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Bone infection caused by debaryomyces hansenii in a normal host: a case report.

Debaryomyces hansenii was isolated from a cystic lesion in the distal tibia of a healthy 23-year-old woman. Ascospores were demonstrated when the organism was incubated at 25 but not at 30 degrees C. Electron microscopy was necessary to demonstrate the warty surface of the ascospore wall. Growth was inhibited in vitro by low concentrations of amphotericin B, 5-fluorocytosine, miconazole, and ketoconazole. Four previous cases of infection caused by Debaryomyces spp. and one caused by the related fungus Torulopsis candida were reviewed.

Adult↗

Xylitol production from wood hydrolyzates by entrapped Debaryomyces hansenii and Candida guilliermondii cells.

Debaryomyces hansenii cells were entrapped in Ca-alginate beads and used for producing xylitol from wood hydrolyzates. Batch experiments showed that bioconversion was severely hindered when Ca-alginate beads were hardened with Al3+ solutions. As an alternative to Al3+ hardening, the improvements in both mechanical stability of bioparticles and fermenting ability of the immobilized system derived from using increased concentrations of sodium alginate were assessed. The best results were obtained using a 4% (w/v) Na-alginate solution in the gelification step. This concentration was selected to perform continuous fermentations in a packed-bed reactor using raw or charcoal-treated hydrolyzates (15.5 g of xylose/L) with two different yeasts: Candida guilliermondii and Debaryomyces hansenii. With a final cell concentration of about 50 g of cells/L (0.075 g of cells/g of beads), the volumetric productivities reached with these yeasts in media made from charcoal-treated hydrolyzates were 0.58 and 0.91 g/L.h, respectively.

Journal Article↗

A beta-glucuronidase-based agar medium for the differential detection of the yeast Debaryomyces hansenii from foods.

A selective and differential solid medium, Debaryomyces differential medium (DDM), was used for the isolation of Debaryomyces hansenii. This medium is formulated to allow detection of the beta-glucuronidase enzyme using the chromogenic substrate magenta-glucuro.CHA (5Br-6Cl-3indolyl-beta-D-glucuronide, cyclohexylammonium salt). Of the more than 120 microorganisms tested, including yeasts, bacteria, and a filamentous fungus, only D. hansenii produced violet colonies, thus permitting its easy discrimination from other organisms. When quality assessment tests were performed, optimal productivity and selectivity were obtained for D. hansenii. The medium was also satisfactory when used to test naturally contaminated food products.

Chromogenic Compounds↗

["Oxygen regulation" of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress].

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide- and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by "the hypoxia genes" and activated (induced) under microaerobic conditions.

Anaerobiosis↗

Flocculation of the yeast Candida famata (Debaryomyces hansenii): an essential role for peptone.

Aggregation of Candida famata (Debaryomyces hansenii) is consistent with being a form of lectin-mediated yeast flocculation. Flocculation of C. famata is unusual in that it requires the presence of peptone, either in the growth medium or added later to harvested cells in buffer. Flocculation after peptone addition was rapid, being largely complete within 10 min. Heat-killed cells also flocculated, arguing for direct participation of peptone in the flocculation binding mechanism. Flocculent C. famata cells progressively lost the ability to flocculate when washed with EDTA. Flocculation was fully restored by peptone addition; calcium addition was without effect. C. famata cells were able to agglutinate erythrocytes in the presence or absence of peptone. Pronase E-treated yeast lost both the ability to haemagglutinate and self-flocculate. Haemagglutination was not diminished by progressive EDTA washing, suggesting that surface lectins remained present and active on the yeast cell walls. Non-flocculating C. famata cells mutually flocculated with non-flocculent Schizosaccharomyces pombe cells, shown to have surface-exposed galactose residues. Mutual flocculation was lost following treatment of C. famata with Pronase E. It was concluded that the cell wall of C. famata contains lectins enabling haemagglutination and mutual flocculation but lacks carbohydrate receptors for these lectins. This yeast self-flocculates only via bridging multi-valent carbohydrates; these being present in peptone.

Abrin↗

The linear plasmid pDHL1 from Debaryomyces hansenii encodes a protein highly homologous to the pGKL1-plasmid DNA polymerase.

Both the linear plasmids, pDHL1 (8.4 kb) and pDHL2 (9.2 kb), of Debaryomyces hansenii TK require the presence of a third linear plasmid pDHL3 (15.0 kb) in the same host cell for their replication. A 3.5 kb Bam HI-PstI fragment of pDHL1 strongly hybridized by Southern analysis to the 3.5 kb NcoI-AccI fragment of pDHL2, suggesting the importance of this conserved region in the replication of the two smaller pDHL plasmids. The 4.2 kb pDHL1 fragment containing the above hybridized region was cloned and sequenced. The results showed that the cloned pDHL1 fragment encodes a protein of 1000 amino acid residues, having a strong similarity to the DNA polymerase coded for by ORF1 of the killer plasmid pGKL1 from Kluyveromyces lactis. The catalytic and proof-reading exonuclease domains as well as terminal protein motif were well conserved as in DNA polymerases of pGKL1 and other yeast linear plasmids. Analysis of the cloned fragment further showed that pDHL1 encodes a protein partly similar to the alpha subunit of the K. lactis killer toxin, although killer activity was not known in the DHL system. Analysis of the 5' non-coding region of the two above pDHL1-ORFs reveal the presence of the upstream conserved sequence similar to that found upstream of pGKL1-ORFs. The possible hairpin loop structure was also found just in front of the ATG start codon of the pDHL1-ORFs like pGKL1-ORFs. Thus the cytoplasmic pDHL plasmids were suggested to possess a gene expression system comparable to that of K. lactis plasmids.

Amino Acid Sequence↗

Cloning and sequencing of a cDNA encoding a copper-zinc superoxide dismutase enzyme from the marine yeast Debaryomyces hansenii.

Cu-Zn superoxide dismutase (SOD-1) is a ubiquitously occurring eukaryotic enzyme with a variety of important effects on respiring organisms. A gene (dhsod-1) encoding a Cu-Zn superoxide dismutase of the marine yeast Debaryomyces hansenii was cloned using mRNA by the RT-PCR technique. The deduced amino-acid sequence shows approximately 70% homology with that of cytosolic superoxide dismutase from Saccharomyces cerevisiae and Neurospora crassa, as well as lower homologies (between 55 and 65%) with the corresponding enzyme of other eukaryotic organisms, including human. The gene sequence encodes a protein of 153 amino acids with a calculated molecular mass of 15-92 kDa, in agreement with the observed characteristics of the purified protein from D. hansenii.

Amino Acid Sequence↗

Copper-zinc superoxide dismutase from the marine yeast Debaryomyces hansenii.

We have isolated the cytosolic form of Cu-Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii. This enzyme has a subunit mass of 18 kDa. The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5.14-4.0 and 1.6-1.8. The enzyme preparation had a remarkably strong stability at pH 6.0-7.0, surviving boiling for 10 min without losing more than 60% of activity. On Western blots, this enzyme was recognized by antibodies raised in rabbits against D. hansenii extracts, while only a weak cross-reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu-Zn SODs. In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S. cerevisiae Cu-Zn SOD.

Amino Acid Sequence↗

Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1Delta strain of Saccharomyces cerevisiae.

The HOG1 gene encodes a MAP kinase that plays an essential role in maintaining water homeostasis in the yeast Saccharomyces cerevisiae. A gene homologous to S. cerevisiae HOG1 has been isolated from a highly salt-tolerant yeast, Debaryomyces hansenii, by phenotypic complementation. DNA sequencing of the clone revealed the presence of an open reading frame encoding a protein 387 amino acids long. The deduced amino acid sequence showed very high similarity with homologous genes identified from S. cerevisiae, Candida albicans and Zygosaccharomyces rouxii. In addition, it has also TGY motif characteristics of hyperosmolarity-activated MAP kinases. The Genbank Accession No. of this sequence is AF185278.

Amino Acid Sequence↗

Subcellular level variation in protein accumulation of the halophylic yeast Debaryomyces hansenii grown under conditions of high osmolarity.

We have analyzed electrophoretic profiles of polypeptides extracted from various cell compartments of the yeast Debaryomyces hansenii, cultured under high osmolarity and under control conditions. We tested the effect of high concentrations of solutes with an osmotic component (sorbitol), and with osmotic and ionic components combined (NaCl or KCl). Densitometric analyses of the extracted polypeptides indicated that the stressing solutes had a differential effect on the relative concentration of total proteins as well as in proteins extracted from three subcellular compartments. Sorbitol caused a significant decrease in the concentration of various polypeptides associated with the mitochondria and the cytoplasm. By contrast, sodium ions elicited marked increases in concentration in four cytoplasmic polypeptides. KCl did not have a major effect in any of the subcellular compartments. Polypeptides were grouped as having a general osmotic response, or as having a response apparently modulated by the particular ionic environment of the growth medium. In all treatments, the number of polypeptides with an increase in their relative concentration was roughly similar to the number of polypeptides with a decrease in concentration, both relative to controls. Our results agree with previous observations on the complexity of the osmoregulatory response involving proteins whose concentration depends on the solute causing the stress. The results also indicate that subcellular compartments respond differently to stressors.

Culture Media↗

Influence of temperature and pH on xylitol production from xylose by Debaryomyces hansenii.

The production of xylitol from concentrated synthetic xylose solutions (S(o) = 130-135 g/L) by Debaryomyces hansenii was investigated at different pH and temperature values. At optimum starting pH (pH(o) = 5.5), T = 24 degrees C, and relatively low starting biomass levels (0.5-0.6 g(x)/L), 88% of xylose was utilized for xylitol production, the rest being preferentially fermented to ethanol (10%). Under these conditions, nearly 70% of initial carbon was recovered as xylitol, corresponding to final xylitol concentration of 91.9 g(P)/L, product yield on substrate of 0.81 g(P)/g(S), and maximum volumetric and specific productivities of 1.86 g(P)/L x h and 1.43 g(P)/g(x) x h, respectively. At higher and lower pH(o) values, respiration also became important, consuming up to 32% of xylose, while negligible amounts were utilized for cell growth (0.8-1.8%). The same approach extended to the effect of temperature on the metabolism of this yeast at pH(o) = 5.5 and higher biomass levels (1.4-3.0 g(x)/L) revealed that, at temperatures ranging from 32-37 degrees C, xylose was nearly completely consumed to produce xylitol, reaching a maximum volumetric productivity of 4.67 g(P)/L x h at 35 degrees C. Similarly, both respiration and ethanol fermentation became significant either at higher or at lower temperatures. Finally, to elucidate the kinetic mechanisms of both xylitol production and thermal inactivation of the system, the related thermodynamic parameters were estimated from the experimental data with the Arrhenius model: activation enthalpy and entropy were 57.7 kJ/mol and -0.152 kJ/mol x K for xylitol production and 187.3 kJ/mol and 0.054 kJ/mol x K for thermal inactivation, respectively.

Carbon↗

Study of inulase from Debaryomyces phaffii Capriotti.

Debaryomyces phaffii possesses an inulinase activity which can be induced by growth on beta-fructosidase and particularly on inulin. The enzyme is located in the cell wall but is easily excreted into the culture medium. Maximum activity on inulin is observed at pH 4 and 50 degrees C. The Km on inulin is 1.2 X 10(-2) M. The enzyme breaks down inulin by splitting off terminal fructosyl units and it is active on sucrose and raffinose.

Ascomycota↗

Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+).

A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.

Amino Acid Sequence↗

Sodium and potassium transport in the halophilic yeast Debaryomyces hansenii.

Debaryomyces hansenii, a halophile yeast found in shallow sea waters and salty food products grows optimally in 0.6 M of either NaCl or KCl, accumulating high concentrations of Na(+) or K(+). After growth in NaCl or KCl, a rapid efflux of either accumulated cation was observed if the cells were incubated in the presence of KCl or NaCl, respectively, accompanied by a slower accumulation of the cation present in the incubation medium. However, a similar, rapid efflux was observed if cells were incubated in buffer, in the absence of external cations. This yeast shows a cation uptake activity of both (86)Rb(+) and (22)Na(+) with saturation kinetics, and much higher affinity for (86)Rb(+) than for (22)Na(+). The pH dependence of the kinetics constants was similar for both cations, and although K(m) values were higher at pH 8.0, there was also an increase in the V(max) values. The accumulation of (22)Na(+) was found to be increased in cells grown in the presence of 0.6 M NaCl. (86)Rb(+) was also accumulated more in these cells, but to a slightly greater extent. The inhibition kinetics of the uptake of (22)Na(+) by K(+), and that of (86)Rb(+) by Na(+) was found to be non-competitive. It can be concluded that Na(+) in D. hansenii is not excluded but instead, its metabolic systems must be resistant to high salt concentrations.

Culture Media↗

Candida famata (Debaryomyces hansenii) DNA sequences containing genes involved in riboflavin synthesis.

Previously cloned Candida famata (Debaryomyces hansenii) strain VKM Y-9 genomic DNA fragments containing genes RIB1 (codes for GTP cyclohydrolase II), RIB2 (encodes specific reductase), RIB5 (codes for dimethylribityllumazine synthase), RIB6 (encodes dihydroxybutanone phosphate synthase) and RIB7 (codes for riboflavin synthase) were sequenced. The derived amino acid sequences of C. famata RIB genes showed extensive homology to the corresponding sequences of riboflavin synthesis enzymes of other yeast species. The highest identity was observed to homologues of D. hansenii CBS767, as C. famata is the anamorph of this hemiascomycetous yeast. The D. hansenii CBS767 RIB3 gene encoding specific deaminase was cloned. This gene successfully complemented riboflavin auxotrophy of the rib3 mutant of flavinogenic yeast, Pichia guilliermondii. Putative iron-responsive elements (potential sites for binding of the transcription factors Fep1p or Aft1p and Aft2p) were found in the upstream regions of some C. famata and D. hansenii RIB genes. The sequences of C. famata RIB genes have been submitted to the EMBL data library under Accession Nos AJ810169-AJ810173.

Amino Acid Sequence↗

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.

Adenosine Diphosphate↗

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations.

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.

Fungal Proteins↗

Glycolytic sequence and respiration of Debaryomyces hansenii as compared to Saccharomyces cerevisiae.

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.

Alcohol Dehydrogenase↗