Microbial Ecology

TWiM #316: Food Addiction and the Gut Microbiome

Podcast and Annotation Information
  • Annotation by Annotation by Jadon A. Demeritte, Gabriel E. Knight, Samyya A. Vandenburg, Madeline Ganshert, Gabi Rant, Kyra O’Brien, Lizzie Proctor, Jeremy Ritzert, Rebecca Seipelt-Thiemann, and Mary E. Allen
  • Podcast audio by TWiM: Listen to TWiM #316 Podcast
  • Podcast transcript by Otter.ai and edited by Rebecca Seipelt-Thiemann and Laurel Thompson: Access Podcast Transcripts
  • Papers Discussed:
    • Samulėnaitė S, García-Blanco A, Mayneris-Perxachs J, Domingo-Rodríguez L, Cabana-Domínguez J, Fernàndez-Castillo N, Gago-García E, Pineda-Cirera L, Burokas A, Espinosa-Carrasco J, et al. 2024. Gut microbiota signatures of vulnerability to food addiction in mice and humans. Gut. 73(11):1799-1815. doi: 10.1136/gutjnl-2023-331445.
    • Backman T, Latorre SM, Symeonidi E, Muszyński A, Bleak E, Eads L, et al. 2024. A phage-tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria. Science. 384, eado0713. doi: 10.1126/science.ado0713.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:55 minutes

The Most Interesting Things (according to students)

Gut bacteria are connected to food addiction in both mice and humans. As people who can be picky eaters, it made us wonder how much our gut microbes influence what we crave or avoid and whether shifting our microbiome could help with things like food addiction or overeating.

“Objective: Food addiction is a multifactorial disorder characterised by a loss of control over food intake that may promote obesity and alter gut microbiota composition. We have investigated the potential involvement of the gut microbiota in the mechanisms underlying food addiction. Design: We used the Yale Food Addiction Scale (YFAS) 2.0 criteria to classify extreme food addiction in mouse and human subpopulations to identify gut microbiota signatures associated with vulnerability to this disorder. Results: Both animal and human cohorts showed important similarities in the gut microbiota signatures linked to food addiction. The signatures suggested possible non-­ beneficial effects of bacteria belonging to the Proteobacteria phylum and potential protective effects of Actinobacteria against the development of food addiction in both cohorts of humans and mice. A decreased relative abundance of the species Blautia wexlerae was observed in addicted humans and of Blautia genus in addicted mice. Administration of the non-­ digestible carbohydrates, lactulose and rhamnose, known to favour Blautia growth, led to increased relative abundance of Blautia in mice faeces in parallel with dramatic improvements in food addiction. A similar improvement was revealed after oral administration of Blautia wexlerae as a beneficial microbe. Conclusion: By understanding the crosstalk between this behavioural alteration and gut microbiota, these findings constitute a step forward to future treatments for food addiction and related eating disorders.” (Samulėnaitė et al., 2024, no changes)

1.2. Main paper; discussion starts at 22:40 minutes

The Most Interesting Things (according to students)

  • Strains of Pseudomonas bacteria use phage-derived tailocins to compete with each other in the plant microbiome. The precision of killing allows different Pseudomonas strains to coexist rather than one dominating the rest. It was fascinating that these mechanisms have been conserved for over 200 years and even seem to co-evolve with the strain that produces them. The idea that something so old and naturally occurring could become a modern therapeutic tool shows the complexity of microbial interactions.
  • Targeting bacterial tailocins may be another potential method of treating bacterial infections outside of traditional antibiotics.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact the survival and growth of microbes.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 25 (ASM_25): Microbes are used as models that provide fundamental knowledge about life processes.
  • Fundamental Statement 3 (ASM_3):  The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 5 (ASM_5):  The structure and function of microbes are revealed by the use of microscopy, culture, metabolic analyses, molecular methods, and bioinformatic tools.
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Describe behavioral characteristics used to define food addiction in mouse models.
  • Define gut dysbiosis and describe its potential impact on food addiction.
  • Identify the study conclusions regarding microbiome composition and diet altering compounds.
 S L
  • Predict the microbiome composition and addictive behavior for other organisms using the conclusions of gut microbiome on food addiction behavior in mouse model experiments.
 S H
  • Describe the structure, function, and origin of tailocins.
  • Identify the role tailocins play in bacterial competition in the plant microbiome.
 M L
  • Compare and contrast tailocins and antibiotics and propose ways tailocin therapy might be applied as an alternative to antibiotics in agriculture or medicine, based on the study’s findings.
  • Explain why Pseudomonas strains can coexist on Arabidopsis thaliana without a single dominant strain.
 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

  • Behavioral Tests on Mice (6:50–9:50): Mouse behavior can be measured.  The researchers measured persistence on how often mice self-administer food, motivation for the effort to earn food pellets, and compulsivity in walking across an electrified floor for chocolate pellets.
  • Microbiome Sequences/Analysis (9:15–13:47 in mice) (14:52 in Humans): Using metagenomic sequencing methods the microbiome composition was measured.  Here, the researchers compared gut microbiota between food-addicted and non-addicted mice and humans.
  • Diet Supplementation and Behavioral Test in Mice (13:47–17:34): Diet supplementation is when something is added to the diet and its effects measured.  The researchers gave mice rhamnose and lactulose sugars and observed changes in food-seeking behaviors.

4.2. Main Paper

  • Field Sampling (26:25–28:12): This is a method of collecting native bacterial species as compared to laboratory strains.  The researchers collected Pseudomonas strains from Arabidopsis plants.
  • Genome Sequencing (28:17–30:30): This is a technique used to sequence an entire genome.  The researchers analyzed 1,524 Pseudomonas genomes and found viral sequences in 99.3% of these bacterial genomes.
  • Phylogenetic Analysis (28:17; 33:56; 44:44–47:38): This type of analysis generally uses genetic similarity and differences to infer evolutionary relationships.  Here, the researchers determined evolutionary relationships between Pseudomonas strains and found that tailocin genes and Pseudomonas coevolved.
  • Transmission Electron Microscopy (31:49–end time):  This is a type of high resolution microscopy using electrons.  The researchers viewed the structure under the microscope to confirm tailocins.
  • Mass Spectrometry (MS) (33:56–end time): This is a technique that is used to determine the molecular identity of a molecule.  The researcher used MS to confirm tailocin identity.
  • Competition Assay (38:50–41:54): These types of assays are used to determine strains that perform best in the context of other strains.  The researchers used this assay to determine whether tailocins are harmful to other strains of Pseudomonas or the strain that produces them.
  • Transposon Mutagenesis (42:28–43:16): A technique where transposable elements are integrated into bacterial strains such that within a pool of the bacteria (library) all genes are disrupted.  Here, the researchers used this to identify genes involved in resistance to tailocin.

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

5.1. Snippet Paper

  • Human Microbiome (0:00–17:00): The gut microbiome is the community of bacteria, fungi, and viruses present and their abundance in the gut. The gut microbiome is known to play roles in nutrient uptake and immune function. The research compares gut microbiota in both mice and humans, highlighting its role in behavior and metabolic regulation.
  • Gut Dysbiosis (1:30–16:30): Dysbiosis is when a microbiome composition is altered from the composition found in healthy individuals.  The study directly examines how differences in gut microbiota composition correlate with food addiction in both mice and humans.
  • Catabolic and Anabolic Reactions (9:15–17:00): Catabolic and anabolic reactions are molecule-degrading and molecule-building reactions, respectively.  Rhamnose is a sugar that mice cannot metabolize, but certain gut microbes like Blautia can catabolize it. This means Blautia breaks rhamnose down to harvest energy. The fermentation products might impact gut chemistry or even signaling pathways to the brain.
  • Epidemiology and Public Health (9:15–22:03): Epidemiology is the study of disease distribution, spread, and cause in the natural world, which contributes to public health information. The study suggests that gut bacteria, such as Blautia, may influence food addiction behavior.

5.2. Main Paper

  • Mechanisms of Pathogenesis (23:18–26:25): Pathogenesis is the study of disease development whose mechanisms vary by pathogen.  The research focused on how Pseudomonas populations evolve to persist in plant hosts, balancing virulence with coexistence.
  • Plant Microbiome (23:18–29:10): A microbiome is the community of bacteria, fungi, and viruses present and their abundance in a particular location. The study investigates Pseudomonas bacteria in Arabidopsis plants, emphasizing microbial interactions in plant environments.
  • Gram-Positive and Gram-Negative Cell Envelope (31:49–35:45): Bacteria are often categorized by by cell wall structure using a Gram stain test;  Gram-positive bacteria have a single membrane and thick cell wall while gram-negative bacteria have two membranes and a thin cell wall.  The hosts explain how tailocins recognize and attack specific bacterial strains by binding to their outer membrane, especially the lipopolysaccharide (LPS). These structural differences in gram-negative cell envelopes influence which strains are susceptible or resistant to tailocin killing.
  • Loss of Function Mutations (36:45–41:54): Loss of function mutations are mutations that reduce function of a protein such that they are essentially null.  They are used to study the normal function of a gene.  Here, the researchers used a loss of function mutation to determine that tailocins, which are phage-derived structures, are being used by the bacteria for competition.
  • Phage Therapy (51:15–52:45 ): Bacteriophages (phages) are bacterial viruses and can be used therapeutically to kill specific bacteria.  The study highlights the potential use of tailocin therapy in agriculture and medicine as an alternative to antibiotics. This is similar to phage therapy because they target specific bacteria without harming the rest of the microbiome.

6. Podcast Questions

  1. Which of the following is/are behavioral characteristic(s) used to define food addiction in mouse models? [pick all that apply]
    1. Persistence
    2. Motivation
    3. Compulsivity
    4. Coordination
  2. Gut dysbiosis refers to ________.
    1. a decrease in normal nutrient absorption in the gut.
    2. a decrease in motility resulting from dietary changes.
    3. increased short-chain fatty acids in the small intestines.
    4. the shift in a microbiome away from a healthy state.
  3. What was a key difference in microbiota composition between food-addicted and non-addicted mice?
    1. Both addicted mice and non-addicted mice had near-identical gut microbiomes.
    2. Addicted mice had a more diverse gut microbiome than non-addicted mice.
    3. Non-addicted mice had a higher proportion of Blautia present than addicted mice.
    4. Addicted mice had a higher proportion of Blautia present than non-addicted mice.
  4. How did rhamnose supplementation affect mice in food addiction studies?
    1. It eliminated all harmful, but none of the beneficial gut bacteria.
    2. It increased gut microbial diversity, but had no behavioral effect.
    3. It altered gut microbiota and decreased food-seeking behavior.
    4. It suppressed compulsive eating without affecting the microbiome.
  5. Rats and mice are close evolutionary relatives, so they likely share biological pathways.  Knowing this, what would you predict for food addictive behaviors if food-addicted rats were fed rhamnose?
    1. Food-addicted rats fed rhamnose would show a reduction in food addictive behaviors compared to food-addicted rats that were not fed rhamnose.
    2. Food-addicted rats fed rhamnose would show food addictive behaviors at a similar level to the food-addicted rats that were not fed rhamnose.
    3. Food-addicted rats fed rhamnose would show food addictive behaviors at a higher level than the food-addicted rats that were not fed rhamnose.
    4. Neither the food-addicted rats fed rhamnose nor the food-addicted rats that were not fed rhamnose would show food addictive behaviors.
  6. Shrews and mice are close evolutionary relatives, so they likely share biological pathways.  Knowing this, what would you predict for the abundance of Blautia species if food-addicted shrews were fed lactulose?
    1. Food-addicted shrews fed lactulose would have lower Blautia abundance compared to food-addicted shrews that were not fed lactulose.
    2. Food-addicted shrews fed lactulose would have increased Blautia abundance compared to the food-addicted shrews that were not fed lactulose.
    3. Food-addicted shrews fed lactulose would show similar Blautia abundance to the food-addicted shrews that were not fed lactulose.
    4. Both the food-addicted shrews fed lactulose and the food-addicted shrews that were not fed lactulose would show high Blautia abundance.
  7. If you were to feed mice an antibiotic that specifically kills Blautia species, how would you expect their behavior to change?
    1. You would see equal food-addictive behavior in antibiotic-treated mice compared to untreated mice.
    2. You would see less food-addictive behavior in antibiotic-treated mice compared to untreated mice.
    3. You would see more food-addictive behavior in antibiotic-treated mice compared to untreated mice.
    4. You would see no food-addictive behaviors in mice treated with the antibiotic or those left untreated.
  8. Which characteristic(s) of tailocins makes them a promising tool for sustainable pathogen control, especially in comparison to traditional antibiotics? Pick all that apply.
    1. They suppress the immune response in host organisms.
    2. They are highly specific and may cause less dysbiosis.
    3. They have caused an decrease in antimicrobial resistance.
    4. They are fast-acting and widespread in their function.
  9. Which of the following best explains what tailocins are and their role in the plant microbiome?
    1. Tailocins are antibiotic molecules produced by plants that eliminate all bacterial pathogens on their surface.  This leads to a single dominant Pseudomonas species on the plant.
    2. Tailocins are phage tail-like structures that target weak microbes and promote natural selection among Pseudomonas strains on Arabidopsis thaliana.  This leads to co-evolving species.
    3. Tailocins are phage tail-like structures repurposed by bacteria to target and kill specific competitors, promoting coexistence among Pseudomonas strains on Arabidopsis thaliana.
    4. Tailocins are proteins secreted by plant roots that feed beneficial microbes while suppressing pathogens.  This leads to a co-evolving group of Pseudomonas in the root microbiome.
  10. Match the following statements as true of tailocins or traditional antibiotics: (T = tailocin; A = traditional antibiotic)
    1. _______ Overuse promotes increased resistance
    2. _______ Overuse does not promote increased resistance
    3. _______ Has bactericidal activity
    4. _______ Originates from bacteriophages
    5. _______ Sensitivity involves something produced by the bacterium
    6. _______ Resistance involves something produced by the bacterium
    7. _______ Functions against a broad range of species
    8. _______ Functions against a narrow range of strains from the same species
  11. If three Pseudomonas strains possessing tailocins were found in equal abundance in a plant’s microbiome.  What would you conclude about the similarity/difference in their genomes and why?
    1. The strains are genetically similar enough that they are unable to kill each other.
    2. The strains are genetically different enough that they are unable to kill each other.
    3. The strains are genetically similar enough that they are able to kill each other.
    4. The strains are genetically different enough that they are able to kill each other.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 3BCE).

7.1.  First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify features of a schematic figure.
  • Define and identify operant conditioning.
  • Distinguish between the behaviors of addicted and non-addicted mice.
  • Apply knowledge of statistical significance to determine whether the differences are likely due to random chance.
  • Distinguish between dependent and independent variables in a study.
Experimental Background (Samulėnaitė et al. 2024, Figure 1)

Addictions, including food addiction, affects the lives of many people, has many contributing factors, and is not well-understood at this point in time.  The researchers for this study, Samulėnaitė et al. (2024) used a mouse model and human to explore the role of gut microbiome in how and why some individuals develop compulsive eating habits. First, the researchers trained genetically identical mice to press a lever to receive chocolate-flavored pellets through a process called operant conditioning or rewards-based learning.  Operant conditioning has training and reinforcing components.  Near the end of the conditioning regime, mice were categorized as “addicted” and “non-addicted” mice based on their behavioral responses in three categories: persistence (panel B), motivation (panel C), and compulsivity (panel D). Persistence of response (panel B) is when a mouse continues to press the lever when pellets are not available, measured in 10 minutes. Motivation (panel C) is when a mouse works harder to earn pellets, i.e., the number of continuous unsuccessful presses before stopping, measured in 5 hours. Compulsivity (panel D) is assessed by how many times a mouse tolerated a painful artificial stimulus to access the chocolate pellets in 50 minutes. Mice having two or three of the three addiction criteria were considered addicted while mice having no or one addiction criteria were considered not addicted. To determine the effects on mouse overall health, pellet intake (panel E) and body weight (panel F) were also compared.

Schematic of tests on mice population, with bar plots showing outcome on addiction criteria, intake, and body weight.
Figure 1  “Characterisation of extreme subpopulations of addicted and non-addicted mice. (A) Timeline of the procedure of the operant behaviour mouse model. Mice were trained during the first 6 days in operant behaviour sessions of 1 hour at a fixed ratio (FR) 1 schedule of reinforcement, followed by 92 daily sessions of FR5. The addiction-like criteria (persistence of response, motivation, and compulsivity) were evaluated in the late period to categorise mice as addicted and non-addicted. (B–D). Behavioural tests for the three addiction-like criteria in the late period (individual values with IQR) in the addicted and non-addicted groups. (B) Persistence of response (t test, ***p<0.001). (C) Motivation (Mann–Whitney U test, ***p<0.001). (D) Compulsivity (Mann–Whitney U test, ***p<0.001). (E) Pellet intake and (F) body weight for those mice classified as addicted (A) and non-addicted (NA) (n=11mice as A and n=13 as NA mice, trained with chocolate pellets). Statistical details are included in online supplemental table S1. The selected subset of mice belongs to a previous publication with a large cohort of male JAX C57BL/6 J mice to evaluate miRNA signatures associated with vulnerability to food addiction [García-­ Blanco A, Domingo-­ Rodriguez L, Cabana-­ Domínguez J, et al. miRNA signatures associated with vulnerability to food addiction in mice and humans. J Clin Invest 2022;132:e156281].” (Samulėnaitė et al. 2024, no changes).

7.1.2. Questions

  1. The schematic illustrates how mice were trained using operant conditioning and assorted into a food-addicted or non-addicted group (panel A).  How many conditioning (training) sessions were used to train the mice?  How many additional reinforcement sessions were used to solidify the addiction behaviors?
    1. 6; 72
    2. 6; 92
    3. 72; 98
    4. 92; 98
  2. The schematic illustrates how mice were trained using operant conditioning and assorted into a food-addicted or non-addicted group (panel A). In the compulsivity measurement, what stimulus was tolerated to obtain food pellets?
    1. Electric shock
    2. Pellet-free time
    3. Lever pushing
    4. Pedestal climb
  3. Which statement best describes the definition of operant conditioning in the context of behavioral experiments?
    1. Learning through observation of others’ actions.
    2. Natural response to environmental stimuli.
    3. Natural reflex response by pairing multiple stimuli.
    4. Learning that is based on rewards or punishments.
  4. Which of the following is an example of operant conditioning?
    1. Mice choose between two types of food pellet shapes.
    2. Mice press a small lever to receive flavored fruit juices.
    3. Mice eat whenever they are hungry from a full food tray.
    4. Mice show an increased heart rate in response to a sound.
  5. Persistence, motivation, and compulsivity were compared for the addicted and non-addicted mice groups (panels B-D). What do the asterisks above each bar graph indicate?
    1. The difference in the trait between addicted and non-addicted mice is statistically significant at the p <0.001 level.
    2. The difference in the trait between addicted and non-addicted mice is statistically significant at the p <0.05 level.
    3. The difference in the trait between addicted and non-addicted mice is statistically significant at the p <0.01 level.
    4. The difference in the trait between addicted and non-addicted mice is not statistically significant.
  6. If the difference in pellet intake (panel E) between addicted and non-addicted mice had a p-value of 0.45, what would that indicate?
    1. The difference in pellet intake between addicted and non-addicted mice is statistically significant; so we should reject the null hypothesis.
    2. The difference in pellet intake between addicted and non-addicted mice is statistically significant; we should not reject the null hypothesis.
    3. The difference in pellet intake between addicted and non-addicted mice is not statistically significant; so we should not reject the null hypothesis.
    4. The difference in pellet intake between addicted and non-addicted mice is not statistically significant; so we should reject the null hypothesis.
  7. Identify the dependent variable in each addiction criterion graph (panel B–D).
    1. ____persistence
    2. ____motivation
    3. ____ compulsivity
  1. Difference in ending and starting weights
  2. Quantification of food choice preferences
  3. Average total food consumed per day
  4. Number of “no food” lever presses in 10 minutes
  5. Number of food delivered lever presses in 5 minutes
  6. Number of shocks delivered in 50 minutes
  7. Days required for the mouse to learn the task
  8. Number of increasing lever presses to earn a pellet

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Define tailocin.
  • Interpret results of a zone of inhibition assay.
  • Describe how tailocins reduce competition among bacteria in a community.
  • Explain how the phylogenetic relationships among strains relate to their susceptibility to tailocin-mediated killing.
  • Predict how experimental results would change if the experiment was modified.
Experimental Background (Backman et al. 2024, Figure 3BCE)

Tailocins are phage-derived bacterial components that are used by bacteria as selective weapons in microbial competition.  They suppress competitors without affecting unrelated members of the microbiome, so they are of interest to researchers studying pathogenesis, the microbiome, and antibiotic resistance.  Having previously identified that a diverse set of co-occurring Pseudomonas strains, rather than a single dominant strain, were present in the Arabidopsis thaliana microbiome, Backman et al (2024) were interested in whether these Pseudomonas species might utilize tailocin or tailocin-like activities to explain these population dynamics, no single dominant Pseudomonas strain.

In their first experiments of this study they identified a conserved viral gene cluster in the genomes of the P. viridiflava clade ATUE5 and confirmed production using electron microscopy to identify a phage tail-like structure. Having confirmed the Pseudomona strains produced a tailocin, The researchers next needed to investigate whether these proteins could kill other Pseudomonas strains.  To do this they extracted the purified tailocin proteins from one strain, p25.A12, and assayed for killing of 83 other Pseudomonas strains, including members of the its own ATUE5 clade (panel B, ATUE5) and other less evolutionarily-related Pseudomonas strains (panel B, other OTU).

Having confirmed that supernatants of this Pseudomonas strain (p25.A12) could in fact kill other Pseudomonas strains, the researchers wanted to confirm the putative tailocin and not some other protein in the mixture was responsible.  To do this they constructed two strains: one strain was missing its genomic copy of the tail fiber assembly (TFA) gene (panel C, ΔTFA) and a second strain was missing the genomic copy of TFA but also had  the wild-type TFA gene added back on a plasmid (panel C,  ΔTFA + TFA). They compared the ability of these constructed strains to the wild-type (WT) strain’s ability to kill a sensitive strain (p25.C2) using a soft agar- zone of inhibition assay.

Finally, since these Pseudomonas strains normally inhabit the plant surface and their abundances vary in that environment, they wanted to determine how the strains interact in those native plant environment communities.  For this, the researchers co-inoculated either wild-type (p25.A12 WT) or the TFA mutant (p25.A12 mutant) with a luciferase-tagged, sensitive, target strain (p25.C2) in different ratios (panel E).  The luciferase value is a measure of surviving target strain (p25.C2) since that is the only luciferase-tagged strain.

7.2.2. Questions

  1. Which of the following best explains what tailocins are and their role in the plant microbiome?
    1. Tailocins are antibiotic molecules produced by plants that eliminate all bacterial pathogens on their surface.  This leads to a single dominant Pseudomonas species on the plant.
    2. Tailocins are phage tail-like structures that target weak microbes and promote natural selection among Pseudomonas strains on Arabidopsis thaliana.  This leads to co-evolving species.
    3. Tailocins are phage tail-like structures repurposed by bacteria to target and kill specific competitors, promoting coexistence of similar Pseudomonas strains on Arabidopsis thaliana.
    4. Tailocins are proteins secreted by plant roots that feed beneficial microbes while suppressing pathogens.  This leads to a co-evolving group of Pseudomonas in the root microbiome.
  2. What lethality level is noted by the yellow notation and what does the pattern of yellow within the ATUE5 clade suggest?
    1. Yellow indicates strongly killed.  Strains in the ATUE5 clade are resistant to p25.A12 tailocin-mediated killing.
    2. Yellow indicates strongly killed.  Strains closely related to p25.A12 are more likely to be sensitive to its tailocin.
    3. Yellow indicates weakly killed.  The p25.A12 tailocins target only very distantly related strains outside ATUE5.
    4. Yellow indicates weakly killed.  All OTUs, including the ATUE5 clade are equally affected regardless of lineage.
  3. A phylogenetic tree is included alongside the lethality heatmap (panel B).  What additional information does this allow you to see?
    1. The mutation rate in tail fiber genes across the ATUE5 clade and other OTUs.
    2. To categorize strains by metabolic rate so that we can consider it in lethality.
    3. To indicate which strains produce tailocins and which do not produce tailocins.
    4. The relationship between sensitivity to tailocins and evolutionary relatedness.
  4. Which strains show evidence of killing the target strain and which strains do not show evidence of killing the target strain in the soft agar-zone of inhibition assay? How can you tell? Pick all that apply.
    1. WT has a zone of inhibition.
    2. WT has no zone of inhibition.
    3. ΔTFA has a zone of inhibition.
    4. ΔTFA has no zone of inhibition.
    5. ΔTFA + TFA has a zone of inhibition.
    6. ΔTFA + TFA has no zone of inhibition.
  5. Which statement best describes the purpose of producing and testing the TFA mutant strains (panel C)?
    1. The researchers needed to be sure that the tailocin and not some co-purifying protein was responsible for the killing activity, so they removed a tailocin gene (which should restore the killing) and then added it back (which should stop killing).
    2. The researchers needed to be sure that a co-purifying protein was the protein complex responsible for the killing activity, so they added a tailocin gene (which should restore the killing) and then removed it again (which should stop killing).
    3. The researchers needed to be sure that the viral gene cluster (VC2) was wholly responsible for the killing activity, so they removed the entire complex (which should restore the killing) and then added it back (which should stop killing).
    4. The researchers needed to be sure that the tailocin and not some co-purifying protein was responsible for the killing activity, so they removed a tailocin gene (which should stop the killing) and then added it back (which should restore killing).
  6. Why does co-infection of the target strain with the mutated competitor strain not result in killing the target strain (panel E)?
    1. The competitor strain cannot assemble the tailocin, so is not harmed.
    2. The target strain is not entirely susceptible to the competitor tailocin.
    3. The competitor strain is not closely enough related to the target strain.
    4. The target strain evolved resistance to the competitor’s tailocin.
  7. If the researchers had used a different strain’s tailocin in the experiment for panel B.  What would you predict for the results?
    1. They would be identical  in lethality and relatedness since the tailocin-based killing is not strain-specific.
    2. They would be different in lethality and relatedness since the tailocin-based killing is strain-specific.
    3. They would be different in specific lethality, but related strains would still be more sensitive to killing.
    4. They would be opposite in specific lethality with closely related strains being more resistant to killing.

8. Paper Information and Licensing

8.1. Snippet paper

  • Samulėnaitė S, García-Blanco A, Mayneris-Perxachs J, Domingo-Rodríguez L, Cabana-Domínguez J, Fernàndez-Castillo N, Gago-García E, Pineda-Cirera L, Burokas A, Espinosa-Carrasco J, et al. 2024. Gut microbiota signatures of vulnerability to food addiction in mice and humans. Gut. 73(11):1799-1815. doi: 10.1136/gutjnl-2023-331445.
  • This article is licensed for Creative Commons use using CC BY NC 4.0, which allows noncommercial re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.

8.2. Main paper

License

Icon for the Creative Commons Attribution 4.0 International License

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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