Investigating the Role of Uric Acid Metabolism in

Saccaromycotina Species Competitiveness Keifer Kurtz*, Nicole Allen*, Kelly Thomasson, and Stephen Proulx

University of California, Santa Barbara – Department of Ecology, Evolution, and Marine Biology

Introduction

Abstract Results

Methods

Discussion

Literature Cited

Acknowledgements

The mechanisms that drive differential fitness within a

community are paramount to understanding community

dynamics, inter-species competition or coexistence, and

even sympatric speciation. Three species of yeast, Candida

californica, Candida zemplinina, and Pichia kluyveri, have

previously been shown to be competitively dominant in

Drosophila melanogaster fecal pools[4]. One of the known

major components of these fecal pools is uric acid[5]. As one

of the main intermediates of the purine catabolic pathway[1],

uric acid can be utilized by many species of yeast as a

nitrogen source[2]. We assayed the growth patterns of a

variety of 6 different species of yeast in a range of growth

media and culturing conditions. By comparing the

exponential growth rates and final growth concentration of

each species we are able to make predictions about

competitive dominance and coexistence. In particular, higher

growth rates in uric acid media of a subset of the species we

measured may be related to their competitive ability in D.

melanogaster fecal pools.

Species Growth Rates in Uric Acid Media: 09-522,

09-529, 09-371, 09-373, 09-378 and SPY021 were grown up

to exponential phase at 25°C on the shaking incubator in 2.5

mM uric acid media (4.5 hours ± 30 minutes). Equal

amounts of species were inoculated into a 96-well plate with

a target initial optical density (OD)λ=600 of 0.10. Species were

allowed to grow for approximately 17 hours in uric acid

(1.25mM or 2.5 mM), synthetic complete, and trace element

media while shaking in a plate reader. ODλ=600 readings

were taken every ten minutes.

Our greatest appreciations go to Dr. Stephen Proulx and

Kelly Thomasson, who mentored us through the entire course

of the project. We would like to thank Dr. Kyria Boundy-Mills of

the Phaff Collection at UC Davis for providing us with the

majority of the yeast strains required to complete this project.

We are also extremely grateful for the contribution of the UC

Santa Barbara Undergraduate Research and Creative

Activities (URCA) unit, as well as the Faculty Research

Assistance Program (FRAP), as this project would have been

impossible without their generous funding. Finally, we thank

our Senior Lab Technician, John McDermott, and our Lab

Manager, Lucien Gendrot, for their valuable support and

feedback throughout the completion of this project.

As a model organism found in ecological communities

worldwide, yeasts interact with organisms of all trophic

levels and functional groups. inter-species competition is of

great importance to the scientific community. Analysis of

differences in competitive ability for life-sustaining resources

of yeasts in the sub-phylum Saccharomycotina can lead to

new discoveries in micro evolutionary patterns. On a larger

scale, examining the mechanisms behind differential fitness

within a community can lead to a greater understanding of

community dynamics, species coexistence, and possibly

even sympatric speciation.

Uric acid is involved in the purine metabolic pathway,

which eventually converts the nucleotides adenine and

guanine into carbon dioxide and ammonia molecules[1].

Since ammonia can account for up to 80% of yeasts’

nitrogen source[2], purine catabolism would appear to be an

important pathway for yeast survival. Many species of

yeasts are capable of utilizing uric acid as a nitrogen

source[3], which indicates when competition for uric acid is

introduced, a divergence in competitive ability occurs.

Candida californica, Candida zemplinina, and Pichia kluyveri

have been previously shown to be competitively dominant in

Drosophila melanogaster fecal pools[4]. D. melanogaster

excretes uric acid as their main form of nitrogenous waste.[5],

This excretion may contribute to the apparent dominance of

C. californica, C. zemplinina, and P. kluyveri in fly fecal pools

In order to test the uric acid catabolism hypothesis for

yeast species dominance, the growth patterns of a variety of

six different species of yeast in a range of growth media and

culturing conditions were assayed. In addition to the

dominant P. kluyveri (09-523), and C. californica (09-378)

species, the competitive abilities of Saccharomyces

cerevisiae (SPY 021), Pichia anomala (09-373),

Hanseniaspora uvarum (09-371) and Pichia

membranifaciens (09-529) were examined throughout this

experiment. Several concentrations of uric acid growth

media were used in order to test its effects on competitive

abilities. Cultures were examined by optical density. This

allowed for the creation of linear plots showing their growth.

Predictions about each strains’ competitive ability were

made through analysis of their exponential growth rates and

final growth concentration.

1: Nelson D, Cox M. Principles of Biochemistry.. 6: 921 p.

2: Eelko G, et al. The concentration of ammonia regulated

nitrogen metabolism in Saccharomyces cerevisiae.

3: LaRue T, Spencer J. The utilization of purines and

pyrimidines by yeasts.

4: Stamps J, et al. Drosophila regulate yeast density and

increase yeast community similarity in a natural substrate.

5: Etienne R, et al. Mechanisms of urea tolerance in urea-

adapted populations of Drosophila melanogaster.

Figure 2: Species Final Optical Density. All six of the tested species

had varying optical densities when grown on synthetic complete media,

2.5mM uric acid media, 1.25mM uric acid media, and vitamin-only media.

Optical density readings were taken using a plate reader at a wavelength

of 600nm. Final optical densities were calculated by taking the mean of

the last five time points (see figure 5).

Figure 3: Species Maximum Growth Rate. Figure 2 was used to

determine data for each of the species growth rates on synthetic

complete media, 2.5mM uric acid media, 1.25mM uric acid media, and

vitamin-only media. Growth rates were plotted along a logarithmic scale.

Figure 4: Example Growth Curve. Shows an example graph of a yeast

growth curve with time in minutes versus optical density at a wavelength

of 600nm. The two example species used were P. kluyveri (09-523) and

H. uvarum (09-371).

Figure 5: Example Regression Analysis. Shows an example graph of

a yeast growth curve with time in seconds versus the log of the optical

density readings from Figure 4. Linear regression analysis was

performed on each set of data. The two example species used were P.

kluyveri (09-523) and H. uvarum (09-371).

Figures 1.1 – 1.3: In-Progress Competition Experiment. Shows possible signs of competitive advantage of certain species based on a qualitative analysis of S. cerevisiae density. There is a decrease in S. cerevisiae density in 2.5mM uric acid media compared to the initial conditions in the synthetic complete media. Colonies were visualized using GFP fluorescence.

The study of inter-species interactions are of great

importance to the scientific community, as these interactions

can have significant impacts on the population dynamics of their

communities. Exploitative competition, in which one species

more efficiently utilizes a resource and therefore depletes

resource availability, is one example of a seemingly-small

interaction that can have large-scale consequences. Yeast

species are found in ecological communities worldwide and

interact with organisms of all trophic levels and functional

groups, making them a prime candidate for the study of inter-

species competition. In this project, it was hypothesized that the

previously-studied competitive dominance of three yeast

species, C. californica, C. zemplinina, and P. kluyveri in D.

melanogaster fecal pools[4] could be attributed to their ability to

better utilize uric acid as a nitrogen source. In order to test this

hypothesis, six different species of yeast from the subphylum

Saccaromycotina – including C. californica (09-378), S.

cerevisiae (SPY 021), P. kluyveri (09-523), P. anomala (09-

373), and H. uvarum (09-371) – were grown in varying types of

liquid media and then analyzed using absorbance readings

taken at 600nm in a plate reader.

Our findings show that uric acid may play a role in the

competitive advantage of 09-523 on a fly fecal substrate, but

cannot be the sole contributory factor. Using R, a mixed model

was utilized to compare the differences in the maximum growth

rate among species on a nutrient rich media source, SCM, and

2.5 mm uric acid media as the sole nitrogenous food source. In

the model, replicates of each treatment were utilized as random

effects while the treatments themselves were modeled as fixed

events. In comparison to 09-371 in uric acid media, three

species appeared to have declined maximum growth rates:

SPY 021 (Est. Std. = -49.59, z= -3.284, p=1.02×10-3 ,

σx=15.10), 09-378 (Est. Std.= -38.67, z= -2.608, p=9.12×10-3,

σx=14.83), and 09-529 (Est. Std.= -32.86, z= -2.243,

p=2.49x10-2 , σx=14.65). Two species, 09-373 (Est. Std. =

41.67, z= 2.810, p=4.95×10-3, σx=10.48) and 09-523 (Est. Std.=

42.98, z= 2.933, p=3.362×10-3 , σx=14.66) showed positive

effects in relation to growth rates. To compare growth in SCM

and uric acid media, the linear model showed that the complete

media source had a positive effect on all species tested (Est.

Std. = 92.16, z= 8.772, p< 2×10-16 , σx=10.51). In this media

type, 09-529 (Est. Std. = -70.90, z= -4.718, p< 2.38×10-6 ,

σx=15.03), 09-378 (Est. Std. = -96.01, z= -6.389, p<=1.66×10-10

, σx=15.03), and 09-523 (Est. Std. = -48.86, z= -3.061, p<

2.204×10-4 , σx=10.51) all showed negative effects in relation to

09-371 on SCM. In contrast, the model showed SPY 021 (Est.

Std. = 52.26, z= 3.420, p< 6.27×10-4 , σx=15.28) had a positive

effect on SCM compared to population standard 09-371. In

contrast, 09-373 09-373 (Est. Std. = -14.60, z= -0.993, p=0.35 ,

σx=15.65) did not display any significant differences from 09-

371, indicating its maximum growth rate was likely similar.

In relation to the population as a whole, 09-523 displayed

higher growth rates compared to all species, except 09-373.

Furthermore, once complete medium was introduced into the

experiment, 09-523 lost its significant advantage and showed

decreased relative maximum growth compared to the

population standard. These indicators suggest 09-523 may

derive its competitive advantage through initial colonization

ability in a uric acid rich environment. In contrast, 09-378

showed the lowest maximum growth rates and final optical

densities in each media type. These findings suggest 09-378

may be more resilient in the fecal pool substrates. However, in

regards to uric acid utilization, future experiments must be

performed to determine the reason for 09-378’s competitive

advantage.