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PubMed2004

NADP-glutamate dehydrogenase activity is increased under hyperosmotic conditions in the halotolerant yeast Debaryomyces hansenii.

Glutamate plays an important role in osmoprotection in various bacteria. In these cases, increased intracellular glutamate pools are not attributable to the NADP-dependent glutamate dehydrogenase (NADP-GDH) or the glutamate synthase, which do not increase their activities under hyperosmotic conditions, but rather to changes in other enzymes involved in glutamate metabolism. We performed a study which indicates that, as opposed to what happens in bacteria, the activity of NADP-GDH is fivefold higher when the halotolerant yeast Debaryomyces hansenii is grown in the presence of 1 M NaCl, compared with growth in media with no added salt. Since purified NADP-GDH activity in vitro was not enhanced by the presence of salt and was more sensitive to ionic strength than the two isoenzymes from S. cerevisiae, increased enzyme synthesis is the most plausible mechanism to explain our results. We discuss the possibility that increased NADP-GDH activity in D. hansenii plays a role in counteracting the inhibitory effect of high ionic strength on the activity of this enzyme.

Ammonium Chloride↗

PubMed2005

Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

Gene Expression Regulation, Fungal↗

PubMed2002

The mitochondrial genome can be altered or lost without lethal effect in the petite-negative yeast Debaryomyces (Schwanniomyces) occidentalis.

The nature of mutations affecting several cytochrome-deficient mutants of Debaryomyces (Schwanniomyces) occidentalis has been characterized. The DR12 mutant, which is deficient in cytochrome b, and the B10Mn mutant, which is deficient in cytochromes b and a, a3, are deleted in the mitochondrial CYB and COX1 genes respectively. The B10 strain, which is partially deficient in cytochrome b, has no detectable change in its mitochondrial DNA and possibly carries nuclear lesion(s). These three mutants, unlike the rho(-) and rho degrees "petite" mutants of Saccharomyces cerevisiae, can still grow on non-fermentable substrates, due to the development of a salicylhydroxamic acid (SHAM)-sensitive alternative pathway linked to phosphorylation at site 1. A gly(-) mutant lacking mtDNA and respiratory capacity has been isolated. For the first time, it is demonstrated that mtDNA can be altered or even lost without lethal consequence in D. occidentalis, although this yeast was classified as a petite-negative species.

Cell Division↗

PubMed2005

Salt-dependent expression of ammonium assimilation genes in the halotolerant yeast, Debaryomyces hansenii.

Debaryomyces hansenii is adapted to grow in saline environments, accumulating high intracellular Na(+) concentrations. Determination of the DhGDH1-encoded NADP-glutamate dehydrogenase enzymatic activity showed that it increased in a saline environment. Thus, it was proposed that, in order to overcome Na(+) inhibition of enzyme activity, this organism possessed salt-dependent mechanisms which resulted in increased activity of enzymes pertaining to the central metabolic pathways. However, the nature of the mechanisms involved in augmented enzyme activity were not analyzed. To address this matter, we studied the expression of DhGDH1 and DhGLN1 encoding glutamine synthetase, which constitute the central metabolic circuit involved in ammonium assimilation. It was found that: (1) expression of DhGDH1 is increased when D. hansenii is grown in the presence of high NaCl concentrations, while that of DhGLN1 is reduced, (2) DhGDH1 expression in Saccharomyces cerevisiae takes place in a GLN3- and HAP2,3-dependent manner and (3) salt-dependent DhGDH1 and DhGLN1 expression involves mechanisms which are limited to D. hansenii and are not present in S. cerevisiae. Thus, salt-dependent regulation of the genes involved in central metabolic pathways could form part of a strategy leading to the ability to grow under hypersaline conditions.

Base Sequence↗

PubMed2005

Debaryomyces hansenii, a highly osmo-tolerant and halo-tolerant yeast, maintains activated Dhog1p in the cytoplasm during its growth under severe osmotic stress.

The HOG pathway is an important mitogen-activated protein kinase (MAPK) signal transduction pathway in Saccharomyces cerevisiae that mediates adaptation of cells to hyper-osmotic stress. Activation of this pathway causes rapid but transient, phosphorylation of the MAPK Hog1p. Phosphorylated Hog1p is rapidly transported to the nucleus that results in the transcription of target genes. The HOG pathway appears to be ubiquitous in yeast. Components of HOG pathway have also been identified in Debaryomyces hansenii, a highly osmotolerant and halotolerant yeast. We have studied activation of HOG pathway in D. hansenii under different stress conditions. Our experiments demonstrated that the pathway is activated by high osmolarity, oxidative and UV stress but not by heat stress. We have provided evidence, for the first time, that D. hansenii maintains phosphorylated Dhog1p in the cytoplasm during its growth under severe osmotic stress.

Cytoplasm↗

PubMed2006

Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata.

Base Sequence↗

PubMed2004

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress.

The comparative analysis of growth, intracellular content of Na+ and K+, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na+ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.

Ascomycota↗

PubMed2006

Colonization of the gastrointestinal tract of the farmed South African abalone Haliotis midae by the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1.

Viable cell counts and/or in situ hybridization were used to determine whether the probionts Vibrio midae SY9, Cryptococcus sp. SS1, and Debaryomyces hansenii AY1 can colonize the gastrointestinal tract of the South African abalone Haliotis midae. The number of culturable probiotic cells reisolated from H. midae fed probiotic-supplemented feed for 3 weeks ranged from 10(6) to 10(7) cfu/g gut material. A significant decrease (P < 0.05) in probiont numbers 2 days after feeding the probiotic-supplemented feed had been halted correlated with a significant decrease (P < 0.05) in intestinal protease and amylase activity. There was a positive correlation between Cryptococcus sp. SS1 and amylase activity (r2= 0.681) and V. midae SY9.8 and protease activity (r2= 0.711) in the H. midae intestine. Although culturable probionts were isolated from abalone that had not been fed probiotic-supplemented feed for a 2-week period, the drop in the number of probiotic cells colonizing the abalone digestive tract 2 days after feeding with the probiotic-supplemented feed had been halted indicates that farmed abalone should be fed probiotic-supplemented feed at least every second day for maximum benefit.

Animal Feed↗

PubMed2004

Regulated expression of green fluorescent protein in Debaryomyces hansenii.

The broad range of environmental conditions under which Debaryomyces hansenii can grow, and its production of lipolytic and proteolytic enzymes, have promoted its widespread use. The present work represents a preliminary characterization of D. hansenii for heterologous expression and secretion of green fluorescent protein (GFP). Six heterologous expression vectors were used to address protein production efficiency under regulated expression conditions. Protein expression in D. hansenii seems to be similar to that in Saccharomyces cerevisiae, with transcription being controlled by almost all of the S. cerevisiae and D. hansenii inducible promoters tested, with the exception of the alcohol dehydrogenase 2 gene promoter from S. cerevisiae. Extracellular protein levels in D. hansenii were lower than in S. cerevisiae when Saccharomyces signal peptides were used.

Alcohol Dehydrogenase↗

PubMed2006

Supplementation requirements of brewery's spent grain hydrolysate for biomass and xylitol production by Debaryomyces hansenii CCMI 941.

The effect of nutrient supplementation of brewery's spent grain (BSG) hydrolysates was evaluated with respect to biomass and xylitol production by Debaryomyces hansenii. For optimal biomass production, supplementation of full-strength BSG hydrolysates required only phosphate (0.5 g l(-1) KH(2)PO(4)), leading to a biomass yield and productivity of 0.60 g g(-1) monosaccharides and 0.55 g l(-1 )h(-1), respectively. Under the conditions studied, no metabolic products other than CO(2) and biomass were identified. For xylitol production, fourfold and sixfold concentrated hydrolysate-based media were used to assess the supplementation effects. The type of nutrient supplementation modulated the ratio of total polyols/total extracellular metabolites as well as the xylitol/arabitol ratio. While the former varied from 0.8 to 1, the xylitol/arabitol ratio reached a maximum value of 2.6 for yeast extract (YE)-supplemented hydrolysates. The increase in xylitol productivity and yield was related to the increase of the percentage of consumed xylose induced by supplementation. The best xylitol yield and productivity were found for YE supplementation corresponding to 0.55 g g(-1) and 0.36 g l(-1 )h(-1), respectively. In sixfold concentrated hydrolysates, providing that the hydrolysate was supplemented, the levels of xylitol produced were similar or higher than those for arabitol. Xylitol yield exhibited a further increase in the sixfold hydrolysate supplemented with trace elements, vitamins and minerals to 0.65 g g(-1), albeit the xylitol productivity was somewhat lower. The effect of using activated charcoal detoxification in non-supplemented versus supplemented sixfold hydrolysates was also studied. Detoxification did not improve polyols formation, suggesting that the hemicellulose-derived inhibitor levels present in concentrated BSG hydrolysates are well tolerated by D. hansenii.

Alcoholic Beverages↗

PubMed2006

Cell wall involvement in the glycerol response to high osmolarity in the halotolerant yeast Debaryomyces hansenii.

Osmotic stress was studied through the induction of the gene coding for glycerol 3-phosphate dehydrogenase (DhGPD1) in the halotolerant yeast Debaryomyces hansenii. This yeast responded to modifications in turgor pressure by stimulating the transcription of DhGPD1 when exposed to solutes that cause turgor stress (NaCl or sorbitol), but did not respond to water stress mediated by ethanol. In contrast to what has been documented to occur in Saccharomyces cerevisiae, D. hansenii protoplasts did not show induction in the transcription of DhGPD1 showing a limitation in their response to solute stress. The results presented indicate that the presence of the cell wall is of significance for the induction of DhGPD1 and hence for osmotic regulation in halotolerant D. hansenii. It appears that the main osmosensor that links high osmolarity with glycerol accumulation may be of a different nature in this yeast.

Ascomycota↗

PubMed2005

Breakthrough invasive infection due to Debaryomyces hansenii (teleomorph Candida famata) and Scopulariopsis brevicaulis in a stem cell transplant patient receiving liposomal amphotericin B and caspofungin for suspected aspergillosis.

An allogeneic stem cell transplant recipient developed pulmonary infiltrates and Aspergillus antigenemia during prophylactic low-dose liposomal amphotericin B. No response to therapy was observed after increasing the dose of liposomal amphotericin B and addition of caspofungin, and breakthrough candidemia developed. Therapy switch to voriconazole did not prevent the development of lethal septic shock. Shortly before death, Scopulariopsis brevicaulis was cultured from bronchial secretions, and positive blood cultures demonstrated persistent candidemia due to Debaryomyces hansenii, teleomorph of Candida famata.

Aged↗

PubMed1978

Properties of alkaline phosphatase of the halotolerant yeast Debaryomyces hansenii.

The molecular weight of a partially purified alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) from the halotolerant yeast Debaryomyces hansenii was estimated to 110,000 by gel filtration. The isoelectric point determined by electrofocusing was at approximately pH 4.4. The enzyme had a broad specificity against phosphomonoesters and also attacked some acid anhydrides. Arsenate, molybdate, and orthophosphate acted as competitive inhibitors. Various metal-binding agents inhibited enzyme activity. A zinc addition almost completely reversed the EDTA inhibition. Magnesium stimulated enzyme activity and was required for maintenance of activity at high concentrations of Na+. Increasing glycerol concentration increased the value of the Michaelis constant (Km) and decreased the maximum velocity (V). Solutions equimolar in KCl and NaCl stimulated enzyme activity by increasing V, whereas the Km was almost unaffected by salt concentration. Enzyme extracted from cells cultured at low salinity was indistinguishable from that of cells grown in the presence of 2.7 M NaCl with respect to several criteria.

Alkaline Phosphatase↗

PubMed1996

The combined effects of environmental conditions on lipolysis of pork fat by lipases of the meat starter culture organisms Staphylococcus xylosus and Debaryomyces hansenii.

The effects of environmental conditions on lipolysis by cell-free extracts from the meat starter culture organisms Staphylococcus xylosus and Debaryomyces hansenii were studied using pork fat emulsions as model systems. For the individual effects of temperature and pH it was found that the optimal conditions for the lipolysis by S. xylosus lipase were 37 degrees C and pH 7.0, and 37 degrees C and pH 6.5 for the lipolysis by D. hansenii lipase. For the combined effects of conditions relevant to meat fermentation, i.e. 10-30 degrees C, pH 4.7-6.0, 2.5-7.5% (w/v) NaCl and incubation times of 2-6 days, the empirical models indicated that temperature, pH and incubation time had important effects on total lipolysis whereas NaCl concentration had little effect. For both cultures lipolysis was strongly inhibited at conditions of meat fermentation compared to optimal conditions. For any set of the conditions which were examined the total lipolysis caused by D. hansenii lipase was lower than that caused by S. xylosus lipase.

Animals↗

PubMed1990

Purification and characterization of glycerol-3-phosphate dehydrogenase (NAD+) in the salt-tolerant yeast Debaryomyces hansenii.

The NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of the salt-tolerant yeast Debaryomyces hansenii was purified by poly(ethylene glycol) precipitation and a combination of chromatographic procedures. The enzyme existed in two forms with different ionic characters and specific activity. On SDS-polyacrylamide gel electrophoresis, both forms yielded one predominant band with an apparent molecular weight of 42,000. The specific activity of the enzyme was dependent on the concentration of the enzyme and on the ionic strength of the dissolving medium. All ions tested stimulated the enzyme activity in the ionic strength range 0-100 mM, with glutamate yielding the highest activity. Above these concentrations, the dehydrogenase showed high tolerance for glutamate in concentrations up to 0.9 M, whereas malate, sulfate and chloride were inhibitory. Enzyme activity showed little sensitivity to the type of cation present and was only slightly affected by 5 M glycerol. The true Km values for the substrates were 6.6 microM for NADH, 130 microM for dihydroxyacetone phosphate, 0.3 mM for NAD and 1.2 mM for glycerol-3-phosphate, and the enzyme showed specificity for these four substrates only. It is proposed that the enzyme functions in cellular osmoregulation by providing glycerol 3-phosphate for the biosynthesis of glycerol, the main compatible solute in D. hansenii, and that the enzyme is well adapted to function in yeast cells exposed to osmotic stress.

Cations↗

PubMed1990

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.

Centrifugation, Density Gradient↗

PubMed2004

Stress responses of linear plasmids from Debaryomyces hansenii.

The triplet linear plasmids pDHL1/2/3 from the salt-tolerant yeast Debaryomyces hansenii TK are localized in the cytoplasm and characterized by a unique feature that they require environmental stressors (0.3 M NaCl or solutes such as sorbitol with equivalent osmolarity) for stable replication and maintenance. The degree of osmolarity dependence of pDHLs was greatly affected by growth temperature of the host cells: the stability of pDHLs was maintained in the absence of osmolarity in cells growing at 25 degrees C, and required osmorarity equivalent to 0.3-1.0 M NaCl on shifting to 30-35 degrees C. Although to less extent, similar osmolarity dependence at high temperatures was observed with another system of D. hansenii linear plasmids. Short-term conditioning of cells to heat or high osmolarity resulted in significant improvement in the plasmid stability, suggesting possible involvement of stress proteins and/or high glycerol level in the stabilization process.

Heat-Shock Response↗

PubMed2005

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface.

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.

Cell Adhesion↗