DebaryOmics: an integrative -omics study to understand the halophilic behaviour of Debaryomyces hansenii.

Debaryomyces hansenii is a non-conventional yeast considered to be a well-suited option for a number of different industrial bioprocesses. It exhibits a set of beneficial traits (halotolerant, oleaginous, xerotolerant, inhibitory compounds resistant) which translates to a number of advantages for industrial fermentation setups when compared to traditional hosts. Although D. hansenii has been highly studied during the last three decades, especially in regards to its salt-tolerant character, the molecular mechanisms underlying this natural tolerance should be further investigated in order to broadly use this yeast in biotechnological processes. In this work, we performed a series of chemostat cultivations in controlled bioreactors where D. hansenii (CBS 767) was grown in the presence of either 1M NaCl or KCl and studied the transcriptomic and (phospho)proteomic profiles. Our results show that sodium and potassium trigger different responses at both expression and regulation of protein activity levels and also complemented previous reports pointing to specific cellular processes as key players in halotolerance, moreover providing novel information about the specific genes involved in each process. The phosphoproteomic analysis, the first of this kind ever reported in D. hansenii, also implicated a novel and yet uncharacterized cation transporter in the response to high sodium concentrations.

A physiological characterization in controlled bioreactors reveals a novel survival strategy for Debaryomyces hansenii at high salinity.

Debaryomyces hansenii is traditionally described as a halotolerant non-conventional yeast and has served as a model organism for the study of osmotolerance and salt tolerance mechanisms in eukaryotic systems for the past 30 years. However, unraveling of D. hansenii's biotechnological potential has always been difficult due to the persistent limitations in the availability of efficient molecular tools described for this yeast. Additionally, there is a lack of consensus and contradictory information along the recent years that limits a comprehensive understanding of its central carbon metabolism, mainly due to a lack of physiological studies in controlled and monitored environments. Moreover, there is little consistency in the culture conditions (media composition, temperature, and pH among others) used by different groups, which makes it complicated when trying to get prevalent conclusions on behavioral patterns. In this work, we present for the first time a characterization of D. hansenii in batch cultivations using highly controlled lab-scale bioreactors. Our findings contribute to a more complete picture of the central carbon metabolism and the external pH influence on the yeast's ability to tolerate high Na+ and K+ concentrations, pointing to a differential effect of both salts, as well as a positive effect in cell performance when low environmental pH values are combined with a high sodium concentration in the media. Finally, a novel survival strategy at very high salinity (2 M) is proposed for this yeast, as well as potential outcomes for its use in industrial biotechnology applications. TAKE AWAY: High salt concentrations stimulate respiration in Debaryomyces hansenii. Sodium exerts a stronger positive impact on cell performance than potassium. µmax is higher at a combination of low pH, high salt, and high temperature. Concentrations of 2 M salt result in slower growth but increased biomass yield. The positive effect of salts is enhanced at low glucose concentration.

Overlapping responses between salt and oxidative stress in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast of importance in basic and applied research. Previous reports hinted about possible links between saline and oxidative stress responses in this yeast. The aim of this work was to study that hypothesis at different molecular levels, investigating after oxidative and saline stress: (i) transcription of seven genes related to oxidative and/or saline responses, (ii) activity of two main anti-oxidative enzymes, (iii) existence of common metabolic intermediates, and (iv) generation of damages to biomolecules as lipids and proteins. Our results showed how expression of genes related to oxidative stress was induced by exposure to NaCl and KCl, and, vice versa, transcription of some genes related to osmotic/salt stress responses was regulated by H2O2. Moreover, and contrary to S. cerevisiae, in D. hansenii HOG1 and MSN2 genes were modulated by stress at their transcriptional level. At the enzymatic level, saline stress also induced antioxidative enzymatic defenses as catalase and glutathione reductase. Furthermore, we demonstrated that both stresses are connected by the generation of intracellular ROS, and that hydrogen peroxide can affect the accumulation of in-cell sodium. On the other hand, no significant alterations in lipid oxidation or total glutathione content were observed upon exposure to both stresses tested. The results described in this work could help to understand the responses to both stressors, and to improve the biotechnological potential of D. hansenni.

Lipolysis and aroma generation as mechanisms involved in masking boar taint in sodium reduced fermented sausages inoculated with Debaryomyces hansenii yeast.

BACKGROUND: The use of boar back fat for processing of fermented sausages may cause the presence of abnormal odours. In dry-cured products, ripening time is essential to develop the sensory characteristics. Yeast has been proposed as an alternative to mask boar taint odour through its metabolic activity but it is necessary to elucidate which mechanisms are involved. The aim is to study the effect of Debaryomyces hansenii inoculation on the lipolysis process and generation of aroma compounds in fermented sausages manufactured with boar back fat at two different ripening times. RESULTS: D. hansenii inoculated sausages had a higher degree of lipolysis as demonstrated by higher content of free fatty acids, ester compounds and branched aldehydes which contribute the fruity odour. The increase in lipolysis produced by D. hansenii inoculation was not followed by an increase in oxidation during processing possibly due to the metabolic activity of yeast. The effect of back fat type was scarcely appreciated whereas ripening time had a stronger effect on sausage. Boar sausages were characterised by a lower polyunsaturated fatty acid profile and lesser lipolysis than gilt sausages. CONCLUSION: Yeast inoculation with D. hansenii and long ripening time were appropriate strategies to limit the perception of boar taint in dry fermented sausages. © 2017 Society of Chemical Industry.

Vacuolar control of subcellular cation distribution is a key parameter in the adaptation of Debaryomyces hansenii to high salt concentrations.

Debaryomyces hansenii is a halotolerant and Na+-includer yeast that can be isolated from different food and low-water activity products. It has also been defined as a marine-occurring yeast but key aspects for this salt tolerant behavior are far from being understood. Here, we searched for clues helping to elucidate the basis of this ability. Our results on growth, Rb+ transport, total K+ and Na+ content and vacuolar fragmentation are compatible with a yeast species adapted to cope with salt stress. On the other hand, we confirmed the existence of D. hansenii strategies that are generally observed in sensitive organisms, such as the production of glycerol as a compatible solute and the efficient vacuolar sequestration of Na+. We propose a striking role of D. hansenii vacuoles in the maintenance of constant cytosolic K+ values, even in the presence of extracellular Na+ concentration values more than two orders of magnitude higher than extracellular K+. Finally, the ability to deal with cytosolic Na+ levels significantly higher than those found in S. cerevisiae, shows the existence of important and specific salt tolerance mechanisms and determinants in D. hansenii.

Monovalent cations regulate expression and activity of the Hak1 potassium transporter in Debaryomyces hansenii.

Debaryomyces hansenii was able to grow in a medium containing residual amounts of K(+), indicating the activity of high affinity K(+) transporters. Transcriptional regulation analysis of the genes encoding the two potassium uptake systems in D. hansenii revealed that while DhTRK1 is not regulated at transcriptional level, expression of DhHAK1 required starvation in the absence of K(+) and Na(+) and was not affected by changes in membrane potential. Rb(+) transport in cells expressing DhHAK1 was activated by external Na(+) or acidic pH and inhibited by high pH. We propose a K(+)-H(+) symporter that, under certain conditions may work as a K(+)-Na(+) transporter, as the mechanism driving K(+) influx mediated by DhHak1p.

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport.

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na(+) or K(+)) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K(+) during growth at high concentrations of KCl. However, it accumulated low amounts of Na(+) and high amounts of K(+) when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na(+)/K(+) on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na(+) and rapid uptake of K(+) and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na(+) excluder yeast and it has an efficient transport system for sodium extrusion.

Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1.

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.

Studies on the time course of the effects of the probiotic yeast Saccharomyces boulardii on electrolyte transport in pig jejunum.

Orally administered Saccharomyces boulardii (synonym Saccharomyces cerevisiae Hansen CBS 5926) has already been shown to affect relevant functions of the mucosa in pig jejunum such as lowering the secretory response to theophylline or stimulating sodium/glucose cotransport, but knowledge of time-dependent relationship is minimal. In this study we examined the effects of S. boulardii on sodium (Na+) and chloride (Cl-) transport in pig jejunum under nonstimulated (basal) and stimulated (secretory) conditions. For this purpose the conventional Ussing chamber method was used for measuring electrical parameters (short circuit currents, Isc; tissue conductances, G(T)) and electrolyte transport of isolated intact jejunal epithelia in the absence and presence of the secretagogue theophylline (10 mM, serosal side). Time profiles of the mucosa response were assessed by treating animals perorally with S. boulardii for 0 (control), 3, 8, and 16 days. Intestinal tissues were obtained from growing pigs in the weight range between 25 and 40 kg. All animals were fed twice daily and received 1.0-1.6 kg/day of a standard diet avoiding probiotics as food additives. After a 9- to 10-day adaptation period the diets for treated animals were supplemented with approximately 1.8 x 10(7) colony forming units (CFU)/g feed of the probiotic. Whereas basal tissue conductances were not affected by treatment duration, basal Isc values decreased significantly during 8 days of treatment, by 26%, indicating a lower electrogenic net ion transport, which, however, was reconstituted after 16 days. This effect could be explained by almost the same reduction of basal Jms of Na+ during 8 days of treatment, whereas respective flux rates in the opposite direction remained stable. Under basal conditions unidirectional and net flux rates of Cl- were not affected by S. boulardii. Induction of secretory conditions by theophylline revealed pronounced increases in net Cl- secretion but this effect was more than 60% lower after 8-day S. boulardii application, and this was reflected by a respectively lower Isc stimulation. Interestingly, this inhibitory effect on the secretory response could no longer be observed in the 16-day group. And this was reflected by a respectively lower Isc stimulation. A similar effect could be observed regarding net Na+ flux rates. Residual fluxes were affected neither by S. boulardii nor by theophylline, therefore, Isc values can be explained completely by respective Na+ and Cl- fluxes. In conclusion, S. boulardii has specific duration-dependent effects on the secretory response of the pig jejunal mucosa which developed during 8-day treatment but disappeared during further application. Thus, this study supports the concept that probiotics may exert beneficial effects in the gastrointestinal tract.

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.

Drug effects on intracellular mycobacteria determined by mass spectrometric analysis of the Na(+)-to-K+ ratios of individual bacterial organisms.

The successful establishment of a drug screening system for intracellular cultivable and noncultivable mycobacteria based on the mass spectrometric determination of bacterial viability is described. To compare drug efficacies on intra- and extracellular mycobacteria, the mycobacteria were subjected to drug treatment either after phagocytosis by the mouse macrophage cell line RAW 264.7 or in cell-free medium. After reisolation, their viability was monitored by analyzing the intrabacterial sodium-to-potassium ratios for a limited number of individual organisms. This approach offers a reliable and quick tool for monitoring the influence of intracellular growth and of additional permeation barriers on intracellular drug efficacy and will thus provide useful information for the rational development and testing of optimized antimycobacterial drugs. In particular, the methodology is applicable to the noncultivable species Mycobacterium leprae, because the mass spectrometric analysis of the intrabacterial sodium-to-potassium ratio allows the determination of bacterial viability independent from their ability to multiply in vitro. Because of the improved metabolic activity of intracellularly growing M. leprae compared with that of extracellularly growing M. leprae, the spectrum of antileprosy drugs that can be tested in vitro could even be extended to those interfering with DNA replication and cell division.

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.

Adreno-cortical function in leprosy.

Adreno cortical function was carried out in 43 cases of leprosy. These cases were further divided into tuberculoid, borderline, lepromatous and Lepra reaction. Serum and urinary electrolyte, urinary 17-Ketosteroid and 17-Ketogenic steroid and plasma cortisol levels were measured to assess the adrenocortical status in these different forms of leprosy. It was observed that these parameters were within normal limit in tuberculoid leprosy except low value of urinary 17-Ketogenic steroid. The borderline and Lepromatous leprosy cases revealed low values of urinary sodium, potassium and 17-Ketogenic steroid and high level of serum potassium. However, the cases of lepra reaction revealed low value of serum and urinary sodium and potassium, urinary 17-Ketogenic steroid. The basal plasma cortisol level was high in this group but it was statistically insignificant.

The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of L-malate and D-malate by luminal-membrane vesicles.

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J.208, 359-368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na(+) gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na(+)-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl(-)>SO(4) (2-)>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na(+)/substrate coupling ratio 3:1) by an Na(+)-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na(+) in the medium.

Regulation of the potassium to sodium ratio and of the osmotic potential in relation to salt tolerance in yeasts.

By using the isotope pairs (22)Na-(24)Na and (42)K-(86)Rb, the uptake and retention of Na and K was studied in the salt-tolerant Debaryomyces hansenii and in the less tolerant Saccharomyces cerevisiae at NaCl levels of 4 mm and 0.68, 1.35, and 2.7 m in the medium. The ratio of K to Na is much higher in the cells than in the media, and higher in D. hansenii than in S. cerevisiae under comparable conditions. The difference between the two species is due to a better Na extrusion and a better uptake of K in D. hansenii. The kinetics of ion transport show that at about the time when extrusion of Na could be demonstrated in D. hansenii, K-Rb previously lost to an easily washable compartment of the cells was reabsorbed in both organisms. More H(+) was given off from S. cerevisiae than from D. hansenii in the course of these events. The findings fit the working hypothesis tested, which regards salt tolerance as partly dependent on the ability to mobilize energy to extrude Na from the cells and to take up K. The volume changes in S. cerevisiae are greater and are more slowly overcome than those in D. hansenii. The total salt level of the cells is not sufficient to counteract the osmotic potential of the medium, so that additional osmoregulatory mechanisms must be involved in determining halotolerance.