Candida species in tchapalo and bangui, two traditional alcoholic beverages from Côte d'Ivoire.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while ß-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Functional analysis of Debaryomyces hansenii Rpn4 on a genetic background of Saccharomyces cerevisiae.

The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.

The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii.

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li+, Na+ and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

Identification of key residues in Debaryomyces hansenii carbonyl reductase for highly productive preparation of (S)-aryl halohydrins.

Key residues of Debaryomyces hansenii carbonyl reductase in the determination of the reducing activity towards aryl haloketones were identified through combinatorial mutation of conserved residues. This study provides a green and efficient biocatalyst for the synthesis of (S)-aryl halohydrins.

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii.

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.

Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

DhARO4 induction and tyrosine nitration in response to reactive radicals generated by salt stress in Debaryomyces hansenii.

It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NO⁻) in D. hansenii after growing in 2 M NaCl. Results showed the presence of NO⁻ and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4.

The euryhaline yeast Debaryomyces hansenii has two catalase genes encoding enzymes with differential activity profile.

Debaryomyces hansenii is a spoilage yeast able to grow in a variety of ecological niches, from seawater to dairy products. Results presented in this article show that (i) D. hansenii has an inherent resistance to H2O2 which could be attributed to the fact that this yeast has a basal catalase activity which is several-fold higher than that observed in Saccharomyces cerevisiae under the same culture conditions, (ii) D. hansenii has two genes (DhCTA1 and DhCTT1) encoding two catalase isozymes with a differential enzymatic activity profile which is not strictly correlated with a differential expression profile of the encoding genes.

Optimal activity and thermostability of xylose reductase from Debaryomyces hansenii UFV-170.

Xylose reductase (XR) is the enzyme that catalyzes the first step of xylose metabolism. Although XRs from various yeasts have been characterized, little is known about this enzyme in Debaryomyces hansenii. In the present study, response surface analysis was used to determine the optimal conditions for D. hansenii UFV-170 XR activity. The influence of pH and temperature, ranging from 4.0 to 8.0 and from 25 to 55 degrees C, respectively, was evaluated by a 2(2) central composite design face-centered. The F-test (ANOVA) and the Student's t test were performed to evaluate the statistical significance of the model and the regression coefficients, respectively. The NADPH-dependent XR activity varied from 0.502 to 2.53 U mL(-1), corresponding to 0.07-0.352 U mg(-1), whereas the NADH-dependent one was almost negligible. The model predicted with satisfactory correlation (R (2) = 0.940) maximum volumetric activity of 2.27 U mL(-1) and specific activity of 0.300 U mg(-1) at pH 5.3 and 39 degrees C, which were fairly confirmed by additional tests performed under these conditions. The enzyme proved very stable at low temperature (4 degrees C), keeping its activity almost entirely after 360 min, which corresponded to the half-time at 39 degrees C. On the other hand, at temperatures >or=50 degrees C it was lost almost completely after only 20 min.

Purification and characterisation of proteases A and D from Debaryomyces hansenii.

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

N1,N12-diacetylspermine oxidase from Debaryomyces hansenii T-42: purification, characterization, molecular cloning and gene expression.

An FAD-dependent N(1),N(12)-diacetylspermine oxidase (DASpmOX), which seems suitable for enzymatic determination of the tumor marker N(1),N(12)-diacetylspermine (DASpm), was isolated from Debaryomyces hansenii T-42. DASpmOX exhibited the most excellent specificity toward DASpm among all polyamine oxidases found to date, and the specificity for DASpm could be raised by adjusting the pH of the buffer and adding TritonX-100. In potassium phosphate (pH 7.0) with 0.3% TritonX-100, this enzyme did not have any detectable activity toward free polyamines, and the reaction rate of N(1),N(8)-diacetylspermidine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine was only 19%, 7.8%, 7.8%, and 1.0% of that of DASpm, respectively. The gene encoding DASpmOX was cloned and expressed in Escherichia coli. The apparent k(cat) and K(m) values of recombinant enzyme for DASpm were found to be 158 s(-1) and 3.1 x 10(-4) M under the conditions described above, respectively.

APHO1 from the yeast Arxula adeninivorans encodes an acid phosphatase of broad substrate specificity.

The extracellular acid phosphatase-encoding Arxula adeninivorans APHO1 gene was isolated using degenerated specific oligonucleotide primers in a PCR screening approach. The gene harbours an ORF of 1449 bp encoding a protein of 483 amino acids with a calculated molecular mass of 52.4 kDa. The sequence includes an N-terminal secretion sequence of 17 amino acids. The deduced amino acid sequence exhibits 54% identity to phytases from Aspergillus awamori, Asp. niger and Asp. ficuum and a more distant relationship to phytases of the yeasts Candida albicans and Debaryomyces hansenii (36-39% identity). The sequence contains the phosphohistidine signature and the conserved active site sequence of acid phosphatases. APHO1 expression is induced under conditions of phosphate limitation. Enzyme isolates from wild and recombinant strains with the APHO1 gene expressed under control of the strong A. adeninivorans-derived TEF1 promoter were characterized. For both proteins, a molecular mass of approx. 350 kDa, corresponding to a hexameric structure, a pH optimum of pH 4.8 and a temperature optimum of 60 degrees C were determined. The preferred substrates include p-nitrophenyl-phosphate, pyridoxal-5-phosphate, 3-indoxyl-phosphate, 1-naphthylphosphate, ADP, glucose-6-phosphate, sodium-pyrophosphate, and phytic acid. Thus the enzyme is a secretory acid phosphatase with phytase activity and not a phytase as suggested by strong homology to such enzymes.

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii.

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4Delta, restoring the specific activity of the enzyme (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) to wild-type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected.

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.

Role of N-terminal hydrophobic region in modulating the subcellular localization and enzyme activity of the bisphosphate nucleotidase from Debaryomyces hansenii.

3', 5'-Bisphosphate nucleotidase is a ubiquitous enzyme that converts 3'-phosphoadenosine-5'-phosphate to adenosine-5'-phosphate and inorganic phosphate. These enzymes are highly sensitive to sodium and lithium and, thus, perform a crucial rate-limiting metabolic step during salt stress in yeast. Recently, we have identified a bisphosphate nucleotidase gene (DHAL2) from the halotolerant yeast Debaryomyces hansenii. One of the unique features of Dhal2p is that it contains an N-terminal 54-amino-acid-residue hydrophobic extension. In this study, we have shown that Dhal2p exists as a cytosolic as well as a membrane-bound form and that salt stress markedly influences the accumulation of the latter form in the cell. We have demonstrated that the N-terminal hydrophobic region was necessary for the synthesis of the membrane-bound isoform. It appeared that an alternative translation initiation was the major mechanism for the synthesis of these two forms. Moreover, the two forms exhibit significant differences in their substrate specificity. Unlike the cytosolic form, the membrane-bound form showed very high activity against inositol-1,4-bisphosphate. Thus, the present study for the first time reports the existence of multiple forms of a bisphosphate nucleotidase in any organism.

Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources.

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.

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.

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.