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Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress.

Pérez-Torrado R, Oliveira BM, Zemančíková J, Sychrová H, Querol A - Front Microbiol (2016)

Bottom Line: The results show different pattern and level of expression among the different species, especially for STL1.These experiments also revealed a different functionality of the glycerol transporters among the different species studied.All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Department, Systems Biology in Yeast of Biotechnological Interest, Instituto de Agroquímica y Tecnología de los Alimentos, IATA-CSIC Valencia, Spain.

ABSTRACT
Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.

No MeSH data available.


Related in: MedlinePlus

Importance of glycerol import for osmotolerance of S. cerevisiae (T73), S. uvarum (BMV58) S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) in drop test assays. (A) Serial dilutions of the different strains were plated in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with glycerol (1 mM glycerol) (B) Growth of S. cerevisiae BY4741Δstl1Δhog1 strain expressing STL1 alleles from S. cerevisiae (T73), S. uvarum (BMV58) or S. kudriavzevii (IFO1802) was monitored in drop tests on non-stress media (SC), in hyperosmotic stress media (SC with 0.7 M sorbitol or 0.3 M KCl), and in hyperosmotic stress media supplemented with 10 mM glycerol. A representative image of biological triplicates is presented. (C) In the same strains used in (B), intracellular glycerol accumulation was measured collecting samples after 0, 1, or 2 days of growth in SC with 10% glucose. Cells precultured in the same media were inoculated (OD600 = 0.3) and incubated at 25°C in 100 ml flasks. Data in time 0 for each strain was considered 100%. Three independent experiments were performed for each strain and averages ± standard deviation are shown. ANOVA with fisher test (p < 0.05) was performed and significantly different values are labeled with different letters.
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Figure 5: Importance of glycerol import for osmotolerance of S. cerevisiae (T73), S. uvarum (BMV58) S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) in drop test assays. (A) Serial dilutions of the different strains were plated in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with glycerol (1 mM glycerol) (B) Growth of S. cerevisiae BY4741Δstl1Δhog1 strain expressing STL1 alleles from S. cerevisiae (T73), S. uvarum (BMV58) or S. kudriavzevii (IFO1802) was monitored in drop tests on non-stress media (SC), in hyperosmotic stress media (SC with 0.7 M sorbitol or 0.3 M KCl), and in hyperosmotic stress media supplemented with 10 mM glycerol. A representative image of biological triplicates is presented. (C) In the same strains used in (B), intracellular glycerol accumulation was measured collecting samples after 0, 1, or 2 days of growth in SC with 10% glucose. Cells precultured in the same media were inoculated (OD600 = 0.3) and incubated at 25°C in 100 ml flasks. Data in time 0 for each strain was considered 100%. Three independent experiments were performed for each strain and averages ± standard deviation are shown. ANOVA with fisher test (p < 0.05) was performed and significantly different values are labeled with different letters.

Mentions: Since STL1 gene presented important differences in mRNA levels in strains from different Saccharomyces species we wanted to study the possible functional differences of this glycerol importer. For that we first compared the growth of a representative strain of S. cerevisiae (T73), S. uvarum (BMV58), S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) species in conditions where the activity of Stl1 is important (Figure 5A). A drop test with the four strains was performed in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with a very low amount of glycerol (SC with 2 M sorbitol (or 2 M KCl) and 1 mM glycerol). In these conditions, if the cells are able to efficiently import glycerol to the cytosol they have a growth advantage when extracellular glycerol is present, i.e., before they synthesize the necessary amount to counterbalance the external osmotic pressure. The results show that cell growth is affected by hyperosmotic stress conditions proportionally to the osmotic pressure, i.e., more in the presence of 2 M KCl than in the presence of 2 M sorbitol. We can observe that BMV58 is the strain with the lowest and Chr16.2 the highest survival level in both hyperosmotic stress conditions. Interestingly, as shown in the Figure 5A, some strains, as IFO1802 and especially BMV58, benefit from the presence of glycerol in the medium more than others (e.g., T73 and Chr16.2). These results are indicative of different capacity to import glycerol in response to hyperosmotic stress among the studied strains. Is interesting to highlight that osmotolerances in minimal media can be different to complete media (see BMV58 in Figure 1 compared to Figure 5A). This reflects that strains disposition to cope to osmotic stress could be different since complete and minimal media induce very different gene expression programs (Gasch et al., 2000; Miura et al., 2008).


Alternative Glycerol Balance Strategies among Saccharomyces Species in Response to Winemaking Stress.

Pérez-Torrado R, Oliveira BM, Zemančíková J, Sychrová H, Querol A - Front Microbiol (2016)

Importance of glycerol import for osmotolerance of S. cerevisiae (T73), S. uvarum (BMV58) S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) in drop test assays. (A) Serial dilutions of the different strains were plated in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with glycerol (1 mM glycerol) (B) Growth of S. cerevisiae BY4741Δstl1Δhog1 strain expressing STL1 alleles from S. cerevisiae (T73), S. uvarum (BMV58) or S. kudriavzevii (IFO1802) was monitored in drop tests on non-stress media (SC), in hyperosmotic stress media (SC with 0.7 M sorbitol or 0.3 M KCl), and in hyperosmotic stress media supplemented with 10 mM glycerol. A representative image of biological triplicates is presented. (C) In the same strains used in (B), intracellular glycerol accumulation was measured collecting samples after 0, 1, or 2 days of growth in SC with 10% glucose. Cells precultured in the same media were inoculated (OD600 = 0.3) and incubated at 25°C in 100 ml flasks. Data in time 0 for each strain was considered 100%. Three independent experiments were performed for each strain and averages ± standard deviation are shown. ANOVA with fisher test (p < 0.05) was performed and significantly different values are labeled with different letters.
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Related In: Results  -  Collection

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Show All Figures
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Figure 5: Importance of glycerol import for osmotolerance of S. cerevisiae (T73), S. uvarum (BMV58) S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) in drop test assays. (A) Serial dilutions of the different strains were plated in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with glycerol (1 mM glycerol) (B) Growth of S. cerevisiae BY4741Δstl1Δhog1 strain expressing STL1 alleles from S. cerevisiae (T73), S. uvarum (BMV58) or S. kudriavzevii (IFO1802) was monitored in drop tests on non-stress media (SC), in hyperosmotic stress media (SC with 0.7 M sorbitol or 0.3 M KCl), and in hyperosmotic stress media supplemented with 10 mM glycerol. A representative image of biological triplicates is presented. (C) In the same strains used in (B), intracellular glycerol accumulation was measured collecting samples after 0, 1, or 2 days of growth in SC with 10% glucose. Cells precultured in the same media were inoculated (OD600 = 0.3) and incubated at 25°C in 100 ml flasks. Data in time 0 for each strain was considered 100%. Three independent experiments were performed for each strain and averages ± standard deviation are shown. ANOVA with fisher test (p < 0.05) was performed and significantly different values are labeled with different letters.
Mentions: Since STL1 gene presented important differences in mRNA levels in strains from different Saccharomyces species we wanted to study the possible functional differences of this glycerol importer. For that we first compared the growth of a representative strain of S. cerevisiae (T73), S. uvarum (BMV58), S. paradoxus (Chr16.2), and S. kudriavzevii (IFO1802) species in conditions where the activity of Stl1 is important (Figure 5A). A drop test with the four strains was performed in non-stress media (SC), in hyperosmotic stress media (SC with 2 M sorbitol or 2 M KCl) and in hyperosmotic stress media supplemented with a very low amount of glycerol (SC with 2 M sorbitol (or 2 M KCl) and 1 mM glycerol). In these conditions, if the cells are able to efficiently import glycerol to the cytosol they have a growth advantage when extracellular glycerol is present, i.e., before they synthesize the necessary amount to counterbalance the external osmotic pressure. The results show that cell growth is affected by hyperosmotic stress conditions proportionally to the osmotic pressure, i.e., more in the presence of 2 M KCl than in the presence of 2 M sorbitol. We can observe that BMV58 is the strain with the lowest and Chr16.2 the highest survival level in both hyperosmotic stress conditions. Interestingly, as shown in the Figure 5A, some strains, as IFO1802 and especially BMV58, benefit from the presence of glycerol in the medium more than others (e.g., T73 and Chr16.2). These results are indicative of different capacity to import glycerol in response to hyperosmotic stress among the studied strains. Is interesting to highlight that osmotolerances in minimal media can be different to complete media (see BMV58 in Figure 1 compared to Figure 5A). This reflects that strains disposition to cope to osmotic stress could be different since complete and minimal media induce very different gene expression programs (Gasch et al., 2000; Miura et al., 2008).

Bottom Line: The results show different pattern and level of expression among the different species, especially for STL1.These experiments also revealed a different functionality of the glycerol transporters among the different species studied.All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.

View Article: PubMed Central - PubMed

Affiliation: Food Biotechnology Department, Systems Biology in Yeast of Biotechnological Interest, Instituto de Agroquímica y Tecnología de los Alimentos, IATA-CSIC Valencia, Spain.

ABSTRACT
Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.

No MeSH data available.


Related in: MedlinePlus