Microbial Ecology

TWiM #282: At-home Evolution with Yeast

Podcast and Annotation Information
  • Annotation by Sydney Hoffman, Yasmeen Balayah, Anthony Azar, Joanna Habacon, Juan Santiago, Jeremy T. Ritzert, Rebecca Seipelt-Thiemann, and Mel Melendrez-Vallard.
  • Podcast audio by TWiM: Listen to TWiM #282 Podcast
  • Podcast transcript by Otter.ai and edited by Darian Taylor and Nick Bellavia: Access Podcast Transcripts
  • Papers Discussed:
    • Moresi NG, Geck RC, Skophammer R, Godin D, Students Y, Taylor MB, Dunham MJ. 2023. Caffeine-tolerant mutations selected through an at-home yeast experimental evolution teaching lab. MicroPubl Biol. doi: 10.17912/micropub.biology.000749.
    • Wucher BR, Winans JB, Elsayed M, Kadouri DE, Nadell CD. 2023. Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation. Proc Natl Acad Sci U S A. 120(6). doi: 10.1073/pnas.2212650120.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:52 minutes

The Most Interesting Things (according to students)

  • High school students can have the opportunity to be a part of making novel discoveries in microbiology simply by conducting home experiments without any complex resources.
  • The study directly involved high school students in the experimental process and sent their projects to a laboratory to be analyzed. How the morphology of the yeast changes as the caffeine concentration increases, leading them to be less wrinkled in appearance. The authors made many connections based on the morphology such as the likelihood of yeast to undergo flocculation.

“yEvo is a curriculum for high school students centered around evolution experiments in S. cerevisiae. To adapt the curriculum for remote instruction, we created a new protocol to evolve non-engineered yeast in the presence of caffeine. Evolved strains had increased caffeine tolerance and distinct colony morphologies. Many possessed copy number variations, transposon insertions, and mutations affecting genes with known relationships to caffeine and TOR signaling–which is inhibited by caffeine–and in other genes not previously connected with caffeine. This demonstrates that our accessible, at-home protocol is sufficient to permit novel insights into caffeine tolerance.” (Moresi et al. 2023, no changes)

1.2. Main paper; discussion starts at 21:03 minutes

The Most Interesting Things (according to students)

  • This research is applicable to a wide range of medically-related topics such as antibiotic resistance. The larger implications of this paper could be great in developing greater techniques to identify and determine medications to use in infections. It was interesting how much the structuring of the co-culture affected the overall results of the study. For example, low density colonies allowed for greater survival of co-cultures against predation, however high density colonies led to the destruction of both species.
  • It was interesting that when E. coli and V. cholerae are mixed, V. cholerae has a slightly lower survival rate upon exposure to Bdellovibrio in comparison to its survival rate alone.

“Biofilm formation, including adherence to surfaces and secretion of extracellular matrix, is common in the microbial world, but we often do not know how interaction at the cellular spatial scale translates to higher-order biofilm community ecology. Here we explore an especially understudied element of biofilm ecology, namely predation by the bacterium Bdellovibrio bacteriovorus. This predator can kill and consume many different Gram-negative bacteria, including Vibrio cholerae and Escherichia coli. V. cholerae can protect itself from predation within densely packed biofilm structures that it creates, whereas E. coli biofilms are highly susceptible to B. bacteriovorus. We explore how predator–prey dynamics change when V. cholerae and E. coli are growing in biofilms together. We find that in dual-species prey biofilms, E. coli survival under B. bacteriovorus predation increases, whereas V. cholerae survival decreases. E. coli benefits from predator protection when it becomes embedded within expanding groups of highly packed V. cholerae. But we also find that the ordered, highly packed, and clonal biofilm structure of V. cholerae can be disrupted if V. cholerae cells are directly adjacent to E. coli cells at the start of biofilm growth. When this occurs, the two species become intermixed, and the resulting disordered cell groups do not block predator entry. Because biofilm cell group structure depends on initial cell distributions at the start of prey biofilm growth, the surface colonization dynamics have a dramatic impact on the eventual multispecies biofilm architecture, which in turn determines to what extent both species survive exposure to B. bacteriovorus.” (Wucher et al 2023, no changes)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often from complex communities
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define resistance in a microbiological context.
  • Identify study variables.
 S L
  • Predict how changes in the methods used would affect the experiment.
 S H
  • Define biofilm.
  • Recall how biofilm formation and sensitivity is applicable to humans and in the medical field.
  • Identify the study’s conclusion about Vibrio cholera‘s mono-culture survival of predation.
 M L
  • Predict the outcome of a hypothetical experiment on this system based on the study’s results.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Yeast Culture Methods (6:38): Microbes have different requirements for growth.  Here, students were given cultures of budding yeast and they transferred them into media with increasing concentrations of caffeine. The longer the strains were passed and the higher the caffeine concentration, the difference in colony morphologies/variants became more apparent.
  • Whole Genome Variant Analysis (6:56-7:40 and throughout the podcast): This is a method for sequencing the genome of evolved organisms, comparing it to a reference genome, and identifying variations in genes responsible for a changed “phenotype.”  Here, yeast colonies were evolved by increasing exposures to caffeine and sent to the University of Washington for whole genome sequencing where they determined which genes were mutated and thus involved in caffeine tolerance.

4.2. Main Paper

  • Bdellovibrio Culture Methods (23:06): Microbes have different requirements for growth, alone and in co-culture.  Here, the researchers grew of E. coli into a thick suspension with low nutrients and then added a small dilution of Bdellovibrio and after 3 days it cleared.
  • Confocal Microscopy (31:10): This is a type of fluorescent microscopy that allows 3-dimensional structures to be visualized.  Here, it was used to visualize the biofilms in vivo.
  • Bacterial Co-culture Methods (33:40–33:55): Microbes have different requirements for growth, alone and in co-culture. For the co-culture group, V. cholerae was inoculated at a greater proportion than E. coli (90% and 10%). This allowed for the high packing of the V. cholerae and for the E. coli to embed themselves in it.
  • FLAG Tag (40:35- 41:05): FLAG is a synthetic amino acid sequence that can be encoded onto a gene so the encoded protein can be purified or detected using an antibody to FLAG (anti-FLAG).  Here, it was used to purify proteins/peptides. The peptide is either the amino (N) or carboxyl (C) terminus cloned to match the protein of interest.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Experimental Process (4:00–4:15): The podcast describes that the snippet introduces high school students to the scientific process, quantitative reasoning, and communicating and collaborating within an experimental setting.
  • Yeast as a Model Organism (4:27): Organisms that are already present and used within our society and have been studied for a long time and are an ideal experimental system that is safe in terms of organisms and reagents.
  • Mutation and Genetic Variation (8:10–10:00): Gain/loss of chromosomes, insertion/missense mutations, chromosomal mutations in genes associated with resistance to caffeine (shown previously).
  • Flocculation (13:25–13:39): This term describes when a group of organisms clump together within a liquid solution. This was seen in the evolved populations of yeast which lead to greater caffeine resistance.

5.2. Main Paper

  • Biofilm (19:00; 26:40–30:52; 42:50): A community of microorganisms that attach to a surface and are encased in a matrix of extracellular polymeric substances. Biofilms were the main research topic today in understanding how their composition helps in defense against microbial predators. Ordered versus non-ordered (homogeneously mixed) biofilms which depend on input numbers (low numbers support ordered; high numbers lead to non-ordered mixed biofilms).
  • Emergent Properties (19:09–19:35; 38:39). Defined and described using salt as an example (Na+ and Cl- together versus apart).
  • Predator–Prey Interactions (21:24–21:45): Predator cultures are bacteria that are able to kill/consume other bacterial species, in this study it was B. bacteriovorus. Prey cultures refer to those bacteria that aren’t able to kill/consume other bacterial species, in this study it was V. cholerae and E. coli.
  • Microbial Diversity (21:40): History and introduction presented for Bdellovibrio, an obligate parasite with two different phases–attack phase and intra-periplasmic phase.
  • Capsules (22:46)–Capsules are not protective from Bdellovibrio.
  • Type IV Pili (25:35–25:48) get in through the cell membrane then alter the cell membrane behind them so other Bdellovibrio cannot get in.
  • Type VI Secretion System (31:20) – V. cholerae has this system in place. Described as molecular switchblade knives. They knocked that out.

6. Podcast Questions

  1. What is resistance, in the context of microbiology?
    1. The ability to survive the presence of toxic or harmful substances in the environment
    2. The inability to replicate DNA in the presence of one or more antimicrobial agents
    3. When the growth or survival of a colony is increased in the presence of toxins or chemicals
    4. When colony morphology is changed due to the presence of a toxin or harmful substance
  2. What is the independent variable measured in this study?
    1. Yeast colony size
    2. Yeast colony morphology
    3. Caffeine concentration
    4. Yeast genome sequence
  3. The students evolved yeast by exposing them to increasing concentrations of caffeine over eight weeks.  What would have happened if a student accidentally started with the highest caffeine concentration? What is your reasoning?
    1. A larger difference would be observed because the stress treatment was higher.
    2. No yeast would have grown for this student because high caffeine is toxic to yeast.
    3. Differences would have occurred faster since the exposure would have started higher.
    4. Yeast would have altered their morphology to grow tall to avoid the higher caffeine levels.
  4. What is a biofilm?
    1. A complex surface-attached stable microbial community
    2. The lab environment built to mimic a natural environment.
    3. A group of nonrelated species coexisting in an environment
    4. An environmental structure where a single species lives.
  5. Why is understanding biofilm composition and sensitivity important to medicine?
    1. It is useful for tracking microbial growth, improving hospital sanitation methods
    2. It is important for identifying cell structures, enhancing imaging technologies
    3. It is essential for studying tissue repair, supporting wound healing research
    4. It is helpful in understanding microbial resistance, enabling better treatments
  6. How was the Vibrio cholerae mono-culture able to survive predation?
    1. The bacteria were able to consume the predator.
    2. The bacteria formed highly structured groups.
    3. The bacteria neutralized the predator using a toxin.
    4. The bacteria were able to swarm and evade.
  7. You have a hypothetical small molecule that binds to V. cholerae membrane and reduces its ability to form ordered structures.  What is the most likely effect on V. choerae predation by Bdellovibrio bacteriovorus?
    1. V. cholerae and B. bactertiovorus will likely switch prey-predator roles.
    2. You will see less predation because the V. cholerae membrane is protected.
    3. You will see more predation because V. cholerae structures are disrupted.
    4. You will not see predation because B. bactertiovorus no longer recognizes prey.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1AB) and the other from the main paper (Figure 1ACDE).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in a stacked bar chart.
  • Identify experimental design features, such as types of variables.
  • Evaluate the data and make conclusions about caffeine tolerance mutant yeast.
  • Predict colony morphology based on caffeine tolerance.
Experimental Background (Moresi et al., Figure 1AB)

Caffeine is the most globally used stimulant, preventing sleepiness by blocking adenosine receptors.  It is also a purine base analog that is known to disrupt DNA integrity and induce stress responses in budding yeast (Saccharomyces cerevisiae).  It was known prior to this study that high caffeine concentrations are toxic to yeast cells, that specific transporters and cell signaling pathways help them to tolerate it, and some of the affected pathways include those associated with metabolism, stress, and aging.  In this study, Moresi et al. (2023) were interested in using this model eukaryote to identify other genes and proteins that are involved in caffeine stress and tolerance.  To do this, an original parent yeast culture which was inhibited by6.25 mM caffeine was evolved to tolerate 10 mM caffeine.  Then, these partially-tolerant yeast were exposed to increasing concentrations of caffeine over an eight-week period (10mM to 20 mM to 40 mM).  At the end of the evolution period, 26 individual yeast colonies were isolated and a minimum inhibitory concentration (MIC) determined for each of the 26 (panel A). These yeast were also plated onto agar medium and colony morphology scored (panels A B).

Stacked bar chart and photo of colony growth with results color coded (pink smooth, yellow petite, and blue wrinkled).
Figure 1. “Characteristics of caffeine-tolerant Fleischmann’s yeast: A. Minimum inhibitory concentration of caffeine for clones isolated from caffeine-evolved populations. B. Representative colony morphologies of evolved clones. Morphology classification indicated by color scale below image and legend in A….” (Moresi et al. 2023, figure and text cropped to include only panels A and B)

7.1.2. Questions

  1. What type of colony does the pink color represent?
    1. Yellow
    2. Petite
    3. Smooth
    4. Wrinkled
  2. What is noted as the wild-type colony appearance?
    1. Yellow
    2. Petite
    3. Smooth
    4. Wrinkled
  3. In this experiment, the dependent variable was _________ and the independent variable was ________.
    1. yeast colony size; yeast genome sequence
    2. yeast colony morphology; caffeine concentration
    3. caffeine concentration; yeast colony morphology
    4. yeast genome sequence; yeast colony size
  4. In this experiment, the surviving yeast colonies were tested to determine their evolved tolerance to caffeine.  What is the most common appearance as the yeast became the highly tolerant?
    1. Yellow
    2. Petite
    3. Smooth
    4. Wrinkled
  5. In this experiment, the surviving yeast colonies were tested to determine their evolved tolerance to caffeine.  What is the most common appearance of yeast that have the lowest tolerance to caffeine?
    1. Yellow
    2. Petite
    3. Smooth
    4. Wrinkled
  6. What would be the most likely appearance of a yeast colony with a caffeine minimum inhibitory concentration (MIC) of 37.5 mM?
    1. Yellow
    2. Petite
    3. Smooth
    4. Wrinkled

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in line graphs, strip plots, and fluorescent microscopy images.
  • Describe spatial distribution of E. coli and V. cholerae before and after the introduction of B. bacteriovorus.
  • Analyze the data to make conclusions about how how B. bacteriovorus affects the population of each prey species in monoculture and in a dual culture.
Experimental Background (Wucher et al., Figure 1ACDE)

In  nature microbes live in multi-species communities called biofilms, and in these communities they interact in a number of ways, including predator-prey relationships.  In addition, microbes can exhibit different behaviors when grown in communities rather than pure culture (monoculture).  To explore how biofilm communities change in these circumstances, Wucher et al. (2023) investigate how Bdellovibrio bacteriovorus, a predator of proteobacteria, affects two different prey species, Vibrio cholerae and Escherichia coli when grown in monoculture or as a community of two (dual-culture). Each of the three species was engineered to express a distinct fluorescent protein for quantification and visualization purposes; colors are noted in the figure legend. In this experiment, the researchers inoculate each prey species alone or as a dual-culture, allow the prey species to form a biofilm (48 hours), introduce the predator species, and continue the culture for a total of 95 hours.  They quantify each species’ bio-volume at 4 timepoints (panel A).  To quantify predation they calculate the change in bio-volume for each prey species in the monoculture and dual-culture by comparing the bio-volume just before prey introduction and 48 hours after prey introduction (panel B).  Wucher et al. (2023) also show representative fluorescent microscopy images for prior to predator introduction (panel C), as well as 24 hours (panel D) and 48 hours (panel E) after predators were introduced.

Line graphs, strip plots and fluorescent microscopy showing biovolume over time. Predator introduction is marked on the line graph, after which the population of the ec dual culture plummets.
Figure 1. “Population dynamics in monoculture and dual-culture biofilms of V. cholerae (red) and E. coli (yellow) undergoing predation by B. bacteriovorus (cyan). Biofilms of V. cholerae and E. coli were grown for 48 h prior to B. bacteriovorus predator exposure. (A) Population dynamics of each prey species in monoculture and dual-culture biofilm growth (n = 4). (B) Percent change in prey biovolume, a direct proxy for population size, 48 h after predator introduction relative to just prior to predator introduction, both in single species prey biofilm controls and dual-species prey co-culture biofilm conditions. (V. cholerae monoculture n = 10; V. cholerae dual culture n = 9; E. coli monoculture n = 4; E. coli dual culture n = 9). Pairwise comparisons were performed by Mann–Whitney U tests. (C–E) Representative images of dual-culture biofilms at (C) 48 h after initial prey inoculation (just prior to predator introduction; “hpi” denotes “hours post-introduction” of predators), (D) 24 h post-introduction of predators, and (E) 48 h post-introduction of predators. Inset frames show regions with details of predators entering host cells and forming rounded bdelloplasts, indicating active predation. Images are single optical sections just above the glass substratum, showing the bottom layers of the biofilms.” (Wucher et al. 2023, no changes)

7.2.2. Questions

  1. Which organism was engineered to produce a cyan fluorescent protein and what is its role in this experiment?
    1. E. coli, structural prey
    2. B. bacteriovorus, prey
    3. V. cholerae, predator
    4. B. bacteriovorus, predator
  2. What line type and color represents the species population data for V. cholerae in dual culture (panel A)?
    1. red, dashed line
    2. red, solid line
    3. yellow, dashed line
    4. yellow, solid line
  3. At what time after the start of the experiment is the predator introduced to the cultures?
    1. 0 hours
    2. 24 hours
    3. 48 hours
    4. 95 hours
  4. How does the introduction of the predator affect the monoculture E. coli population (panel A)?
    1. The individual cell volumes increase
    2. The individual cell volumes decrease
    3. The population number increases
    4. The population number decreases
    5. The population and cells are unaffected
  5. How does the introduction of the predator affect the monoculture V. cholerae population (panel A)?
    1. The population number increases
    2. The population number decreases
    3. The individual cell volumes increase
    4. The individual cell volumes decrease
    5. The population and cells are unaffected
  6. How does the introduction of the predator affect the dual-culture of V. cholerae and E. coli populations (panel A)?  When bio-volumes are compared for dual-culture and monoculture after predator introduction, the dual-culture V. cholerae population ________  and the dual culture E. coli population ________  when compared to the monoculture.
    1. becomes susceptible to predation; becomes protected from predation
    2. becomes susceptible to predation; is unaffected by the dual culture
    3. is unaffected by the dual culture; becomes protected from predation
    4. becomes protected from predation; becomes susceptible to predation
    5. becomes protected from predation; is unaffected by the dual culture
    6. is unaffected by the dual culture; becomes susceptible to predation
  7. To gain a better idea of how the populations were interacting in the dual-culture biofilm, the authors used fluorescent microscopy.  How are V. cholerae and E. coli generally spatially arranged before introducing B. bacteriovorus (panel C)?
    1. E. coli forms dense clusters, V. cholerae disperses
    2. V. cholerae forms dense clusters, E. coli disperses
    3. E. coli and V. cholerae are both evenly dispersed
    4. Both E. coli and V. cholerae form dense clusters
  8. How did introducing B. bacteriovorus affect the spatial arrangement of the prey species (panel D)?
    1. E. coli is present exclusively within V. cholerae biofilms
    2. E. coli forms clusters away from V. cholerae clusters
    3. E. coli concentrates near the edges of V. cholerae clusters
    4. Both prey spread uniformly throughout the environment
  9. After a longer incubation with predator, what can be concluded about the spatial arrangement of the prey species (panel D)?
    1. E. coli is mainly found only within V. cholerae clusters
    2. E. coli is entirely predated and V. cholerae persisted
    3. E. coli clusters reside at the center of V. cholerae clusters
    4. E. coli clusters persist outside V. cholerae clusters
  10. Taking all of these data into account, what is the likely relationship between the prey in the biofilm structure?
    1. V. cholerae produces a chemoattractant for  E. coli.
    2. V. cholerae dense structures protect it and E. coli within it.
    3. V. cholerae makes a quorum sensor toxin to destroy E. coli
    4. There is no relationship between V. cholerae and E. coli.

8. Paper Information and Licensing

8.1. Snippet paper

  • Moresi NG, Geck RC, Skophammer R, Godin D, Students Y, Taylor MB, Dunham MJ. 2023. Caffeine-tolerant mutations selected through an at-home yeast experimental evolution teaching lab. MicroPubl Biol. doi: 10.17912/micropub.biology.000749
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journals’ website.

8.2. Main paper

  • Wucher BR, Winans JB, Elsayed M, Kadouri DE, Nadell CD. 2023. Breakdown of clonal cooperative architecture in multispecies biofilms and the spatial ecology of predation. Proc Natl Acad Sci U S A. 120(6). doi: 10.1073/pnas.2212650120.
  • This article is licensed for Creative Commons Attribution-NonCommerical-NoDerivatives License 4.0 use using CC BY-NC-ND 4.0, which allows re-use for non-commercial purposes as long as no derivatives are created. See the article’s copyright information on the journal web page.

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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