Glycerol metabolism and osmoregulation in the salt-tolerant yeast Debaryomyces hansenii.

A glycerol-nonutilizing mutant of the salt-tolerant yeast Debaryomyces hansenii was isolated. When subjected to salt stress the mutant produced glycerol, and the internal level of glycerol increased linearly in proportion to increases of external salinity as in the wild-type strain. However, at increased salinity the mutant showed a more pronounced decrease of growth rate and growth yield and lost more glycerol to the surrounding medium than did the wild type. Uptake experiments showed glycerol to be accumulated against a strong concentration gradient, and both strains displayed similar kinetic parameters for the uptake of glycerol. An examination of enzyme activities of the glycerol metabolism revealed that the apparent Km of the sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5) was increased 330-fold for sn-glycerol 3-phosphate in the mutant. Based on the findings, a scheme for the pathways of glycerol metabolism is suggested.     

Growth under alkaline conditions of the salt-tolerant yeast Debaryomyces hansenii IFO10939

A salt-tolerant yeast Debaryomyces hansenii IFO 10939, which is able to grow at pH 10.0, was isolated and characterized. IFO 10939 had the ability of maintaining intracellular pH. The in vivo activation of plasma membrane ATPase was observed in cells grown at pH 6.2 during conditioning in buffer at pH 9.0. Alkalification of growth medium exhibited a significant increase in acetate and propionate production. The results suggested that the regulation of intracellular pH was involved in plasma membrane ATPase pumping protons out of the cells and weak acid formation for the source of the protons in cells growing at high pH. Received: 4 December 2001 / Accepted: 24 January 2002     

Osmoregulation of the salt-tolerant yeast Debaryomyces hansenii grown in a chemostat at different salinities.

The intracellular solute composition of the salt-tolerant yeast Debaryomyces hansenii was studied in glucose-limited chemostat cultures at different concentrations of NaCl (4 mM, 0.68 M, and 1.35 M). A strong positive correlation between the total intracellular polyol concentration (glycerol and arabinitol) and medium salinity was demonstrated. The intracellular polyol concentration was sufficient to balance about 75% of the osmotic pressure of the medium in cultures with 0.68 and 1.35 M NaCl. The intracellular concentration of K+ and Na+, which at low external salinity gave a considerable contribution to the intracellular water potential, was only slightly enhanced with raised medium salinity. However, the ratio of intracellular K+ to Na+ decreased; but this decrease was less drastic in the cells than in the surrounding medium, i.e., the cells were able to select for K+ in favor of Na+. The turgor pressure, which was estimated on the basis of intracellular solute concentrations, was 2,200 kPa in cultures with 4 mM NaCl and decreased when the external salinity was raised, resulting in a value of about 500 kPa in cultures with 1.35 M NaCl. The maintenance of a positive turgor pressure at high salinity was mainly due to an increased production and accumulation of glycerol.     

Genomic exploration of the hemiascomycetous yeasts: 14. Debaryomyces hansenii var. hansenii

By analyzing 2830 random sequence tags (RSTs), totalling 2.7 Mb, we explored the genome of the marine, osmo- and halotolerant yeast, Debaryomyces hansenii. A contig 29 kb in length harbors the entire mitochondrial genome. The genes encoding Cox1, Cox2, Cox3, Cob, Atp6, Atp8, Atp9, several subunits of the NADH dehydrogenase complex 1 and 11 tRNAs were unambiguously identified. An equivalent number of putative transposable elements compared to Saccharomyces cerevisiae were detected, the majority of which are more related to higher eukaryote copia elements. BLASTX comparisons of RSTs with databases revealed at least 1119 putative open reading frames with homology to S. cerevisiae and 49 to other genomes. Specific functions, including transport of metabolites, are clearly over-represented in D. hansenii compared to S. cerevisiae, consistent with the observed difference in physiology of the two species. The sequences have been deposited with EMBL under the accession numbers AL436045-AL438874.     

Cloning and expression of two genes coding for sodium pumps in the salt-tolerant yeast Debaryomyces hansenii

Two genes encoding Na(+)-ATPases from Debaryomyces hansenii were cloned and sequenced. The genes, designated ENA1 from D. hansenii (DhENA1) and DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was expressed in the presence of high Na(+) concentrations, while the expression of DhENA2 also required high pH. A mutant of Saccharomyces cerevisiae lacking the Na(+) efflux systems and sensitive to Na(+), when transformed with DhENA1 or DhENA2, recovered Na(+) tolerance and also the ability to extrude Na(+).     

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.     

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 CBS767^T. 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 CBS767^T. 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 β-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767^T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon. This revised version was published online in August 2006 with corrections to the Cover Date.     

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 CBS767^T. 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 CBS767^T. 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 β-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767^T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon. This revised version was published online in June 2006 with corrections to the Cover Date.     

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

Abstract 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 overproducing phenotype of two isolates was confirmed by chemical analysis.     

Isolation and characterization of an autonomously replicating sequence (ARSD) from the marine yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is known to tolerate salinities ranging from 0 to 24%. As a first step toward the molecular analysis of halotolerance in this organism, we report the isolation of an autonomously replicating sequence (ARS) and its use in the construction of a shuttle vector. The ARS from D. hansenii (ARSD) is 0.4 kbp long, and the function rests in 0.13 kbp of the sequence. Sequence analysis of ARSD shows strong homology to ARS from other organisms, including a 12-bp consensus sequence common to all ARS functional in Saccharomyces cerevisiae.     

Transport and utilization of hexoses and pentoses in the halotolerant yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast species that is known for its halotolerance. This organism has seldom been mentioned as a pentose consumer. In the present work, a strain of this species was investigated with respect to the utilization of pentoses and hexoses in mixtures and as single carbon sources. Growth parameters were calculated for batch aerobic cultures containing pentoses, hexoses, and mixtures of both types of sugars. Growth on pentoses was slower than growth on hexoses, but the values obtained for biomass yields were very similar with the two types of sugars. Furthermore, when mixtures of two sugars were used, a preference for one carbon source did not inhibit consumption of the other. Glucose and xylose were transported by cells grown on glucose via a specific low-affinity facilitated diffusion system. Cells derepressed by growth on xylose had two distinct high-affinity transport systems for glucose and xylose. The sensitivity of labeled glucose and xylose transport to dissipation of the transmembrane proton gradient by the protonophore carbonyl cyanide m-chlorophenylhydrazone allowed us to consider these transport systems as proton symports, although the cells displayed sugar-associated proton uptake exclusively in the presence of NaCl or KCl. When the V(max) values of transport systems for glucose and xylose were compared with glucose- and xylose-specific consumption rates during growth on either sugar, it appeared that transport did not limit the growth rate.     

Unusual flocculation characteristics of the yeast Candida famata (Debaryomyces hansenii)

Candida famata NCYC 576 cells aggregated throughout growth in YEPD. Aggregates were dispersed by Pronase E, EDTA or specific sugars. EDTA-dispersed cells reaggregated after calcium ion addition. Unlike Saccharomyces cerevisiae, C. famata cells lost the ability to flocculate with repeated EDTA washings. These cells regained flocculation when resuspended in the first washing solution after calcium addition. Candida famata NCYC 576 aggregation is consistent with lectin-mediated yeast flocculation, where lectins are not surface-anchored, as in S. cerevisiae but attached to cells only by lectin action.     

Candida mogii and Debaryomyces hansenii yeast development in the presence of metal chlorides

The yeasts Candida mogii 2 and Debaryomyces hansenii 8 isolated from salted fish spawn in the process of its storage were found to be capable of growth in 4 M KCl, 3 M MgCl2, 2.5 M NaCl, 1.5 BaCl2 and 1 M CaCl2. The activity of water (aw) in these solutions varied from 0.983 to 0.719; the maximum osmotic pressure was 380 atm. The cultures grew also in a 3 M sucrose solution, at low concentrations of osmotically active substances and without them. Therefore, they can be regarded as osmotolerant microorganisms. The osmotolerance of the cultures decreased with temperature of solutions. The absence of growth or weak growth in solutions of certain other chlorides should be attributed to toxicity of cations.     

Proteomic changes in response to potassium starvation in the extremophilic yeast Debaryomyces hansenii

In this work, we performed for the first time a proteomic approach to the processes induced by long-term potassium starvation in the halotolerant yeast Debaryomyces hansenii. The proteomic profile under this ionic stress conditions shows that important changes in gene expression take place as an adaptive response. We found a significant protein expression repression as well as metabolic changes such as the inhibition of the upper part of the glycolysis, the amino acid synthesis, and the Krebs cycle. On the other hand, genes related to stress responses, protein degradation, and sterols synthesis were upregulated in response to potassium deprivation. The findings in this study provide important information about how this particular yeast copes with ionic stress at molecular levels, which might further enrich the global understanding of salt tolerance processes in eukaryal systems and moreover highlighting the importance of the 'omics' approaches as a complement to the classical physiological studies.     

Thermokinetic and energetic profiles of the yeast Debaryomyces hansenii in the presence of sodium chloride

Summary Sodium chloride decreased the growth yield with respect to glucose by 60% (at 17% NaCl, w/v), and narrowed the temperature span of growth of Debaryomyces hansenii from 6.8–40.5°C without the salt to 13.2–34.5°C with 16% NaCl. The thermokinetic profile (conjunct display of the Arrhenius plots of the specific rates of growth and thermal death) was dissociative either without or with NaCl, the minimum temperature of thermal death increasing from 42 to 45°C with 10% NaCl.     

Draft genome sequence of the yeast Pachysolen tannophilus CBS 4044/NRRL Y-2460

A draft genome sequence of the yeast Pachysolen tannophilus CBS 4044/NRRL Y-2460 is presented. The organism has the potential to be developed as a cell factory for biorefineries due to its ability to utilize waste feedstocks. The sequenced genome size was 12,238,196 bp, consisting of 34 scaffolds. A total of 4,463 genes from 5,346 predicted open reading frames were annotated with function.     

Draft genome sequence of Rubrivivax gelatinosus CBS

Rubrivivax gelatinosus CBS, a purple nonsulfur photosynthetic bacterium, can grow photosynthetically using CO and N(2) as the sole carbon and nitrogen nutrients, respectively. R. gelatinosus CBS is of particular interest due to its ability to metabolize CO and yield H(2). We present the 5-Mb draft genome sequence of R. gelatinosus CBS with the goal of providing genetic insight into the metabolic properties of this bacterium.     

Draft genome sequence of high-siderophore-yielding Pseudomonas sp. strain HYS

We sequenced the genome of the high-siderophore-yielding strain Pseudomonas sp. HYS and then analyzed its iron acquisition systems. The 5.6-Mb draft genome sequence has a special pattern of pyoverdine synthesis clusters and contains an hmuRSTUV heme uptake cluster, which has a homolog only in some strains of the order Enterobacteriales.     

Production and characteristics of Debaryomyces hansenii killer toxin

The optimal conditions for the production of the killer toxin of Debaryomyces hansenii CYC 1021 have been studied. The lethal activity of the killer toxin increased with the presence of NaCl in the medium used for testing the killing action. Production of the killer toxin was stimulated in the presence of proteins of complex culture media. Addition of nonionic detergents and other additives, such as dimethylsulfoxide enhanced killer toxin production significantly. Killer toxin secretion pattern followed the growth curve and reached its maximum activity at the early stationary phase. Optimal stability was observed at pH 4.5 and temperatures up to 20 °C. Above pH 4.5 a steep decrease of the stability was noted. The activity was hardly detectable at pH 5.1.