Information Flow and Genetics

TWiM #249: Phage-Pathogen and Toxin-Antitoxin Conflicts

Podcast and Annotation Information
  • Annotation by Adchayan Santhirasegaran, Jisel Miranda-Santana, Kirsten Nicole Bleming, and Michaela Gazdik Stofer
  • Podcast audio by TWiM: Listen to TWiM #249 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Hannah Harris: Access Podcast Transcripts
  • Papers Discussed:
    • LeGault KN, Hays SG, Angermeyer A, McKitterick AC, Johura FT, Sultana M, Ahmed T, Alam M, Seed KD. 2021. Temporal shifts in antibiotic resistance elements govern phage-pathogen conflicts. Science. 373(6554):eabg2166. doi: 10.1126/science.abg2166
    • Mager A, Safran T, Engelberg-Kulka H. 2021. Intracellular Localization of the Proteins Encoded by Some Type II Toxin-Antitoxin Systems in Escherichia coli. mBio. 31;12(4):e0141721. doi: 10.1128/mBio.01417-21.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:54 minutes

The Most Interesting Things (according to students)

  • Phage therapy is a technique that involves bacteriophage targeting the “bad” cells, in this case possible Vibrio cholerae, which can help fight/prevent further cholera outbreaks within hosts. It is interesting that phage therapy could be used to treat bacterial infections in place of antibiotics.
  • Bacteriophage from stool obtained from patients at earlier or later dates could not infect V. cholera clinical isolates. Only phage extracted from stool around the same time as the clinical isolates were present were able to infect them.

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 read the article on PubMed.

1.2. Main paper; discussion starts at 24:59 minutes

The Most Interesting Things (according to students)

  • Fluorescent microscopy can be used to observe the movement of specific proteins inside of the bacterial cells. I have seen images of this work in eukaryotic cells like human cells, but bacteria are so small I was surprised you could study the movement of proteins within them.
  • Toxin MazF localizes at the poles of a cell but the introduction of antitoxin to a cell caused the toxin to disperse throughout the cell. Most researchers believed that the antitoxin prevented toxin function by binding to it and inhibiting its enzyme activity. Instead researchers showed that the antitoxin can actually change the localization of MazF, which may play a role in preventing it from being toxic.

“Bacterial toxin-antitoxin (TA) systems encode a toxin and an antitoxin that counteracts the toxin. Such TA systems are found abundantly on bacterial chromosomes and on extrachromosomal genetic elements. The toxin is always a protein. Based on the nature of the antitoxin (protein or RNA) and on their mode of regulation, they are classified into six groups (I to VI). In the group II TA systems, both the toxin and the antitoxin are proteins, and the gene specifying the antitoxin precedes the gene specifying for the toxin. Here, we studied the intracellular localization in Escherichia coli cells of the proteins specified by the following type II TA modules: mazEF, chpBIK, mqsRA, and rnlAB. We visualized the localization of these proteins by fusing them with the fluorescent protein mCherry using recombinant DNA technology. We used fluorescence microscopy and image analysis software to obtain and quantify protein distribution data. With the exception of the chpBIK TA module, we found that the localization of each toxin-antitoxin complex was different from the localization of the toxin itself. Our results demonstrate clearly that the presence of the antitoxin shifts the localization of its respective toxin toward the middle of the cell, which could contribute to the reduction of cellular toxicity” (Mager et al 2021, no changes)

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
  • Structure and Function (V&C_SF)
  • Information Flow and Genetics (V&C_IFG)
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 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 9 (ASM_9):The replication of viruses is determined by their unique structures, DNA or RNA genomes, and the cells they infect.
  • Fundamental Statement 5 (ASM_5):The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define the purpose of the lytic bacteriophage replication and infection time-flow assays.
  • Describe how phage resistance in Vibrio cholerae is accomplished.
 S L
  • Identify the role of orbA in Vibrio cholera-phage infection and predict the consequence of it being mutated.
 S H
  • Describe the relationship between toxin and antitoxin proteins.
  • Explain how the fluorescent mCherry protein is used to study specific features of toxin-antitoxin systems.
 M L
  • Defend the importance of localizing the components of toxin-antitoxin interactions.
 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

  • Time-Shift Assay (9:48–11:30): These assays are used to study coevolutionary dynamics in host-parasite or antagonistic interactions. They compare the interactions of host cells with parasites from a given time point compared to those from the past or future..
  • Plaque Assays (10:08–11:30): A plaque assay measures how many virus particles are in a sample. In this experiment plaque assays were used to determine what strains of bacteriophages could infect and lyse V. cholerae clinical isolates
  • Comparative Genomics (12:00–13:00): In this methodology, genome sequences are compared to find differences and commonalities.  Comparison of genome differences between the V. cholerae isolates from different time points allowed researchers to identify how isolates were protected from bacteriophage infection.

4.2. Main Paper

  • Fluorescence Imaging using mCherry Protein (27:03–28:32): This is a microscopy method to follow a protein’s location by having a fusion of the protein of interest and a fluorescent one, here, mCherry. Tagging toxin-antitoxin proteins with the mCherry fluorescent protein allowed researchers to visualize their intracellular location inside of bacterial cells; this was used to track movement of proteins inside the cell to determine toxin-antitoxin interactions.

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

5.1. Snippet Paper

  • Viral Reproduction (3:25–4:42): Lytic vibriophages lytically infect V. cholera clinical isolates. Lytic ability of the bacteriophage is observed through plague assays.
  • Horizontal Gene Transfer (12:05–14:05) :Genetic elements like ICE are used by Vibrio cholerae to acquire resistance genes through transfer of genetic material between different bacterial strains.
  • Phage Therapy (22:48–24:31): Bacteriophage targeting of Vibrio cholerae could be used as a therapy against cholera outbreaks in environments where antibiotic resistance is present

5.2. Main Paper

  • Toxin-antitoxin (25:00–26:02): An antitoxin inhibits the activity of its corresponding toxin, typically by direct binding which prevents the toxin from exerting damaging effects. This research showed that in some TA systems, the binding of the antitoxin changes the localization of the toxin in the cell. (Ex: MazF changes localization from poles of the bacterial cell to localization at the center of the cell when the antitoxin is bound).
  • Plasmids (25:00–26:02): Toxin-antitoxin systems were first discovered associated with plasmid DNA and have a  role in maintaining plasmids in cells. The loss of the plasmid leads to loss of the antitoxin, so the cell is killed by the remaining toxin.
  • Biofilm vs Planktonic Growth Rate (37:12–38:05): RNases associated with toxin-antitoxin systems can degrade mRNA that is not helpful for biofilm formation. This can switch bacteria from planktonic (floating) growth to biofilm development, which can facilitate survival in hostile environments.

6. Podcast Questions

  1. Which of the following best describes what a time-shift assay is?
    1. An experiment that measures the survival rates of sensitive and resistant bacterial strains over time without any external factors.
    2. A technique that examines coevolution by comparing bacterial infection rates between bacteria and phage isolated at different time points.
    3. A  method for determining the exact color and reflectance of bacterial cell membranes using confocal fluorescent microscopy techniques.
    4. An assay that analyzes DNA sequences for mutant types collected from bacteria isolated at different time points, over a long period of time.
  2. What allowed Vibrio cholerae bacteria to prevent infection by bacteriophages?
    1. They changed their genetic code through mutations that prevented bacteriophage binding and infection.
    2. They produce toxins under specific stress conditions that repel bacteriophages by binding phage tails.
    3. They obtain a ICE element containing a BREX system through horizontal gene transfer which restricts phages.
    4. They form symbiont relationships with other bacteria to both block and defend against the bacteriophages.
  3. What role does the protein OrbA play in the coevolution of V. cholerae and vibriophages?
    1. OrbA promotes the integration of ICE elements into the V. cholerae genome, allowing the bacteria to increase resistance to the bacteriophage. The phage would be more resistant and the bacteria would be infected.
    2. OrbA is found in the bacteriophage and acts as a novel anti-BREX inhibitor, allowing phage to overcome the resistance mechanism of the bacteria. If mutant, the phage would not be able to infect V. cholerae
    3. OrbA increases bacterial resistance to antibiotics by increasing levels of horizontal gene transfer in V. cholerae.  Mutating orbA would reduce horizontal gene transfer and the phage population would decline.
    4. OrbA is found in the bacteria and enhances the function of restriction enzymes and degradation of the viral genome.  Mutating orbA would cause the bacterium to be more susceptible to phage infection.
  4. What is the main function of the antitoxin in a bacterial toxin-antitoxin system?
    1. It neutralizes a specific toxin
    2. It damages the outer cell wall.
    3. To increases antibiotic resistance
    4. It replicates the phage genome.
  5. Why is the fluorescent mCherry protein used in studies of toxin-antitoxin systems?
    1. It reduces the toxicity of the toxin protein.
    2. It increases the bacteria’s fitness and survival.
    3. It is a way to visualize protein locations in cells.
    4. It deactivates and degrades the antitoxin protein.
  6. Why is the study of the intracellular location of toxin-antitoxin proteins important?
    1. It aids in finding new treatments for antibiotic-resistant, clinical bacteria strains
    2. It confirms that all bacteria have toxin-antitoxin systems, but some are very weak
    3. It shows plasmids encode the toxin and antitoxin, thus defining a therapeutic target
    4. It provides insights into the biological roles and location of these systems within cells

7. Figure Reading Exercises

The following are two figure reading exercises, the first from the main paper (Figure 2A-C) and the second from the snippet paper (Figure 1A-D).

7.1. First Figure Reading Exercise 

7.1.1. Learning Objectives

Students will be able to:

  • Identify important features in fluorescent microscopy images.
  • Describe the intracellular localization of each component of the mazEF module.
  • Analyze the data to conclude how the presence of MazE alters the spatial distribution of MazF within bacterial cells
  • Defend the experimental approach of using fluorescence tagging to study protein localization
Experimental Background (Mager, et al., Figure 2A-C)

Toxin-antitoxin (TA) systems in bacteria involve two different genes, one that encodes a toxin and another that encodes an antitoxin, which counters the effect of the toxin. There are six different types of TA systems based on the type of antitoxin and method of regulation. The researchers in this paper are studying type II TA systems. Type II systems encode a stable toxic protein and a protein antitoxin that neutralizes the toxin’s effects. Mager et al. (2021) investigate how each component of the mazEF module might influence the cellular localization of the other. The authors tagged each protein separately (MazE in panel A, MazF in panel B) or a fusion of the two (MazEF in panel C) with a fluorescent protein called Cherry (red) to visualize the relative position of TA system proteins inside cells.

Fluorescent micrograph images.

Figure 2: “The intracellular localization of the protein products of the mazEF TA module in Escherichia coli. (A) Two localizations of the toxin MazF were observed, polar (indicated by white arrows) and nonpolar (indicated by yellow arrows). (B) The antitoxin MazE is dispersed in the cells. (C) MazEF (in which antitoxin MazE and toxin MazF are combined) is mostly localized in the middle of the cells (indicated by white arrows). (D) In the presence of the overexpressed antitoxin MazE, the toxin MazF is mostly localized in the middle of the cells (indicated by white arrows). Cell membranes were labeled with FM 1-43FX (green). Bar, 2 μm. Magnified (2×) sections of the images are shown at the corners.” (Mager et al, 2021, no changes)

7.1.2. Questions

  1. What do the white and yellow arrows highlight in the microscopy image in panel A ?
    1. The natural fluorescent differences in the toxin-antitoxin system
    2. The polar and non-polar locations of the MazF protein, respectively
    3. The differing locations of the MazF and MazE proteins, respectively
    4. The presence of true and background fluorescent signals, respectively
  2. Which localization pattern is observed for the antitoxin MazE in E. coli cells?
    1. Dispersed throughout the cytoplasm
    2.  Localized at the poles of the cells
    3.  Concentrated at the mid-cell
    4. Homogeneously distributed across the cell membrane
  3. What patterns do you observe by comparing the individual localizations with the fused MazEF complex in E. coli?
    1. The fusion now localizes at the poles of the bacterial cells.
    2. The fusion is homogeneously distributed in the cytoplasm.
    3. The fusion shifts MazE to the center of the bacterial cells.
    4. The fusion disrupts the localization of the cell’s cytoskeleton.
  4. Which observation suggests that MazE may neutralize the toxic effects of MazF?
    1. MazE remained evenly dispersed in the cell but MazF localization changes.
    2. MazEF localize at the poles when combined together, changing their position.
    3. MazF localization remains unchanged when MazE is also expressed.
    4. MazEF localization is different than both MazF and MazE individually.
  5. Why is the use of mCherry fluorescence tagging critical in this study?
    1. It enables the visualization of protein localization within the cell.
    2. It inhibits the toxicity of MazF and allows the cell to survive.
    3. It enhances the fluorescence of MazF and MazE proteins.
    4. It increases the stability of proteins under stress conditions.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify important features of data visualizations.
  • Identify regions of importance in DNA elements conferring phage resistance.
  • Analyze data to conclude patterns of phage susceptibility in clinical Vibrio cholerae isolates and vibriophages.
  • Predict how phage infection affects the spread of SXT ICEs.
Experimental Background (LeGault et al., Figure 1A-D)

In natural environments, bacterial populations encounter and are attacked by bacteriophages. Bacteriophage predation results in up to 40% bacterial mortality, so phage predation is a driver of bacterial evolution and community composition. Bacteria and phage often coevolve, with bacteria evolving defense mechanisms against phage predation and the phage evolving mechanisms to avoid those defenses. In this study, LeGault et al. (2021) examine how phage drive pathogen evolution using Vibrio cholerae clinical isolates and Vibrio cholerae bacteriophages (vibriophages) isolated from patient stool.  To examine how the bacteria-phage may have co-evolved they performed time-shift assays (schematic in panel A) by infecting V. cholera isolates obtained in different years with phage that were obtained: 1) in the same year as the bacterium (“contemporaneous phage”), 2) earlier in time than the bacterial isolate (“past phage”), or 3) after the time the bacterium was isolated (“future phage”).  The ability of each phage (denoted as ICP1 phage) to infect each clinical isolate was determined and the results displayed as a susceptibility matrix (panel B).  When investigating the genomic differences among resistant strains, the researchers discovered some of these bacteria contained DNA elements called SXT integrative and conjugative elements (SXT ICE; panel B).  SXT-ICE are typically clustered in five regions called hotspot regions.  To investigate the contribution of the bacterial SXT hotspot region 5 (HS5) to phage infection, the researchers conjugated two clinical strains (VchInd5 and VchInd6) that were resistant to all phages to a strain that was sensitive to all phage (SXT-) and tested infection (panel C).  They also mutated the hot spot region for each clinical strain (d-HS5) and tested for infection (panel C).  The genome organization for strains of V. cholerae that are toxigenic and non-toxigenic is compared (panel D).

  • Legault et al, 2021 is not licensed for Creative Commons use, so the figure cannot be copied here. Please see Figure 1 in the article at the journal’s web page.  This paper is available on Pubmed at: https://pmc.ncbi.nlm.nih.gov/articles/PMC9064180/

7.2.2. Questions

  1. Which color square denotes that plaques were found, indicating successful infection in panel B?
    1. purple
    2. gray
    3. teal
    4. white
    5. black
  2. Which feature distinguishes SXT( – ) isolates in the study?
    1. They resist all phage infections regardless of time period.
    2. They are susceptible to all phages from all time periods.
    3. They have no difference in resistance compared to SXT(+) isolates.
    4. They restrict phages using the same mechanisms as SXT ICEs.
  3. What was different between contemporaneous and temporally mismatched Vibrio cholerae isolates and phage pairs?
    1. Contemporaneous pairs were both from clinical isolates while temporally mismatched pairs were always isolated from environmental samples.
    2. Contemporaneous pairs were isolated from patients during the same date range while temporally mismatched pairs were from different date ranges.
    3. Contemporaneous pairs were isolated from the same patient while temporally mismatched pairs came from one patient and one environmental sample.
    4. Contemporaneous pairs were both from environmental samples while temporally mismatched pairs mixed clinical and environmental samples.
  4. What do the time-shift assays reveal about phage susceptibility in Vibrio cholerae isolates?
    1. V. cholerae is resistant to nearly all phages from a specific time period.
    2. V. cholerae in a mismatched pairs were the most susceptible to infection.
    3. V. cholerae isolates are most susceptible to contemporaneous phages.
    4.  Vibriophages infect V. cholerae strains equally regardless of time period.
  5. What conclusion can be drawn from the genomic distribution of the antiphage hotspot 5 of SXT ICEs?
    1. The antiphage genes are only found on SXT ICEs from Vibrio cholerae strains.
    2. SXT ICEs encode phage defense systems in toxigenic strains of Vibrio cholerae.
    3. The antiphage genes are found on SXT ICEs in genomes of diverse bacterial taxa.
    4. SXT ICEs are limited to regions where cholera is endemic and antibiotic resistant.
  6. SXT ICEs are mobile genetic elements and V. cholerae can perform conjugation naturally. Hypothesize how phage infection may influence the transfer of SXT ICEs.
    1. It has no effect on SXT ICE transfer because genomes are stable.
    2. It reduces the conjugation rate of DNA elements between bacteria.
    3. It stimulates high-frequency conjugative transfer of SXT ICEs.
    4. It prevents the dissemination and increase of antibiotic resistance.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Mager A, Safran T, Engelberg-Kulka H. 2021. Intracellular Localization of the Proteins Encoded by Some Type II Toxin-Antitoxin Systems in Escherichia coli. mBio. 31;12(4):e0141721. doi: 10.1128/mBio.01417-21.
  • 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/10.1128/mbio.01417-21

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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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