Microbial Ecology

TWiM #255: Fleaing the Plague

Podcast and Annotation Information
  • Annotation by Alex Bray, Harshita Sharma, Grace Helle, Nancy Boury, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #255 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Aidan Sasaoka: Access Podcast Transcripts
  • Papers Discussed:
    • Kehe J, Ortiz A, Kulesa A, Gore J, Blainey PC, Friedman J. 2021. Positive interactions are common among culturable bacteria. Science Advances. 7(45). doi: 10.1126/sciadv.abi7159
    • Bland DM, Miarinjara A, Bosio CF, Calarco J, Hinnebusch BJ. 2021. Acquisition of yersinia murine toxin enabled Yersinia pestis to expand the range of mammalian hosts that sustain flea-borne plague. PLOS Pathogens. 17(10):e1009995. doi: 10.1371/journal.ppat.1009995

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:41 minutes

The Most Interesting Things (according to students)

It was interesting how a majority of the bacteria used in the kChip co-cultures prioritized their symbiotic mutualism over their competitiveness.

“Interspecies interactions shape the structure and function of microbial communities. In particular, positive, growth-promoting interactions can substantially affect the diversity and productivity of natural and engineered communities. However, the prevalence of positive interactions and the conditions in which they occur are not well understood. To address this knowledge gap, we used kChip, an ultrahigh-throughput coculture platform, to measure 180,408 interactions among 20 soil bacteria across 40 carbon environments. We find that positive interactions, often described to be rare, occur commonly and primarily as parasitisms between strains that differ in their carbon consumption profiles. Notably, nongrowing strains are almost always promoted by strongly growing strains (85%), suggesting a simple positive interaction–mediated approach for cultivation, microbiome engineering, and microbial consortium design.” (Kehe et al. 2021)

1.2. Main paper; discussion starts at 21:12 minutes

The Most Interesting Things (according to students)

It is interesting to think about how a difference in the rodent species might have made a huge difference in the number of deaths due to Bubonic Plague.

“Yersinia murine toxin (Ymt) is a phospholipase D encoded on a plasmid acquired by Yersinia pestis after its recent divergence from a Yersinia pseudotuberculosis progenitor. Despite its name, Ymt is not required for virulence but acts to enhance bacterial survival in the flea digestive tract. Certain Y. pestis strains circulating in the Bronze Age lacked Ymt, suggesting that they were not transmitted by fleas. However, we show that the importance of Ymt varies with host blood source. In accordance with the original description, Ymt greatly enhanced Y. pestis survival in fleas infected with bacteremic mouse, human, or black rat blood. In contrast, Ymt was much less important when fleas were infected using brown rat blood. A Y. pestis Ymt− mutant infected fleas nearly as well as the Ymt+ parent strain after feeding on bacteremic brown rat blood, and the mutant was transmitted efficiently by flea bite during the first weeks after infection. The protective function of Ymt correlated with red blood cell digestion kinetics in the flea gut. Thus, early Y. pestis strains that lacked Ymt could have been maintained in flea-brown rat transmission cycles, and perhaps in other hosts with similar blood characteristics. Acquisition of Ymt, however, served to greatly expand the range of hosts that could support flea-borne plague.” (Bland et al. 2021).

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • Evolution (V&C_E)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement (ASM_12): Bacteria and Archaea exhibit extensive metabolic diversity, including nitrogen fixation, methane production, and anoxygenic photosynthesis, many of which are unique to these two domains
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 20 (AMS_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • 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.
  • Fundamental Statement 23 (ASM_23):  The health of the environment and all organisms (microbes, plants, humans, other animals) are closely linked and interdependent, as described by the One Health paradigm.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Differentiate between the different symbiotic relationships.
  • Identify the technology improvement that allowed the researchers to test 150,000 interactions.
  • Identify the general conclusions of the research.
 S L
  • Propose experiments that the researchers can use to address the limitations of the study mentioned by the podcasters.
 S H
  • Recall the disease caused by Yersinia pestis.
  • Identify the research that led the researchers to explore species as a factor in this study.
 M L
  • Make a recommendation based on the conclusion in this study that could help prevent future pandemics of Bubonic Plague.
 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

  • Co-culturing (5:30): The process of rapidly growing multiple species of bacteria (often included in pairs) to determine bacterial relationships in a given microbiome under controlled, predetermined conditions.
  • kChip (5:32): A method of rapidly testing and culturing multiple bacterial species concurrently and in the same environment.
  • Biolog System (8:02): A system that allows bacteriologists to easily identify strains of both gram-positive and gram-negative bacteria from a known database.

4.2. Main Paper

  • Fluorescent Microscopy of Tagged Bacteria (28:50): Fluorescence is often used to locate proteins  within cells or tissues using a “tag” that emits light when excited by a laser.  Here, the researchers use a fluorescently tagged bacterium to follow/locate it within the flea.
  • Feeding Chamber (31:55): This technique is used to mimic how fleas feed from a mammalian host.  Fleas are put on a glass chamber covered by parafilm or rodent skin and allowed to feed from the blood in the glass chamber.

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

5.1. Snippet Paper

  • Mutualism (4:02): A type of symbiotic relationship between two species in which both species involved are benefitted.
  • Co-culture (5:44): The process of growing two or more species of bacteria in the same culture.
  • Bacterial Competition (15:29): Different species of bacteria competing over resources, often outgrowing one another in a parasitic relationship.
  • Black Queen and Red Queen Hypotheses (18:01; 36:36): Black Queen hypothesis involves  the adaptation strategy by loss of “expensive” genes by a specific community member so long as the community provides what is needed in the loss.  The Red Queen hypothesis involves the adaptation strategy of constant competition for survival.  Here, the researchers found more evidence of the Black Queen hypothesis since so many relationships were positive (benefit to each) rather than negative (competition).
  • Genetic Drift (20:33): The variation of genetic frequencies within a specific gene pool caused by various evolutionary factors.

5.2. Main Paper

  • Yersinia pestis (21:57): The causative bacterial agent of the bubonic plague.
  • Plasmid (24:47): A carrier of bacterial genetic information outside of the bacterial chromosome that can transfer genetic material to external organisms, such as during horizontal gene transfer.
  • Bactericidal Agents (37:24): Specific antibiotics that inhibit the function or formation of bacterial cell walls.

6. Podcast Questions

  1. Match the relationship with its description. [1 = neutralism; 2 = parasitism; 3 = commensalism; 4 = amensalism; 5 = mutualism]
    1. ______ A relationship in which both species benefit.
    2. ______ A relationship in which neither species benefits or is harmed.
    3. ______ A relationship in which one species benefits and the other is not harmed.
    4. ______ A relationship in which one species is harmed and the other is not harmed.
    5. ______ A relationship in which one species benefits and the other is harmed.
  2. The researchers used the ______ to enable them to test 150,000 co-culture interactions, which is an improvement over the _____ system which used microtiter plates.
    1. Biolog; kCulture
    2. kChip; Biolog
    3. Biochip; kChip
    4. kCulture; Biochip
  3. The podcasters and researchers note that the co-culture experiment has its limitations. Match the experiment with the limitation it will address.
Limitation Experiment: The researchers should also …
a. ____ Only two bacterial orders were tested 1. grow bacteria on rich medium.
b. ____ Only soil-derived bacteria were tested 2. grow bacteria with polymers as the carbon source.
c. ____ Bacteria were initially grown on minimal medium 3. test bacteria from other environments.
d. ____ Bacteria were initially grown on glucose medium 4. grow bacteria using other sugars.
e. ____ Bacteria were grown using only soluble carbon sources 5. test bacteria from other taxonomic groups
  1. According to the podcasters, what were the major conclusions of the co-culture study? [pick all that apply]
    1. Pairs of bacteria that grew weakly individually usually had positive relationships in co-culture.
    2. Pairs of bacteria that grew well individually usually had negative relationships in the co-culture.
    3. Pairs of bacteria where one grew slow and one grew well usually had positive relationships.
    4. Pairs of bacteria where one grew in glucose and one in galactose usually had positive relationships.
  2. Yersinia pestis causes the infectious disease called ______ .
    1. Bubonic plague
    2. Tetanus
    3. Influenza
    4. Tuberculosis
  3. What discrepancy in findings caused the researchers to focus on the species from which the blood came as a major factor?
    1. Researchers found that fleas feed on blood from the dorsal skin of brown rats, but ventral in mice.
    2. One group found that brown rats had greater numbers and more aggressive fleas than mice had.
    3. Researchers found that all rodents except brown rats have an antitoxin called Ymt in their blood.
    4. One group found that Ymt is required for survival in the flea, but another group found it wasn’t.
  4. Based on the podcast discussion, which rodent species should be the most relevant to control to prevent future Bubonic Plague pandemics?
    1. Mouse
    2. Brown rat
    3. Black rat
    4. All of the above

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3AD) and one from the main paper (Figure 5).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in stacked bar charts.
  • Analyze data to draw conclusions about interaction differences based on carbon source.
  • Analyze data to draw conclusions about interaction differences based on taxonomic group.
Experimental Background (Kehe et al., Figure 3AD)

In nature bacteria interact with each other as co-cultures, which is unlike the laboratory where we grow bacteria in pure cultures (mono-cultures).  While laboratory conditions give us a viewpoint of the bacterial characteristics under those conditions, it is certainly limited.  Learning more about how they interact with each other, and also how they grow and what they might produce, is important for both nature conservation and how we might co-opt their life-style to produce compounds or limit growth of pathogenic species.  To learn more about and characterize bacteria-bacteria interactions, Kehe et al. (2021) employed a new technology called the kChip to broadly characterize interactions as commensalistic (light blue), mutualistic (dark blue), parasitic (purple), competitive (red), amensalistic (pink), or neutralistic (gray).  Briefly, they co-cultured pairs of soil bacteria from two taxonomic orders where one bacterium of the pair was labeled with green fluorescent protein and its growth quantified.  They investigated interactions when the co-culture was performed with different carbon sources (panel A) and also characterized what types of interactions were most prevalent when pairs did or didn’t differ in their taxonomic group: En, Enterobacterales. Ps, Pseudomonadales.

stacked bar chars
Figure 3. “Positive interactions depended strongly on strain properties.(A) Interaction classification by carbon source organized into biochemical categories. Bar colors indicate interaction classification (legends are provided in Fig. 2A). (B) Average interaction type by number of carbon atoms. The dot color indicates the biochemical class. (C) Interaction classification by phylogenetic relatedness of the taxonomic pairs. En, Enterobacterales. Ps, Pseudomonadales. (D) Interaction type by phylogenetic relatedness of the taxonomic pairs, averaged across all carbon sources. (E) Interaction classification by pairwise Euclidean metabolic distance (binned). Bin 0 represents with self-interactions. Bins 1 to 8 each contain roughly equal numbers of bidirectional interactions. (F) Interaction type by metabolic distance. All data are at 72 hours.” (Kehe et al. 2021)

7.1.2. Questions

  1. Which color represents the relative abundance of commensal interactions?
    1. Light blue
    2. Purple
    3. Pink
    4. Dark blue
  2. Which group in panel A shows the co-culture with sugar alcohols as the carbon source?
    1. First group from the left; gold lettering
    2. Second group from the left; pink lettering
    3. Third group from the left; green lettering
    4. Fourth group from the left; blue lettering
  3. When trehalose (tre) disaccharide is the carbon source, which type of interaction is observed most frequently?
    1. Mutualistic
    2. Commensalistic
    3. Parasitic
    4. Competitive
  4. Of all the carbon sources used for the co-cultures, which interaction is most common?
    1. Competitive
    2. Mutualistic
    3. Parasitic
    4. Commensalistic
  5. Which carbon source supports the greatest amount of mutualistic interactions?
    1. TCA succinate
    2. Uridine
    3. Amino acid Ala
    4. TCA fumarate
  6. Interaction types between and across the two taxonomic orders were quantified in panel D.  What can you conclude about “between” and “across” interactions? [pick all that apply]
    1. There are more parasitic interactions among bacteria within the same taxonomic group than across groups.
    2. There are more parasitic, mutualistic, and commensalistic interactions across groups than among a group.
    3. There are more competitive interactions among bacteria within the same taxonomic group than across groups.
    4. There are more amensal and neutral interactions within the Enterobacterales than within Pseudomonadales.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of line graphs and their associated notations.
  • Identify the positive and negative controls in an experiment.
  • Analyze the data and draw conclusions regarding the effects of blood source, flea species, and ymt-status on transmission and flea infection.
Experimental Background (Bland et al., Figure 5)

Yersinia pestis is the bacteria responsible for causing Bubonic Plague, which caused at least three pandemics, one in the 500 AD, one in the mid-1300s, and one in the late 1800s.  The most deadly to date was the one in the 1300s which killed 1-2 million people worldwide.  Y. pestis is a recent relative of another bacterium, Y. pseudotuberculosis, which is a mild pathogen.  Studies comparing these identified that only five mutations could change the mildly pathogenic Y.  pseudotuberculosis into a highly pathogenic bacterium much like Y. pestis.  One of these changes was in Yersinia murine toxin (Ymt), a factor that until now had been ignored due to conflicting results about its effects/role in infection.  Bland et al. (2021) used these conflicting results to hypothesize that the blood source, specifically, the mammal species would play a major role.  In these studies they were interested in determining the effect of the blood source (Brown rat or mouse) and flea species (Xenopsylla cheopis or Odontopodisma montana) on transmission of wild-type (KIM6+) or ymt-deficient (KIM6+ymtH188N) Y. pestis.  They started by infecting each flea species using mouse or brown rat blood inoculated with wild-type or the ymt-deficient strain.  They quantified transmission by determining the number of living bacteria that could be isolated from the flea feeding apparatus (panels A and B) as well as infection by quantifying the number of fleas that became infected (panels C and D) over 30 days.  They also recorded the number of fleas with a blocked feeding apparatus of the total number fed (numbers at points in panels A and B).

line graphsFigure 5. “Rodent fleas can transmit Ymt mutant Y. pestis for at least 3 weeks when infected using brown rat blood. Y. pestis transmission dynamics were monitored for 3 to 4 weeks for groups of 150–267 X. cheopis (A) or O. montana fleas infected using 3.4 x 108−1.9 x109 CFU/ml KIM6+ or KIM6+ymtH188N Y. pestis (B) in either mouse (blue) or brown rat (red) blood and subsequently maintained on sterile blood of the same type. Numbers in parentheses indicate the total number of fleas that fed followed by the number of fleas with evidence of foregut obstruction (partially or fully blocked). Roughly equivalent numbers of male and female fleas were used for transmission assays. Infection rate was determined for groups of 10–20 female C) X. cheopis or D) O. montana at various times following infection. https://doi.org/10.1371/journal.ppat.1009995.g005 “(Bland et al. 2021, no changes)

7.2.2. Questions

  1. What is the notation for bacteria that are deficient in ymt and what type of mutation is this?
    1. KIM6+ymtH188N; Gain of function
    2. KIM6+; Loss of function
    3. KIM6+ymtH188N; Loss of function
    4. KIM6+; Gain of function
  2. Which panel and which line shows the number of living bacteria found in the X. cheopis fleas fed the brown rat blood inoculated with the wild-type bacteria?
    1. A; red closed circles
    2. A; red open circles
    3. B; red closed circles
    4. B; blue open circles
  3. In the experiment shown in panel C, what is the positive control?
    1. The wild-type bacteria inoculated into mouse blood
    2.  The ymt-deficient bacteria inoculated into rat blood
    3. The ymt-deficient bacteria inoculated into mouse blood
    4. The wild-type bacteria inoculated into brown rat blood
  4. What conclusions can you make regarding the effect of Ymt presence/absence on flea transmission from brown rat blood and mouse blood in X. cheopis? Pick all that apply.
    1. CFU steadily increased in rat blood regardless of whether Ymt is present, so the longer fleas are infected, the more transmission.
    2. When Ymt is absent, CFU is reduced in mouse blood compared to rat blood, so transmission from mice would be less than from rats.
    3. CFU is increased for all bacteria regardless of blood type or Ymt presence, so both bacteria from both bloods are transmitted well.
    4. When Ymt is absent, CFU is low for both mouse and brown rat blood, so transmission from mice or from rats is dependent on Ymt.
  5. Based on the data, which type of flea, bacteria, and blood-feed species should you be most concerned about in stopping the next Bubonic Plaque pandemic?
    1. Rat; ymt-deficient; X. cheopis
    2. Mouse; wild-type; both fleas
    3. Rat; both bacteria; both fleas
    4. Mouse; ymt-deficient; O. montana

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

License

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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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