[Show abstract][Hide abstract] ABSTRACT: The effects of acidified-nitrite stress on the growth initiation and intracellular pH (pH(i)) of individual cells of Debaryomyces hansenii and Candida zeylanoides were investigated. Our results show that 200 microg/ml of nitrite caused pronounced growth inhibition and intracellular acidification of D. hansenii at an external pH (pH(ex)) value of 4.5 but did not at pH(ex) 5.5. These results indicate that nitrous acid as such plays an important role in the antifungal effect of acidified nitrite. Furthermore, both yeast species experienced severe growth inhibition and a pH(i) decrease at pH(ex) 4.5, suggesting that at least some of the antifungal effects of acidified nitrite may be due to intracellular acidification. For C. zeylanoides, this phenomenon could be explained in part by the uncoupling effect of energy generation from growth. Debaryomyces hansenii was more tolerant to acidified nitrite at pH(ex) 5.5 than C. zeylanoides, as determined by the rate of growth initiation. In combination with the fact that D. hansenii was able to maintain pH(i) homeostasis at pH(ex) 5.5 but C. zeylanoides was not, our results suggest that the ability to maintain pH(i) homeostasis plays a role in the acidified-nitrite tolerance of D. hansenii and C. zeylanoides. Possible mechanisms underlying the different abilities of the two yeast species to maintain their pH(i) homeostasis during acidified-nitrite stress, comprising the intracellular buffer capacity and the plasma membrane ATPase activity, were investigated, but none of these mechanisms could explain the difference.
Applied and Environmental Microbiology 07/2008; 74(15):4835-40. · 3.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of organic acids (lactic and acetic) and extracellular pH (pHex) on the intracellular pH (pHi) of Saccharomyces cerevisiae and Candida milleri during co-fermentation with lactobacilli were investigated by using Fluorescence-Ratio-Imaging-Microscopy (FRIM). Yeasts were grown in a system that partially mimics sourdough composition, using individual fermentation and combinations with lactic acid bacteria. Fermentations were carried out at 25 °C for 22 h at an initial pH of 5.3. The two yeast species grew equally well during the co-fermentations with lactobacilli. Our results reveal large differences in pHi values between the two yeast species, primarily in relation with pHex changes, while the concentration of organic acids did not seem to affect the pHi. Moreover, the pHi of S. cerevisiae seemed to be affected by maltose consumption. The pH gradient (difference between internal and external pH) of S. cerevisiae remained rather constant, ranging from 2.0 to 2.5. C. milleri instead exhibited a higher pHi, that remained constant throughout the experiments and was unaffected by pHex and/or sugar consumption. Thus, the pH gradient of C. milleri varied much more than that of S. cerevisiae, ranging from 2.3 to 3.8. Our results suggest that the two yeast species have different pHi regulation mechanisms.
LWT - Food Science and Technology. 01/2008; 41(9):1610-1615.
[Show abstract][Hide abstract] ABSTRACT: Ammonia production by yeasts may contribute to an increase in pH during the ripening of surface-ripened cheeses. The increase in pH has a stimulatory effect on the growth of secondary bacterial flora. Ammonia production of single colonies of Debaryomyces hansenii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Geotrichum candidum was determined on glycerol medium (GM) agar and cheese agar. The ammonia production was found to vary, especially among yeast species, but also within strains of D. hansenii. In addition, variations in ammonia production were found between GM agar and cheese agar. Ammonia production was positively correlated to pH measured around colonies, which suggests ammonia production as an additional technological parameter for selection of secondary starter cultures for cheese ripening. Furthermore, ammonia appeared to act as a signaling molecule in D. hansenii as reported for other yeasts. On GM agar and cheese agar, D. hansenii showed ammonia production oriented toward neighboring colonies when colonies were grown close to other colonies of the same species; however, the time to oriented ammonia production differed among strains and media. In addition, an increase of ammonia production was determined for double colonies compared with single colonies of D. hansenii on GM agar. In general, similar levels of ammonia production were determined for both single and double colonies of D. hansenii on cheese agar.
Journal of Dairy Science 12/2007; 90(11):5032-41. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effects of four types of plastic surfaces and four pre-incubation media, containing high/low glucose and +/- amino acids, on adhesion of Saccharomyces cerevisiae BY4742 wild type and Deltaflo11 mutant (strain background S288c) were investigated. No difference in adhesive ability between the two yeast strains was observed in any of our experiments, thus confirming that FLO11 is not operational in the S. cerevisiae S288c strain background. The adhesive abilities of both yeast strains depended on the plastic type and pre-incubation conditions. The poorest adhesion was observed on hydrophilic polystyrene, whereas hydrophobic polystyrene resulted in moderate adhesion. The best adhesion of both yeast strains was observed on polystyrene surfaces with combined hydrophilic/hydrophobic domains. When amino acids were present in the pre-incubation media, lack of glucose increased the cell surface hydrophobicity and enhanced the adhesion to all four types of polystyrene. Lack of amino acids in the pre-incubation media increased the cell surface hydrophobicity and enhanced the adhesion especially to polystyrene surfaces with combined hydrophilic/hydrophobic domains. Our results suggest that glucose and amino acid starvation induces other genes than FLO11 in S. cerevisiae S288c coding for hydrophobic cell surface constituents with adhesive properties to especially moderately hydrophobic plastic surfaces.
[Show abstract][Hide abstract] ABSTRACT: To identify the main amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to the polystyrene surface PolySorp.
Using a combination of phage display and competitive elution revealed that 12-mer peptides of phages from competitive panning with S. cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11Delta mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more abundant in the peptides of phages from competitive elution with FLO11 cells than in those from competitive elution with flo11Delta cells. Furthermore, two phages with hydrophobic peptides containing 1 or 2 tryptophan, and 3 or 5 proline, residues inhibited the adhesion of FLO11 cells to PolySorp more than a phage with a hydrophobic peptide containing no tryptophan and only two proline residues.
Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface.
Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments.
Journal of Applied Microbiology 11/2007; 103(4):1041-7. · 2.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The proteome of the highly NaCl-tolerant yeast Debaryomyces hansenii was investigated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), and 47 protein spots were identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) followed by mass spectrometry (MS). The influence of NaCl on the D. hansenii proteome was investigated during the first 3 h of NaCl exposure. The rate of protein synthesis was strongly decreased by exposure to 8% and 12% (w/v) NaCl, as the average incorporation rates of l-[(35)S]methionine within the first 30 min after addition of NaCl were only 7% and 4% of the rate in medium without NaCl. In addition, the number of protein spots detected on 2D gels prepared from cells exposed to 8% and 12% (w/v) NaCl exceeded less than 28% of the number of protein spots detected on 2D gels prepared from cells without added NaCl. Several proteins were identified as being either induced or repressed upon NaCl exposure. The induced proteins were enzymes involved in glycerol synthesis/dissimilation and the upper part of glycolysis, whereas the repressed proteins were enzymes involved in the lower part of glycolysis, the route to the Krebs cycle, and the synthesis of amino acids. Furthermore, one heat shock protein (Ssa1p) was induced, whereas others (Ssb2p and Hsp60p) were repressed.
FEMS Yeast Research 04/2007; 7(2):293-303. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims: To examine the relationship between the growth and pH gradients of Debaryomyces hansenii at a single-cell level.Methods and Results: Using bioimaging techniques, the cell areas and early pH gradients (ΔpH10), i.e. the pH gradients determined 10 min after initiation of experiments, were determined for single cells of two D. hansenii strains in fluid and on solid (agar) substrate with and without 8% (w/v) NaCl. The combination of NaCl and solid substrate prolonged the growth initiation of both D. hansenii strains additively. In all our experiments, primarily two groups of cells existed; a vital group consisting of growing single cells with intact early pH gradients, and a group of dead cells without early pH gradients.Conclusions: Our results show that growth initiation of the D. hansenii cells is severely affected by NaCl and to a lesser extent by the type of substrate in an additive and strain dependent way. Moreover, the early pH gradient of a vital D. hansenii cell cannot be correlated with the rate of its subsequent growth.Significance and Impact of the Study: Our study reveals new knowledge on the growth and pH gradients of D. hansenii on solid surfaces in the presence of NaCl.
[Show abstract][Hide abstract] ABSTRACT: The effects of NaCl stress on cell area and intracellular pH (pHi) of individual cells of two Debaryomyces hansenii strains were investigated. Our results show that one of the strains was more NaCl tolerant than the other, as determined by the rate of growth initiation. Whereas NaCl stress caused similar cell shrinkages (30-35%), it caused different pHi changes of the two D. hansenii strains; i.e., in the more NaCl-tolerant strain, pHi homeostasis was maintained, whereas in the less NaCl-tolerant strain, intracellular acidification occurred. Thus, cell shrinkage could not explain the different intracellular acidifications in the two strains. Instead, we introduce the concept of yeasts having an intracellular pKa (pK(a,i)) value, since permeabilized D. hansenii cells had a very high buffer capacity at a certain pH. Our results demonstrate that the more NaCl-tolerant strain was better able to maintain its pK(a,i) close to its pHi homeostasis level during NaCl stress. In turn, these findings indicate that the closer a D. hansenii strain can keep its pK(a,i) to its pHi homeostasis level, the better it may manage NaCl stress. Furthermore, our results suggest that the NaCl-induced effects on pHi were mainly due to hyperosmotic stress and not ionic stress.
Applied Microbiology and Biotechnology 09/2006; 71(5):713-9. · 3.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.