotides) of the D2 domain at the 5'-terminal end of the large subunit ribosomal RNA gene reveal 98.0% identity with Saccharomyces exiguus. Since strains of a particular yeast species usually show less than 1% substitution in the D2 domain, the yeast in question is considered to be a new species. The name Saccharomyces turicensis is proposed indicating the place Zürich (Turicum in Latin) where the yeast had been isolated.

Cytogenetic studies in leprosy patients before and after chemotherapy.

The frequencies of chromosome aberrations and sister chromatid exchanges (SCEs), cell proliferation kinetics and mitotic indices were studied in peripheral blood lymphocyte cultures of leprosy patients both before and after chemotherapy. The differences in the frequencies of chromosome aberrations and SCEs between controls, paucibacillary and multibacillary patients were found to be statistically highly significant (P less than 0.001). The extent of cytogenetic damage seemed to depend on the severity of the disease. Lymphocytes of untreated leprosy patients showed a low mitotic index and a slow rate of cell proliferation. Following combined treatment with dapsone and rifampicin there was an increase, but to a lesser degree (P less than 0.01), in the frequency of SCEs and chromosome aberrations while the drug combination of dapsone, rifampicin and clofazamine had a nonmutagenic effect on chromosomes of the patient. Furthermore, after drug treatment, the cell proliferation rate and mitotic indices in paucibacillary patients were comparable to that of controls. These results indicate the clastogenic potency of Mycobacterium leprae and the remedial effects that follow therapeutic drug treatment.

A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism.

The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed. This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation. Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli. The glycerol overproducting phenotype of two isolates was confirmed by chemical analysis.