Metabolic Pathways

TWiM #177: Microbial Sibling Conflict

Podcast and Annotation Information
  • Annotation by Leonardo Baumgartner, Martin Leyhe, Triston Walsh, Rebecca Seipelt-Thiemann, and Nancy Boury.
  • Podcast audio by TWiM: Listen to TWiM #177 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #199 Transcript
  • Papers Discussed:
    • Lavelle K, Murphy J, Fitzgerald B, Lugli GA, Zomer A, Neve H, Ventura M, Franz CM, Cambillau C, Sinderen DV, Mahony J. 2018. A Decade of Streptococcus thermophilus Phage Evolution in an Irish Dairy Plant. Appl Environ Microbiol. 84(10):e02855-17. DOI: 10.1128/AEM.02855-17
    • Troselj V, Treuner-Lange A, Søgaard-Andersen L, Wall D. 2018. Physiological Heterogeneity Triggers Sibling Conflict Mediated by the Type VI Secretion System in an Aggregative Multicellular Bacterium. mBio. 9(1):e01645-17. DOI: 10.1128/mBio.01645-17

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:11 minutes

The Most Interesting Things (according to students)

Phage are ubiquitous and are often neglected in microbial ecology, while they play an important role in ecosystem dynamics, controlling bacterial populations (lysis by phage infection) and transferring genes (transduction).

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.

1.2. Main paper; discussion starts at 25:29 minutes

The Most Interesting Things (according to students)

Myxobacteria exhibit higher forms of differentiation than other bacteria and Myxococcus xanthus uses type 6 secretion systems to inject toxin into their less productive brethren.

“A hallmark of social microorganisms is their ability to engage in complex and coordinated behaviors that depend on cooperative and synchronized actions among many cells. For instance, myxobacteria use an aggregation strategy to form multicellular, spore-filled fruiting bodies in response to starvation. One barrier to the synchronization process is physiological heterogeneity within clonal populations. How myxobacteria cope with these physiological differences is poorly understood. Here, we investigated the interactions between closely related but physiologically distinct Myxococcus xanthus populations. We used a genetic approach to create amino acid auxotrophs and tested how they interact with a parental prototroph strain. Importantly, we found that auxotrophs were killed by their prototroph siblings when the former were starved for amino acids but not when grown on rich medium or when both strains were starved. This antagonism depended on the type VI secretion system (T6SS) as well as gliding motility; in particular, we identified the effector-immunity pair (TsxEI) as the mediator of this killing. This sibling antagonism resulted from lower levels of the TsxI immunity protein in the starved population. Thus, when starving auxotrophs were mixed with nonstarving prototrophs, the auxotrophs were susceptible to intoxication by the TsxE effector delivered by the T6SS from the prototrophs. Furthermore, our results suggested that homogeneously starving populations have reduced T6SS activity and, therefore, do not antagonize each other. We conclude that heterogeneous populations of M. xanthus use T6SS-dependent killing to eliminate starving or less-fit cells, thus facilitating the attainment of homeostasis within a population and the synchronization of behaviors.” (Troselj et al 2018, no changes)

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology(V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
ASM Fundamental Statements
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.
  • Fundamental Statement 27 (ASM_27): The extent of microbial diversity is largely unknown, and exploration of this diversity is critical to understanding microbes and their role in the biosphere.
  • 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 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Differentiate between lytic and lysogenic phage replication cycles.
  • Identify the source of the phage according to the study.
 S L
  • Predict the source of a phage given test results for ingredients of a fermented product.
 S H
  • Recall Myxobacteria’s two types of motility.
  • Define auxotrophs.
  • Identify the way Myxobacteria recognize non-self/non-kin cells in a mixed culture.
 M L
  • Hypothesize a mechanism that would promote multicellularity.
 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

  • Phage Methods (14:30–15:20): There are many types of assays that can be used to isolate and quantify phages.  Here, a double agar plaque assay was used to isolate and characterize phage.
  • Polymerase Chain Reaction (PCR) (17:07–17:25): This is a technique using a heat-stable DNA polymerase, DNA template, and DNA primers to amplify specific DNA fragments.  Here, multiplex PCR, which used multiple specific primer sets was used to characterize phage isolates.

4.2. Main Paper

  • Selective Media (38:27–39:15): This is a technique that uses different media to characterize specific bacterial features.  Here, the use of selective media and auxotrophs was used to test how the wild-type bacteria would interact with physiologically distinct bacteria.

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

5.1. Snippet Paper

  • Viral Reproduction Cycles (6:20–6:59; 12:45–13:15): There are lytic and lysogenic cycles in phages.
  • Microbial Diversity (8:25–9:25; 13:15–14:00; 21:50–22:20): Phage are ubiquitous.
  • Host Recognition (19:24–20:30): Bacterial host recognition can occur via phage tail proteins.
  • Bioengineering (20:35–21:45): The researchers used cloning/genetic engineering to make resistant strains of bacteria.

5.2. Main Paper

  • Motility (29:00–30:00): Myxobacteria have different forms of motility (adventurous and social).
  • Chemoorganotrophs (29:30–30:30): Myxobacteria are social “hunters.”
  • Genomics (34:00–34:40): Myxococcus xanthus has a 16 million base pair (bp) genome, the largest of any known prokaryote.
  • Cell-Cell Recognition (36:40–37:30): Myxococcus xanthus are capable of cell-cell recognition through exchange of outer membrane.
  • Secretion Systems (39:15–39:40; 36:00; 37:30–38:45): Secretion systems deliver toxins for population control and deliver effectors to targets.
  • Gene Regulation (39:45–42:45): Toxins kill non-growing cells through a system similar to programmed cell death.

6. Podcast Questions

  1. In the podcast, the speakers discuss how a phage destroyed a cheese-producing culture where Michael saw the OD of the culture decrease to 0 in just a few minutes.  Which part of the phage life cycle would you predict the phage is in at that point?
    1. lytic
    2. lysogenic
    3. lytogenic
    4. lysic
  2. What is the likely environmental source of the phage studied in the snippet paper?
    1. the glassware
    2. the water
    3. contact transfer
    4. the whey protein
  3. In the study, the researchers identified a phage that devastated production of a cheese-making facility and identified its likely source.  In this hypothetical situation, you have been contacted by a kimchi manufacturer with the same issue.  So, you follow a similar protocol and test ingredients, but test for a control phage (2877) in addition to the harmful phage (2990). Your results are shown in the table.  What is/are the most likely source(s) of the phage?
Kimchi Ingredient phage 2990 phage 2877
cabbage low low
carrots high low
green onions low low
rice flour low low
radish moderate low
fish sauce low low
garlic low low
ginger low low
sugar low low
chili flakes low low
    1. Sauce and chili flakes have the harmful phage, so likely the spice manufacturer.
    2. Cabbage has the largest mass and surface area of the recipe, so likely it is there.
    3. Root vegetables have the harmful phage, so the source is likely the soil.
    4. Flour and sugar have the harmful phage, so likely purified with high carbohydrates.
  2. In the main paper discussion, the podcasters mention two types of Myxobacteria motility.  What are they?
    1. slime, dry
    2. adventurous, social
    3. fruiting, swarming
    4. spreading, directed
  3. An auxotroph is an organism that ___________ .
    1. requires a nutritional supplement to grow because it lacks the ability to make it
    2. produces all essential nutrients internally without needing any external source
    3. can survive only in environments completely free of organic compounds
    4. obtains energy exclusively from light and does not require chemical nutrients
  4. The podcasters note several ways that different bacteria recognize  “non-kin” or “non-self.”  When you mix different M. xanthus strains, what allows them to tell  non-self?
    1. mimicry of other bacteria
    2. inner membrane exchange
    3. type IV secretion system
    4. evasion by swarming
  5. The podcasters note that developing multicellularity probably requires coordination among cells.  Which of the biological processes or mechanisms might help with this coordination?
    1. quorum sensing
    2. receptor mediated transport
    3. passive diffusion
    4. random mutation

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1 and 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in experimental design for this experiment.
  • Differentiate between prototrophs and auxotrophs.
  • Analyze experimental data to make conclusions about how altering growth conditions impacts swarming.
  • Predict how different media are likely to affect swarming behavior for these strains.
Experimental Background

Myxococcus xanthus is a social bacterium that forms “hunting parties” (swarming) to surround other soil bacteria and secrete digestive enzymes.  In this study, Troselj et al. (2018) focused on the population dynamics of “kin” M. xanthus populations during starvation, which is known to induce swarming. To first characterize the behavior of wild-type M. xanthus bacteria in starvation conditions, they grew wild-type bacteria on nutrient-poor A1 minimal medium in the absence or presence of calcium (panel A). As a note, round colonies are made up of bacteria that do not move (non-motile, non-swarming). Having identified conditions for wild-type swarming behavior, they next engineered different nutritionally mutant “kin” strains (auxotrophs) by deleting genes involved in the biosynthesis of specific amino acids (panel B).  They tested the behavior of these strains by growing them with and without their coordinating nutritional supplement (panel B).

Micrograph photos of cell strains, showing spread and change depending on the medium.
Figure 1: “Phenotypic properties of strains on minimal medium. (A) Cell motility in DK1622 (WT) is blocked on A1 and is restored by addition of 2 mM CaCl2 to A1 (A1 Ca2+). (B) Gene disruptions were made in ORFs predicted to be involved in amino acid biosynthesis, and the resulting mutants were assessed on A1 Ca2+ minimal medium agar (left) and A1 Ca2+ supplemented with 100 mg/liter of the indicated amino acids (right). Stereoscope micrographs were taken after a 96-h incubation. Strain details are provided in Table 1.” (Troselj et al 2018, no changes)

 

7.1.2. Questions

  1. Why was it necessary to add calcium to the A1 minimal media?
    1. The bacteria need to mimic starvation.
    2. The bacteria need it to be able to swarm.
    3. The bacteria need it to induce mutations.
    4. The bacteria need to grow and multiply.
  2. There are images of both auxotrophs and prototrophs in the data panels shown. Which are prototrophs?
    1. The bacteria with mutations in the amino acid pathways where the medium was supplemented.
    2. All of the bacteria grown on medium that was not supplemented with calcium or amino acids.
    3. The bacteria that have mutations in the amino acid pathways and grown on either medium.
    4. The bacteria labeled as wild-type (WT) and grown with or without the additional calcium.
  3. What impact does the addition of arginine to the argE mutant strain have? [pick all that apply]
    1. It supports bacterial growth.
    2. It reduces swarming behavior.
    3. It makes bacteria yellow.
    4. It make bacteria antibiotic resistant.
  4. If the researchers had supplemented the argE auxotroph with tryptophan, you would expect to see ___ of the bacteria.
    1. More growth
    2. Less growth
    3. No growth
    4. Less swarming
  5. If the researchers had used rich media instead of minimal media, they would have likely ___.
    1. seen more swarming of the bacteria
    2. seen less growth of the auxotrophs
    3. Seen less swarming of the bacteria
    4. had smaller, round white colonies
    5. observed no change in growth

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key aspects of experimental design including methods and ways of displaying data.
  • Evaluate experimental data to make conclusions about the impact of altering media conditions on growth, survival, and predation.
Experimental Background

Myxococcus xanthus is a social bacterium that forms “hunting parties” (swarming) to surround other soil bacteria and secrete digestive enzymes. Starvation is known to induce swarming for these predator bacteria and they somehow identify “kin,” which are not to be destroyed, from “non-kin,” which are not destroyed.  In this study, Troselj et al. (2018) investigate their behavior in mixed populations of starving and non-starving cells.  To do this, they mostly co-cultured a prototroph and an auxotroph in a number of ways using five different culture conditions: 1) A1 minimal media, 2) A1 and Ca2+,  3) A1 Ca2+ and histidine 4) rich medium (CTT), or 5) starvation medium (TPM).  Recall that a prototroph can make all of its own amino acids, but an auxotroph cannot and requires supplementation with the missing amino acid. Also, in this system, the researchers had identified that normal swarming behavior required the addition of calcium to the minimal A1 medium.  In their first experiment, they co-cultured a hisC auxotroph and the wild-type prototroph bacteria in a 1:1 ratio for 48 hours in each of the five media types and determine the ratio of the two bacterial types (competitive index; panel A).  To further explore behavior and competition dynamics over time rather than at a single time point, they performed the experiment again in just three of the media types and quantified the ratio of bacterial types every 12 hours for 72 hours total (competitive index; panel B).  To next compare how the growth and survival of the bacterial types differed in monoculture and co-culture, they quantified the living bacteria when grown in two of the media types for monoculture of each bacterium (WT, hisC) and in co-culture (hisC/WT).  Data are reported as colony forming units (CFU) for this experiment (panel C). Note that only hisC counts are reported for the hisC/WT co-culture.  In their final experiment, they tested the competition of the other auxotrophs against wild-type in two media types using the same 1:1 culture ratio and conditions as noted for panel A (panel D).

 

Four-part set of bar and line charts.
Figure 2: “The HisC auxotroph is conditionally antagonized by a prototroph on minimal medium. (A) HisC auxotroph labeled with tdTomato (DW2603) was mixed at a 1:1 ratio with an unlabeled prototroph (WT; DK1622) and plated on the indicated media. After incubation for 48 h, cells were harvested and examined with a microscope to determine the competitive index (prototroph-to-auxotroph ratio). (B) Similar to panel A, except that the competitive index is shown as a function of time on different media. *, no HisC auxotroph cells were detected at this time. (C) A HisC auxotroph (DW2609) and prototroph (DW2412) were plated as monocultures or a mixed culture (hisC/WT) on indicated media, and CFU were determined at 72 h. The dashed line indicates CFU for each culture at 0 h. From the mixed culture, only the hisC CFU (Kmr) is shown. All assays, unless stated otherwise, were done in biological triplicates; error bars indicate the SD. (D) Indicated auxotrophs (unlabeled) were mixed with WT labeled with tdTomato (DW2412) at 1:1 starting ratios on the indicated media and incubated for 48 h. “(Troselj et al 2018, no changes)

 

7.2.2. Questions

  1. The researchers use the competitive index to report their data in three of their panels.  What would a competitive index of 1 indicate?
    1. The two cells types are found in equal proportions.
    2. Wild-type has 100% more cells than the mutant.
    3. The mutant has 100% more cells than wild-type.
    4. The optical density (OD) of the co-culture was 1.0.
  2. In which media types should the hisC auxotroph be able to grow? [pick all that apply]
    1. A1
    2. A1 + Ca
    3. A1 + Ca + his
    4. CTT
    5. TPM
  3. In which condition(s) is/are the prototroph more numerous than the hisC auxotroph (panel A)?
    1. A1
    2. A1 + Ca
    3. A1 + Ca + his
    4. CTT
    5. TPM
  4. The number of living cells was quantified as colony forming units (CFU) for monocultures and the co-culture (WT and hisC) in two media types (panel C). What can you conclude from the data?
    1. hisC grow less well in both media types.
    2. WT grows less well in both media types.
    3. Swarming and predation are not linked.
    4. Swarming and predation are correlated.
  5. Panels A-C all report data about bacteria that cannot make their own histidine (histidine auxotroph).  Looking at panel D, we see that the interactions between the prototroph and other auxotrophs are similar, and thus not specific to just the histidine auxotroph.
    1. True
    2. False

8. Paper Information and Licensing

8.1. Snippet paper

  • Lavelle K, Murphy J, Fitzgerald B, Lugli GA, Zomer A, Neve H, Ventura M, Franz CM, Cambillau C, Sinderen DV, Mahony J. 2018. A Decade of Streptococcus thermophilus Phage Evolution in an Irish Dairy Plant. Appl Environ Microbiol. doi: 10.1128/AEM.02855-17
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page: https://journals.asm.org/doi/10.1128/aem.02855-17

8.2. Main paper

  • Troselj V, Treuner-Lange A, Søgaard-Andersen L, Wall D. 2018. Physiological Heterogeneity Triggers Sibling Conflict Mediated by the Type VI Secretion System in an Aggregative Multicellular Bacterium. mBio. doi: 10.1128/mBio.01645-17
  • 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 https://journals.asm.org/doi/full/10.1128/mbio.01645-17 .

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.

Share This Book