DNA probes specific for the yeast species Debaryomyces hansenii: useful tools for rapid identification

We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences. These sequences were first identified in a survey of D. hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested. Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767. The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese. F01pro and M18pro hybridized to the DNA of all D. hansenii var. hansenii tested, but not to DNA of other yeast species including the closely related strain of D. hansenii var. fabryii CBS 789. Hybridization patterns of F01pro and M18pro on digested genomic DNA of D. hansenii indicated that the sequences were repeated in the genome of all D. hansenii var. hansenii tested, and gave distinct polymorphic patterns. The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme. F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D. hansenii and, together with M18pro, simplifies the study of this species in complex flora.     

Sequence-based identification of species belonging to the genus Debaryomyces

In the present study we assessed the identification by sequence analysis of the 15 species belonging to the genus Debaryomyces. We found that the following species can be identified both quickly and correctly by direct sequence comparison of the ribosomal 5.8S-ITS region: D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae. In contrast, the species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D. pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae showed high sequence similarity in ribosomal regions with one or several species. In these cases, sequence comparison of the ACT1 gene is proposed to ensure unequivocal strain designation.     

PCR-RFLP analysis of the IGS region of rDNA: a useful tool for the practical discrimination between species of the genus Debaryomyces

The amplification by PCR of the intergenic spacer region (IGS) of rDNA followed by restriction fragment length polymorphism (RFLP) analysis was evaluated as a potential method for discriminating the 16 species belonging to the genus Debaryomyces. The digestion of this region with some or all the enzymes used in this study (HapII, HhaI and MboI) produced species-specific patterns that permitted differentiation of the species in the genus. With the exception of Debaryomyces vanrijiae, the technique was also efficient for␣distinguishing the varieties in the species Debaryomyces hansenii (var. hansenii, var. fabryi), Debaryomyces occidentalis (var. occidentalis, var. persoonii) and Debaryomyces polymorphus (var. africanus, var. polymorphus), respectively. PCR-RFLP analysis of the IGS region of rDNA is proposed as a clear and reproducible technique for the practical discrimination of species of the yeast genus Debaryomyces.     

Alcohol-based quorum sensing plays a role in adhesion and sliding motility of the yeast Debaryomyces hansenii

The yeast Debaryomyces hansenii was investigated for its production of alcohol-based quorum sensing (QS) molecules including the aromatic alcohols phenylethanol, tyrosol, tryptophol and the aliphatic alcohol farnesol. Debaryomyces hansenii produced phenylethanol and tyrosol, which were primarily detected from the end of exponential phase indicating that they are potential QS molecules in D.?hansenii as previously shown for other yeast species. Yields of phenylethanol and tyrosol produced by D.?hansenii were, however, lower than those produced by Candida albicans and Saccharomyces cerevisiae and varied with growth conditions such as the availability of aromatic amino acids, ammonium sulphate, NaCl, pH and temperature. Tryptophol was only produced in the presence of tryptophane, whereas farnesol in general was not detectable. Especially, the type strain of D.?hansenii (CBS767) had good adhesion and sliding motility abilities, which seemed to be related to a higher hydrophobicity of the cell surface of D.?hansenii (CBS767) rather than the ability to form pseudomycelium. Addition of phenylethanol, tyrosol, tryptophol and farnesol was found to influence both adhesion and sliding motility of D.?hansenii.     

Characterization of β-glucosidase activity in yeasts of oenological origin

I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40C. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.     

Dynamics and characterization of yeasts during natural fermentation of Italian sausages

A multiphasic approach was used to investigate the yeast ecology in Italian fermented sausages. Culture-dependent and -independent methods were applied to identify the yeast species during the maturation process and to characterize the numerically dominant species. Plating analysis and subsequent molecular identification of the isolates highlighted the dominance of Debaryomyces hansenii, but at least other three yeast species -Candida zeylanoides, Pichia triangularis and Metschnikowia pulcherrima - contributed to the fermentation as well. Direct denaturing gradient gel electrophoresis analysis confirmed that D. hansenii was the main yeast species present and its activity was also demonstrated. No other yeasts species were detected on the direct denaturing gradient gel electrophoresis gels, whereas DNA of Penicillium farinosum, Penicillium viridicatum and Mucor racemosus were present. Molecular characterization by RAPD-PCR analysis of the D. hansenii isolates demonstrated a shift in its population from the beginning to the end of the maturation of the sausages. Strains present during the early stages of the fermentation were grouped in clusters that differed from those isolated in the final phases of the maturation, underlining the genetic differences between these two populations of D. hansenii. However, all the isolates were able to grow in the presence of 3.5% sodium chloride and at 10 degrees C, evidence that these parameters did not select the species present at the end of the maturation period.     

Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.     

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

Biosensor analyzer for BOD index express control on the basis of the yeast microorganisms Candida maltosa, Candida blankii, and Debaryomyces hansenii

The parameters of biosensors based on the yeast strains Candida maltosa VKM Y-2359, Candida blankii VKM Y-2675, and Debaryomyces hansenii VKM Y-2482 for biochemical oxygen demand (BOD) detection are compared. The catalytic activity of the strains was analyzed in relation to the growth phase. The possibility of using D. hansenii as a basis for receptor element of a biosensor for BOD detection in municipal and biotechnological wastewaters was shown.     

Yeasts Associated with Various Amazonian Native Fruits

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Key words: yeast diversity, fruit, Amazonia, PCR-RFLP, 5.8S-ITS     

Oxidative stress sensitivity in Debaryomyces hansenii

Debaryomyces hansenii is an osmotolerant and halotolerant yeast of increasing interest for fundamental and applied research. In this work, we have performed a first study on the effect of oxidative stress on the performance of this yeast. We have used Saccharomyces cerevisiae as a well-known reference yeast. We show that D. hansenii is much more susceptible than S. cerevisiae to cadmium chloride, hydrogen peroxide or 1,4-dithiothreitol. These substances induced the formation of reactive oxygen species (ROS) in both yeasts, the amounts measured being significantly higher in the case of D. hansenii . We also show that NaCl exerted a protective effect against oxidative stress in Debaryomyces , but that this was not the case in Saccharomyces because sodium protected that yeast only when toxicity was induced with cadmium. On the basis of the present results, we raised the hypothesis that the sensitivity to oxidative stress in D. hansenii is related to the high amounts of ROS formed in that yeast and that observations such as low glutathione amounts, low basal superoxide dismutase and peroxidase activities, decrease in ATP levels produced in the presence of ROS inducers and high cadmium accumulation are determinants directly or indirectly involved in the sensitivity process.     

Draft genome sequence of salt-tolerant yeast Debaryomyces hansenii var. hansenii MTCC 234

Debaryomyces hansenii is one of the most halotolerant species of yeast, and the genome sequence of D. hansenii strain CBS767 is already available. Here we report the 11.46-Mb draft genome of D. hansenii strain MTCC 234, which is even more halotolerant than strain CBS767. Comparative analysis of these sequences would definitely provide further insight into the halotolerance of this yeast.     

Debaryomyces psychrosporus sp. nov., a yeast species from a Venezuelan cave

Three yeast strains, which are phenotypically indistinguishable from Debaryomyces hansenii, were recovered from secondary mineral deposits (stalactites and stromatolites) obtained in the Crystal Eyes Cave, Roraima Tepui Mountain, Venezuela. Analyses of the D1/D2 domains of the LSU rRNA gene as well as the concatenated sequences of the nearly entire SSU rRNA gene, the ITS regions and the D1/D2 domains of the LSU rRNA gene confirmed the placement of these strains in the genus Debaryomyces, but relationship with all valid species of D. hansenii complex was distant. Based on the observed considerable sequence divergence the three strains are proposed as a new species, D. psychrosporus sp. nov., with the type strain NCAIM Y.01972(T) (CBS 11845(T), NRRL Y-48723(T)).     

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.     

Fourier-transform infrared microspectroscopy,a novel and rapid tool for identification of yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

A survey of yeasts in traditional sausages of southern Italy

The evolution of the yeast population during manufacturing and ripening of 'salsiccia sotto sugna', a typical salami of the Lucania region (southern Italy), was investigated. Four different batches, produced in four farms in Lucania, were studied. Each batch showed a specific yeast population, and the most frequently isolated yeasts belonged to Debaryomyces hansenii and its anamorph Candida famata, and Rhodotorula mucilaginosa. Yarrowia lipolytica was isolated from three sausage batches. The Y. lipolytica isolates were further characterised, in particular for their lipolytic activity on pork fat. Lipolytic activity was maximal at pH 5.5, with oleic and palmitic acids as major free fatty acids produced. The use of randomly amplified polymorphic DNA-polymerase chain reaction allowed the detection of a high genetic heterogeneity among the isolates phenotypically assigned to the species Y. lipolytica.     

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.