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On the generation of a dose-response curve of fluconazole for two strains of S. cerevisiae and a glycerol dose-response curve

for two strains of H. salinarum. Ashwin Monian, Elizabeth Tsui, Vamsi Yechoor

The Schmid Lab

Magwene Lab

Introduction

Saccharomyces cerevisiae (Baker’s yeast) is a unicellular fungus that forms colonies with other yeasts. Fluconazole is an anti-fungal drug that inhibits the production of phospholipids that form the cellular membrane. We tested the effect of fluconazole on two subspecies of this organism - S288C (a strain mainly used in the laboratory), and a Wild Type strain taken from an oak tree (referred to as Wild Type Oak). Because the Wild Oak Strain is found in nature, we predict that it would have a higher growth rate than S288C when exposed to fluconazole because it would have been exposed to more stressors than the S288C strain. Halobacterium salinarum is a species of archaebacteria found in hypersaline environments. Its metabolic pathways are currently being studied to provide more information as to how archaea have evolved, as metabolic pathways are highly conserved. A previous study done by Schmid et. al shows that 0.08% v/v glycerol results in higher growth than no sugar. However, the roles of glycerol and glucose in the metabolic pathways of these organisms are relatively unclear, and our experiment, therefore, aimed to test the role of glycerol in the metabolic pathway of H. salinarum by varying the concentration of glycerol and measuring growth.

Abstract

In these experiments, our aims were to 1) identify the effects of varied concentrations of fluconazole on two strains of Saccharomyces cerevisiae, S288C and a Wild Type Oak, and to 2) identify the role of glycerol in the metabolic pathways of two strains of Halobacterium salinarum, Δura3 and ΔtrmB. In our first experiment, we exposed these two strains to three sublethal concentrations of fluconazole - 0.5 μg/mL, 1.0μg/mL, and 1.5μg/mL. In our second experiment, we exposed the two strains of halobacteria to solutions of 8% v/v glycerol, 0.8% v/v glycerol, and 0.08% v/v glycerol. For both of these experiments, we quantified growth by measuring optical density over three days. From our yeast experiment, we determined that Wild Type Oak had a higher max growth rate with an increase in the concentration of fluconazole than the S288C lab strain. However, when examining our data for doubling time, we realized that the addition of fluconazole showed no significant results. For our experiments involving halobacteria, our results show that an increase in the concentration of glycerol decreases doubling time for the ΔtrmB strain, however, the doubling time of Δura3 was not shown to be significantly affected by the addition of glycerol. When comparing lag times, we concluded that the ΔtrmB mutant had a shorter lag time than Δura3 across all treatments.

S. cerevisiae H0,1 : The growth of yeast will not change with an alteration of the dose of fluconazole. Ha,1: As the dose of fluconazole increases, the growth of the yeast will decrease. H0,2: The strains S288C and Wild Type Oak will respond equally to fluconazole. Ha,2: The Wild Type Oak strain will demonstrate a higher level of growth than the S288C when exposed to fluconazole.

Hypotheses

H. salinarum NRC-1 H0,1 : The growth of the halobacteria will not change with an alteration of the dose of fluconazole. Ha,1: As the concentration of glycerol increases, the growth of the halobacterium will increase. H0,1: The strains Δura3 and ΔtrmB will respond equally to glycerol. Ha,1: The strains will respond differently to glycerol.

Acknowledgments We would like to thank Dr. Paul Magwene of Duke University, Dr. Schmid of Duke University, Heidi Cederholm, Dr. Rotem Ben-Shachar, Blake Billmyre, and Dr. Zackary Scholl. Andrea Barrett, and Dr. Sheck of the North Carolina School of Science and Mathematics.

Methods

Yeast 1) We pipetted 3 mL of YPD were into two tubes.

2) We placed 3 μL of the S288C strain into one tube and 3μL of the Wild Type Oak into the other.

3) We vortexed, and then pipetted 200 μL of strain+YPD solution into 3 wells per strain.

4) We prepared 0.5 μg/mL fluconazole , 1.0 μg/mL fluconazole, and 1.5 μg/mL fluconazole doses by pipetting fluconazole stock solution (1.0 μg/mL fluconazole) into the remainder of the previous solutions.

5) We finally placed the well plate in the BioScreen and measured the optical density over three days.

Halobacteria

1) We pipetted 1 mL each of Δura3 and ΔtrmB into cuvettes.

2) We measured optical density of each in order to dilute each solution to 0.1 Au by adding base media.

3) We prepared 0.08% glycerol, 0.8% glycerol, and 8.0% glycerol solutions by pipetting glycerol stock solution into the remainder of the previous solutions.

4) Vortex, and then pipette 200 microlitres of strain+base+glycerol solution into wells, then place well plate into BioScreen.

Results

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Figure 1: In this graph, max growth rate, measured in cells per hour, for S288C and Wild Type Oak, is plotted against varying concentrations of fluconazole: Control(no fluconazole), 0.5µg/mL, 1.0µg/mL, and 1.5µg/mL.

Figure 2: This graph depicts doubling time versus differing concentrations of fluconazole: Control(no fluconazole), 0.5µg/mL, 1.0µg/mL, and 1.5µg/mL.

Figure 3: In this graph, doubling time of Δura3 and ΔtrmB is plotted against differing concentrations of glycerol: Control (0%), 0.08% Glycerol, 0.8% Glycerol, and 8.0% Glycerol.

Figure 4: This graph shows lag time of Δura3 and ΔtrmB versus varying concentrations of glycerol: Control (0%), 0.08% Glycerol, 0.8% Glycerol, and 8.0% Glycerol.

Discussion of Results Our data yielded results that both supported and contradicted our hypotheses. From Figure 1, we observed that the Maximum Growth Rate, measured in cells per hour, of the Wild Oak strain, increased with an increase in concentration of fluconazole. While this contradicts our first hypothesis that an increase in fluconazole would decrease the maximum growth rate; it confirms our second hypothesis that the Wild Oak Strain would perform better than S288C at higher concentrations of fluconazole (ρ = .034 and ρ = .040 for fluconazole doses of 1.0 µg/mL and 1.5 µg/mL, respectively). However, as shown in Figure 2, the doubling time is unaffected by varying the concentration of fluconazole both within and when comparing the strains. We also can conclude that the strains do not respond differently to the fluconazole in terms of their doubling time. From Figure 3, we can see that the two strains’ doubling time is only affected at lower concentrations of glycerol, (ρ = .001 and ρ = 0.017 for the concentrations of 0% and 0.08% glycerol, respectively). According to Figure 4, we can conclude that for all concentrations of glycerol, excluding the 8% glycerol sample, Δura3 had a longer lag time than ΔtrmB (ρ < .002, ρ = .003 , ρ = .000089, and ρ = .0515, for Control, 0.08% Glycerol, 0.8% Glycerol, and 8.0% Glycerol, respectively) . Taken together, these results tentatively lead us to the conclusion that there may be a mechanism for a negative feedback loop at high concentrations of glycerol. For our yeast experiment, we were unable to prove our first hypothesis; however, the second hypothesis could also not be proven due to inconsistent results. For our halobacterium experiments, our data pointed to the conclusion that the opposite of our first hypothesis was true. With few exceptions, our second hypothesis was proven to be true.

Pictures of: S. cerevisiae (left) and H. Salinarum (right) – Retrieved from http://www.mimage.uni-frankfurt.de/images/modelsystems/s_cerevisiae_cells.jpg and http://www.contrib.andrew.cmu.edu/~pvan/halo-micrograph.jpg, respectively