Yeast communities and genetic polymorphism of Saccharomyces cerevisiae strains associated with artisanal fermentation in Brazil

Yeast communities and genetic polymorphism of prevalent Saccharomyces cerevisiae strains isolated from the spontaneous fermentation of the sugarcane juice during the production of aguardente in three distilleries in the state of Minas Gerais, Brazil, were studied. S. cerevisiae was the prevalent species during the process of aguardente production, but Schizosaccharomyces pombe was predominant in old fermentations in one distillery. Transient yeast species were found in a variable number, probably due to the daily addition of sugarcane juice, and they were different for each of the three distilleries studied. PFGE and PCR analysis of the predominant strains of S. cerevisiae isolated from the fermented must showed a high degree of genetic polymorphism among the three distilleries. A high molecular variability of S. cerevisae strains was also observed among strains isolated from the same vat at different fermentation ages. Our results showed that there was a succession of geneticly different strains of S. cerevisae during the process of aguardente production.     

Polymorphism detection among wild Saccharomyces cerevisiae strains of different wine origin

In this study, wild Saccharomyces cerevisiae strains, isolated from spontaneously fermenting grapes of different varieties and origins, were submitted to genetic analysis using different molecular techniques, such as amplification of genes coding for cell wall proteins and containing minisatellite-like sequences, karyotyping, mtDNA-RFLP, and analysis of the δ region. The lowest discriminative power was obtained by minisatellites analysis, in particular the amplification of AGA1 genes. Karyotyping and mtDNA-RFLP analysis yielded the same differentiation among the strains, whereas the PCR amplification of δ sequences resulted the best method as it was fast and it showed a very high discriminative power. In any case, it has to be underlined that some strains, showing the same delta profiles, exhibited a different mtDNA restriction profile and electrophoretic karyotype, suggesting that more than one molecular marker is required for reliable strain discrimination. Although the techniques used revealed a different resolution power, they all revealed a genetic relationship among strains isolated from spontaneous fermentation of grapes of different origins. In fact, none of the typing methods was able to discriminate some strains isolated from different areas.     

Saccharomyces bayanus var. uvarum in Tokaj wine-making of Slovakia and Hungary

Using genetic hybridisation analysis and molecular karyotyping we revealed an association of Saccharomyces bayanus var. uvarum species with Tokaj wine-making. Along with identification of Saccharomyces strains isolated by E. Minárik in Slovakia, the composition of Tokaj populations in Hungary was studied. Twenty-eight Hungarian Saccharomyces strains were analysed in terms of karyotype. The majority of strains belong to S. bayanus var. uvarum. Two non-identified Saccharomyces strains were found to be polyploid according to their complex karyotype patterns.     

The molecular genetic differentiation of cultured Saccharomyces strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that baker's yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae and S. bayanus var. uvarum hybrids. The molecular karyotyping of baker's, brewer's, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)5 can be used to study the populations of cultured S. cerevisiae strains.     

Molecular Relationships Between Saccharomyces cerevisiae Strains Involved in Winemaking from Mendoza, Argentina

Three molecular typing techniques were applied to assess the molecular relationships of Saccharomyces cerevisiae strains isolated from winery equipment, grapes, and spontaneous fermentation in a cellar located in “Zona Alta del Río Mendoza” (Argentina). In addition, commercial Saccharomyces strains widely used in this region were also included. Interdelta PCR typing, mtDNA restriction analysis, and microsatellite (SSR) genotyping were applied. Dendrograms were constructed based on similarity among different patterns of bands. The combination of the three techniques discriminated 34 strains among the 35 isolates. The results of this study show the complex relationships found at molecular level among the isolates that share the same ecological environment, i.e., the winemaking process. With a few exceptions, the yeast isolates were generally clustered in different ways, depending on the typing technique employed. Three clusters were conserved independently of the molecular method applied. These groups of yeasts always clustered together and had high degree of similarity. Furthermore, the dendrograms mostly showed clusters combining strains from winery and fermentation simultaneously. Most of the commercial strains included in this study were clustered separately from the other isolates analyzed, and just a few of them grouped with the strains mainly isolated from spontaneous fermentation. Only one commercial strain was clustered repetitively with a noncommercial strain isolated from spontaneous fermentation in the three dendrograms. On the other hand, this study has demonstrated the importance of selecting an appropriate molecular method according to the main objectives of the research.     

Characterization of the genome of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from red berry wines

Using restriction analysis of noncoding rDNA regions, multiplex PCR, and molecular karyotyping, we have examined Saccharomyces strains isolated from red berry wine materials in Russia, Belarus, and Ukraine. According to the molecular analysis, all strains belong to the species S. cerevisiae. A correlation was revealed between microsatellite fingerprints of the strains and the source of their isolation. The strains isolated from juices and from the surface of different berries showed distinct PCR profiles. The genome compositions of interspecific Saccharomyces hybrids of natural and laboratory origin were studied.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

Combined application of methods to taxonomic identification of Saccharomyces strains in fermenting botrytized grape must

AIMS: Although numerous physiological and molecular methods have been proposed for yeast taxonomy, the unambiguous separation of Saccharomyces sensu stricto species in natural samples is still an incompletely resolved issue. In this study the power of various methods was compared in the identification of strains isolated from fermenting botrytized grape musts. METHODS AND RESULTS: Conventional taxonomic and physiological tests and molecular methods developed for rapid identification were used. CONCLUSIONS: None of the methods tested was sufficiently powerful. However, the combination of electrophoretic karyotyping and the PCR-RFLP of MET2 with growth tests at 10 and 37 degrees C provided results sufficient for species identification of Saccharomyces wine strains which were not interspecific hybrids or recombinants. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed combination of molecular and physiological methods allows specific taxonomic identification and separation of Saccharomyces wine strains without extensive genetic and molecular analysis. The proposed combined approach can also identify hybrids and recombinants.     

Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.     

Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.     

Molecular genetic characterization of the yeast Lachancea kluyveri

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the alpha- galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79-100%) of their MEL genes. The phylogenetic analysis of all of the known alpha-galactosidases in the "Saccharomyces" clade showed that the MEL genes of the yeasts L. kluyveri. L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains.

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.     

PCR–DGGE differentiation of strains of Saccharomyces sensu stricto

A quick molecular biology method based on the polymerase chain reaction (PCR) and Denaturing Gradient Gel Electrophoresis (DGGE) was developed for distinguishing strains belonging to the Saccharomyces sensu stricto group. Differentiation was obtained between S. cerevisiae, S. paradoxus and S. bayanus / S. pastorianus although no distinction was possible between S. bayanus and S. pastorianus using the amplification of the ITS regions. The ability to distinguish between different strains of the Saccharomyces sensu stricto group could allow for a better understanding of the ecology of these species on grapes as well as in musts and wines and the method developed can be useful for the quick identification of Saccharomyces sensu stricto strains from numerous isolates.     

Molecular genetic identification of Saccharomyces sensu stricto strains from African sorghum beer

Genetic relationships of 24 phenotypically different strains isolated from sorghum beer in West Africa and the type cultures of the Saccharomyces sensu stricto species were investigated by universally primed polymerase chain reaction (PCR) analysis, microsatellite fingerprinting and PCR-restriction fragment length polymorphism (RFLP) of the ribosomal internal transcribed spacers. The results demonstrate that internal transcribed spacer (ITS) PCR-RFLP analysis with the endonucleases HaeIII, HpaII, ScrFI and TaqI is useful for discriminating S. cerevisiae, S. kudriavzevii, S. mikatae from one another and from the S. bayanus/S. pastorianus and S. cariocanus/S. paradoxus pairs. The sorghum beer strains exhibited the same restriction patterns as the type culture of S. cerevisiae CBS 1171. PCR profiles generated with the microsatellite primer (GTG)(5) and the universal primer N21 were almost identical for all isolates and strain CBS 1171. Despite phenotypic peculiarities, the strains involved in sorghum beer production in Ghana and Burkina Faso belong to S. cerevisiae. However, based on sequencing of the rDNA ITS1 region and Southern hybridisation analysis, these strains represent a divergent population of S. cerevisiae.     

ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus

The present study was designed to evaluate the usefulness of two novel molecular typing methods, amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) and the PCR melting profile (PCR MP), for Staphylococcus aureus strain differentiation. Thirty-seven S. aureus strains isolated from patients with a history of furunculosis were studied. The strains were identified by determining several phenotypic properties and were genotyped using three differentiation methods: macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE), ADSRRS-fingerprinting, and PCR MP technique. In some cases the results obtained showed that the S. aureus isolated from the nose was identical to the one from the furuncle of the same patient. The same genotype was also identified for S. aureus strains isolated from two different members of a family with a history of recurrent furunculosis, although the active lesions were present in only one of them when the investigation was done. Results from strain genotyping illustrated that the recently developed ADSRRS-fingerprinting and PCR MP techniques are useful for studies of intraspecies genetic relatedness of S. aureus strains. They are as effective in discriminating closely related strains as the PFGE method, which is currently considered to be "the gold standard" for epidemiological studies.     

Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards

AIMS: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. METHODS AND RESULTS: A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. CONCLUSION: Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.     

General evidence supporting the hypothesis that Saccharomyces cerevisiae vaginal isolates originate from food industrial environments

Saccharomyces cerevisiae strains isolated from pregnant women were identified and characterized by molecular techniques which disclosed a wide chromosomal variability and possible segregations due to sporulation. The morphological analysis showed that very few strains were able to sporulate and generate pseudohyphae, whereas none produced proteases, raising some doubts on the importance of these characters in strain pathogenicity. The analysis of ethanol production revealed that these strains are quite similar to those found in fermentative plants, suggesting a possible derivation from the food industrial environment. Since the absence of relevant amounts of sugar does not confer selective advantage to strong fermentative metabolisms, these findings suggest that a metabolic adaptation to the vaginal environment did not occur yet.     

Engineering of baker's yeasts, E. coli and Bacillus hosts for the production of Bacillus subtilis Lipase A

Lipases are versatile biocatalists showing multiple applications in a wide range of biotechnological processes. The gene lipA coding for Lipase A from Bacillus subtilis was isolated by PCR amplification, cloned and expressed in Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis strains, using pBR322, YEplac112 and pUB110-derived vectors, respectively. Lipase activity analysis of the recombinant strains showed that the gene can be properly expressed in all hosts assayed, this being the first time a lipase from bacterial origin can be expressed in baker's S. cerevisiae strains. An important increase of lipase production was obtained in heterologous hosts with respect to that of parental strains, indicating that the described systems can represent a useful tool to enhance productivity of the enzyme for biotechnological applications, including the use of the lipase in bread making, or as a technological additive.     

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.     

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques

AIMS: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. METHODS AND RESULTS: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. CONCLUSIONS: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.