The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains.

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.

DhDIT2 Encodes a Debaryomyces hansenii Cytochrome P450 Involved in Benzo(a)pyrene Degradation-A Proposal for Mycoremediation.

Pollutants, such as polycyclic aromatic hydrocarbons (PAHs), e.g., benzo(a)pyrene (BaP), are common components of contaminating mixtures. Such compounds are ubiquitous, extremely toxic, and they pollute soils and aquatic niches. The need for new microorganism-based remediation strategies prompted researchers to identify the most suitable organisms to eliminate pollutants without interfering with the ecosystem. We analyzed the effect caused by BaP on the growth properties of Candida albicans, Debaryomyces hansenii, Rhodotorula mucilaginosa, and Saccharomyces cerevisiae. Their ability to metabolize BaP was also evaluated. The aim was to identify an optimal candidate to be used as the central component of a mycoremediation strategy. The results show that all four yeast species metabolized BaP by more than 70%, whereas their viability was not affected. The best results were observed for D. hansenii. When an incubation was performed in the presence of a cytochrome P450 (CYP) inhibitor, no BaP degradation was observed. Thus, the initial oxidation step is mediated by a CYP enzyme. Additionally, this study identified the D. hansenii DhDIT2 gene as essential to perform the initial degradation of BaP. Hence, we propose that D. hansenii and a S. cerevisiae expressing the DhDIT2 gene are suitable candidates to degrade BaP in contaminated environments.

Xylose and yeasts: A story beyond xylitol production.

Six different yeasts were used to study their metabolism of glucose and xylose, and mainly their capacity to produce ethanol and xylitol. The strains used were Candida guilliermondii, Debaryomyces hansenii, Saccharomyces cerevisiae, Kluyveromyces marxianus, Meyerozyma guilliermondii and Clavispora lusitaniae, four isolated from a rural mezcal fermentation facility. All of them produced ethanol when the substrate was glucose. When incubated in a medium containing xylose instead of glucose, only K. marxianus and M. guilliermondii were able to produce ethanol from xylose. On the other hand, all of them could produce some xylitol from xylose, but the most active in this regard were K. marxianus, M. guilliermondii, C. lusitaniae, and C. guilliermondii with the highest amount of xylitol produced. The capacity of all strains to take up glucose and xylose was also studied. Xylose, in different degrees, produced a redox imbalance in all yeasts. Respiration capacity was also studied with glucose or xylose, where C. guilliermondii, D. hansenii, K. marxianus and M. guilliermondii showed higher cyanide resistant respiration when grown in xylose. Neither xylose transport nor xylitol production were enhanced by an acidic environment (pH 4), which can be interpreted as the absence of a proton/sugar symporter mechanism for xylose transport, except for C. lusitaniae. The effects produced by xylose and their magnitude depend on the background of the studied yeast and the conditions in which these are studied.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Salinity and high pH affect energy pathways and growth in Debaryomyces hansenii.

The physiological behavior of Debaryomyces hansenii in response to saline stress and elevated pH was studied. The combination of 1 M NaCl salt and pH 8.0 was required to produce significant changes in the lag phase of growth and a consequent effect on viability. pH 8.0 in the absence or presence of 1 M NaCl produced changes in physiological functions such as respiration, acidification, rubidium transport, transmembrane potential, and fermentation. Our data indicated a stimulation of the H+-ATPase of the plasma membrane at pH 8.0, which increased the transmembrane potential and favored the entry of Na+; this effect was intensified in the presence of NaCl, so the increased energy expenditure resulting from H+ pumping and the extrusion of excess Na+ affected viability. The gene expression pattern studied by microarrays of cells incubated under saline conditions and high pH revealed a down-regulation in genes related to energy-producing pathways and in some genes involved in the cell cycle and DNA transcription, confirming our experimental hypothesis. Although D. hansenii can tolerate high pH and high salt concentrations, its physiological behavior, is better at pH 6.0 and in the absence of sodium; thus, it is an alkali-halotolerant yeast and not a halophilic yeast as previously proposed by other authors.

Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

The branched mitochondrial respiratory chain from Debaryomyces hansenii: components and supramolecular organization.

The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.

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.

Mitochondria from the salt-tolerant yeast Debaryomyces hansenii (halophilic organelles?).

The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na(+) and 10 mM K(+); these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca(2+) or Mg(2+). However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K(+) and Na(+), and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K(+)/Na(+)-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na(+) or K(+). In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a Delta Psi due to a low AOX activity. In D. hansenii mitochondria K(+) and Na(+) optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.

Activation of fermentation by salts in Debaryomyces hansenii.

The presence of 1.0 M KCl or NaCl during growth of Debaryomyces hansenii results in increased ethanol production. An additional increase of fermentation was observed when the salts were also present during incubation under nongrowing conditions. Extracts of cells grown in the presence of salt showed increased alcohol dehydrogenase and phosphofructokinase activities, indicating that these enzymes are responsible for the increased fermentation capacity. This is confirmed by measurements of the glycolytic intermediates. The increased fermentation capacity of the cells grown with salts seems to enable them to cope with the additional energy required for uptake and/or efflux of cations.

Effects of salts on aerobic metabolism of Debaryomyces hansenii.

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.

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.

[Adaptation strategies of halophilic microorganisms and Debaryomyces hansenii (halophilic yeast)]. / Estrategias de adaptación de microorganismos halófilos y Debaryomyces hansenii (Levadura halófila).

The term halophile is used for all those organisms belonging to hypersaline habitats; they constitute an interesting class of organisms able to compete successfully in salt water and to resist its denaturing effects. A wide diversity of microorganisms, prokaryotic and eukaryotic belong to this category. Halophile organisms have strategies allowing them not only to withstand osmotic stress, but also to function better in the presence of salt, in spite of maintaining high intracellular concentrations of salt, partly due to the synthesis of compatible solutes that allow them to balance their osmotic pressure. We describe the characteristics of some halophile organisms and D. hansenii (halophile yeast), that allow them to resist high concentrations of salt. The interest to know the great diversity microorganisms living in hypersaline habitats is growing, and has begun to be the center of recent investigations, since halophile organisms produce an wide variety of biomolecules that can be used for different applications. In this review we describe some mechanisms with which some halophile organisms count to resist the high concentration of salts, mainly NaCl.