Microbial Ecology

TWiM#265: Antiviral Hotspots and Dessication Tolerance

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:38 minutes

The Most Interesting Things (according to students)

The relationship between satellite phages (like P4) and their helper phages (like P2) can shift from parasitic to mutualistic depending on the environment. Satellite phages bring in antiviral defense systems that protect both the bacterial host and the helper phage from other aggressive phages in a competitive setting. This causes a normally parasitic relationship to turn into a mutually beneficial one.

“Bacteria carry diverse genetic systems to defend against viral infection, some of which are found within prophages where they inhibit competing viruses. Phage satellites pose additional pressures on phages by hijacking key viral elements to their own benefit. Here, we show that E. coli P2-like phages and their parasitic P4-like satellites carry hotspots of genetic variation containing reservoirs of anti-phage systems. We validate the activity of diverse systems and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Antiviral hotspots participate in inter-viral competition and shape dynamics between the bacterial host, P2-like phages, and P4-like satellites. Notably, the anti-phage activity of satellites can benefit the helper phage during competition with virulent phages, turning a parasitic relationship into a mutualistic one. Anti-phage hotspots are present across distant species and constitute a substantial source of systems that participate in the competition between mobile genetic elements.” (Rousset et al. 2022, no changes)

1.2. Main paper; discussion starts at 20:08 minutes

The Most Interesting Things (according to students)

  • We found it fascinating that the researchers identified the hydrophilin proteins DtpA and DtpB in A. baumannii as key components enabling the bacterium to survive in dry conditions, such as those found in hospital settings. This discovery provides a promising start to combating A. baumannii-associated healthcare infections. I also enjoyed learning that studying these proteins has broader applications, such as preserving over-the-counter probiotics and stabilizing refrigerator-dependent mRNA vaccines, which could facilitate global vaccine distribution.
  • Desiccated A. baumannii causes more virulent infections from a few adaptations. Mice infected with the desiccated A. baumannii had higher bacterial loads in their tissues compared to non-desiccated infected mice. There was also increased resistance to immune defenses, all which give it competitive advantage over bacteria that hadn’t been desiccated.

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 8.2.

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)
  • Microbial Ecology(V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology(V&C_ME)
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 favoring the growth and survival of certain variants.
  • Fundamental Statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.
  • 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 favoring the growth and survival of certain variants.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.
  • Fundamental Statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society, and ecosystems.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify at least two bacterial defense mechanisms against phages.
  • Define satellite phages and how they with other types of phages.
 S L
  • Predict the experimental outcome if you were trying to identify other mutualistic phage interactions.
 S H
  • Describe the role of DtpA protein in desiccation tolerance of Acinetobacter baumannii.
  • Recall potential applications for hydrophilin proteins like DtpA in biotechnology or medical settings.
 M L
  • Propose an experiment related to a hypothesis discussed in the podcast.
 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

  • Plaque Assays (3:56–5:06; 6:46–9:25; 10:20–11:26; 14:20 — 16:07; 18:30–18:36): These assays quantify the number of viral plaques on bacterial lawns after phage infection.  Here, they were used to measure the effectiveness of cloned bacterial defense systems.  Here, the role of the Ocr protein in activating the PARIS system (an anti-infection system) was tested by transforming E. coli with plasmids expressing Ocr, infecting with phage T7, and assessing infection efficiency.
  • Cloning (9:45–10:17): These are molecular biology methods to isolate a gene, usually by polymerase chain reaction (PCR) or restriction enzymes and study it from within a plasmid.  Here, defense system genes from E. coli were amplified and cloned into plasmids then transformed into E. coli for testing. Mutants were also constructed and verified.
  • Next Generation Sequencing and Variant Analysis (11:30–13-55): This is a next generation sequencing technique to sequence the genome and compare sequences to identify variants that may be responsible for a phenotype.  Here, phage mutants that overcame bacterial defense were isolated, their DNA was extracted, and mutations were identified.

4.2. Main Paper

  • Desiccation Assay (22:00–23:34; 24:00–25:00): Assays are used to quantify a specific feature related to the study. Here, the ability to survive desiccation was assayed by growing bacterial cultures under controlled conditions. Specifically, desiccated samples were rehydrated at various time points and viable cells were determined.
  • Stress Assays (24:12–25:55): Assays are used to quantify a specific feature related to the study, here the ability to withstand specific environmental stressors. Here, Acinetobacter baumannii was subjected to desiccation, oxidative stress, and heat, then survival was quantified.
  • Strain Generation (26:30–26:50; 33:20–37:31): To study the function of genes, strains lacking or containing extra copies of genes are used.  Here, the researchers used  allelic exchange to create multiple mutants, targeting genes like lon, dtpA, dtpB, bfmR, and katE in Acinetobacter baumannii.
  • Transposon Sequencing (Tn-Seq) (26:55–29:00): Transposon sequencing is a next generation sequencing genomic screening method to identify genes involved in specific phenotypes.  Here, it was employed to identify genes in Acinetobacter baumannii that contribute to desiccation tolerance. The method involved generating a transposon mutant library pool of bacteria with transposon insertions and sequencing those with altered desiccation survival to identify the gene that was disrupted by the transposon.
  • β-galactosidase Assay (30:20–31:10): This is a colorimetric enzymatic assay.  Here the researchers conducted the assay to test DtpA’s ability to stabilize enzymes under stress conditions. They found that DtpA was more effective than the traditional stabilizing protein bovine serum albumin (BSA), confirming its role in protecting enzymes from heat and desiccation stress.

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

5.1. Snippet Paper

  • Bacterial Defense Mechanisms  (6:00–9:00): Bacteria have developed various strategies to defend against phage infections, including restriction enzymes, abortive infection, and nucleotide depletion. These mechanisms cluster in “defense islands,” allowing for efficient expression of defense genes and highlighting the organized nature of bacterial defenses.
  • Phage Evolution and “Moron” Genes (9:00–10:00): Phages evolve alongside bacteria, incorporating non-essential “moron” genes that enhance their host’s fitness. This relationship ensures the phage’s survival is tied to that of the bacterium, reflecting complex evolutionary dynamics.
  • Evolutionary Arms Race (20:00–20:20): The interactions between bacteria and phages highlights an evolutionary arms race where each organism develops strategies to counter the other’s defenses. This dynamic leads to continuous adaptation, influencing microbial communities.

5.2. Main Paper

  • Antimicrobial Tolerance in Pathogens (20:54–31:09): The ability of Acinetobacter baumannii to survive prolonged periods of desiccation poses significant risks in healthcare settings. The study shows that desiccation enhances the ability of A. baumannii to withstand reactive oxygen species, which typically harm bacteria. Understanding these tolerance mechanisms is crucial for controlling infections caused by this resilient pathogen.
  • Interactions Between Genes in Desiccation Response (24:00–37:28): The research explores how various genes interact to confer desiccation tolerance, emphasizing the complexity of bacterial adaptations and highlighting the importance of gene networks in resilience.
  • Role of Hydrophilins in Bacterial Resilience (29:25–30:02; 33:32–36:04; 40:40–41:30): Hydrophilins, particularly the DtpA protein, create protective barriers around cellular components during desiccation, aiding in survival and suggesting potential applications in biotechnology for stabilizing sensitive materials.

6. Podcast Questions

  1.  In studying the phage-bacteria interactions, researchers explored satellites as being dependent on the helper phage’s capsid protein to package their genetic material. In addition to this dependence, what other effect might these satellites have on their helper phages?
    1. increase the helper phage’s reproduction rate
    2. inhibit the reproduction of the helper phage
    3. increase the size of the helper phage genome
    4. inhibit the helper phage from infecting coli cells
  2. The Ocr protein in certain phages has a unique feature that helps it evade bacterial restriction-modification (R-M) systems. How does Ocr achieve this, and what is its specific function?
    1. modifies bacterial DNA directly, preventing it from being cut by restriction enzymes
    2. replaces amino acid in the PARIS system to avoid restriction enzyme recognition
    3. mimics the structure of DNA and inhibits restriction enzymes in the bacterial R-M system
    4. blocks the PARIS system by breaking down bacterial proteins involved in defense
  3. What role does the Ocr protein play in the bacterial defense strategy against phages, and what is the outcome?
    1. bypasses the R-M system; triggers the PARIS leading to abortive bacterial infection
    2. directly attacks the phage, thus causing the self-destruction of the phage particles
    3. enables the bacterial R-M system, allowing the phage to replicate and assemble freely
    4. modifies bacterial DNA, which makes it unrecognizable to phage and phage-like proteins
  4. The speakers discuss how the P2-like phage and P4 satellite relationship involves the satellite utilizing  P2-like phage’s capsid and P4 satellite having functions that  don’t inhibit P2 replication.  This is categorized as a mutualistic relationship.  You want to identify other satellite-phage mutualistic pairs, so you take two bacterial strains, one with a known satellite and one without. You infect both strains with each functional “test” phage, compare levels of the satellite phage particles and the functional phage particles. What results would you expect for a mutualistic interaction?
    1. You should see only satellite phage particles and not functional phage particles.
    2. You should see both satellite phage particles and functional phage particles.
    3. You should see only functional phage particles and not satellite phage particles.
    4. You should see neither satellite phage particles nor functional phage particles.
  5. Which of the following best describes the role of the DtpA protein in Acinetobacter baumannii?
    1. enhances bacterial motility by activating flagella
    2. facilitates DNA replication and DNA repair
    3. promotes bacterial biosynthetic processes
    4. forms protective barriers during desiccation
  6.  Which of the following could be a biotechnological application of hydrophilin proteins like DtpA?
    1. Improving bacterial DNA replication and repair speed
    2. Enhancing enzyme stability in industrial processes
    3. Decreasing levels of bacterial antibiotic resistance
    4. Reducing bacterial infection rates in hospitals and clinics
  7. The podcasters discuss the need to identify whether people have antibodies to the DtpA or similar proteins before they can be used in the clinic.  What type of experiment and what sample source would allow you to identify the answer to this?
    1. Enzyme-linked immunosorbent assay (ELISA); human serum
    2. Mass spectrometry; tissue lysate from human kidney or lung
    3. Electron microscopy; sections of spleen or thyroid tissue
    4. Beta-galactosidase assays; human blood or urine samples

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 7) and one from the main paper (Figure 1) .

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features and functions in the schematic diagrams.
  • Analyze the sequence similarity, gene structure, and gene presence to identify related phages and phage components.
  • Propose a mechanism by which these prophages acquired their anti-phage systems.
Experimental Background (Rousset et al., Figure 7)

Restriction-modification, CRISPR-Cas. and nucleotide depletion are some of the anti-phage systems employed by bacteria to fight viral infection.  Understanding these systems is crucial because they represent mechanisms by which bacteria protect themselves from phage infections.  These studies have implications for bacterial evolution, but also for the development of phage-based therapies. Here, Rousset et al. (2022) identified and characterized a new anti-phage system by drawing on the large abundance of available genomic resources. In their study they identify and validate anti-viral hotspots in E. coli and then look for these same features, as well as known defense system components in prophages of other bacterial genomes, Vibrionales (panel A) and Bacilliales (panel B).

Prophase genome visualized as a diagram.
Figure 7: “Hotspots for anti-phage systems encoded on other prophage genomes. (A and B) Genomic view of hotspots encoded on prophages from Vibrionales (A) and Bacilliales (B). Phage genes are shown with different shades of blue. Gray shades show the percentage of identity between homologous proteins from different genomes. Genome accession numbers and positions are shown on the left.” (Rousset et al. 2022, no changes)

7.1.2. Questions

  1. What color represents the phage genes involved in replication?
    1. red
    2. yellow
    3. dark blue
    4. light blue
  2. Which two Vibrio anti-phage systems have the most similar integrase genes? [pick 2]
    1. Vibrio parahaemolyticus S130 a
    2. Vibrio campbellii ATCC BAA-1116
    3. Vibrio parahaemolyticus J-C2-34
    4. Vibrio crassostreae ZF-91
    5. Vibrio cyclitrophicus FF75
    6. Aliivibrio wodanis AWOD1
  3. Which of the following statements best describes the candidate defense systems shown in the figure?
    1. They are known defense systems identified in previous studies and are marked in red.
    2. They are genes hypothesized to be novel defense systems and are marked in yellow.
    3. They are genes related to replication functions and are marked in dark blue.
    4. They are primarily related to integrase functions and are marked in light blue.
  4. Which of the following is a the most reasonable conclusion for similarity of prophage (phage) genes?
    1. All of the known prophage genes are alike, regardless of the exact bacterial species.
    2. Vibrio prophage genes are more like each other than Bacillus prophage genes are like each other.
    3. Bacillus prophage genes are more like each other than Vibrio prophage genes are like each other.
    4. The end portions of the prophages is highly variable with similarities across bacterial species.
  5. Which of the following is a the most reasonable conclusion for similarity of prophage-encoded defense genes?
    1. The central portion of the prophages (defense genes) is highly variable with similarities across bacterial species.
    2. Vibrio prophage defense genes are more like each other than Bacillus prophage defense genes are like each other.
    3. Bacillus prophage defense genes are more like each other than Vibrio prophage defense genes are like each other.
    4. All of the known defense genes and candidate defense genes are alike, regardless of the exact bacterial species.
  6. Based on the similarity and dissimilarity of phage sequences and genome structures, what is the most likely mechanism by which they acquired the defense systems?  What is your evidence?
    1. random mutation events; they are not similar in the same genus
    2. horizontal gene transfer; they are not similar in the same genus
    3. end joining repair mechanisms; they are genes from the host genome
    4. inherited genome elements; they are most similar in the same genus

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in line and bar plots.
  • Evaluate the data and make conclusions about desiccation survival across species and strains.
  • Analyze host colonization data to make conclusions about differences in bacterial load.
  • Evaluate the data and make conclusions about how desiccation affects A. baumannii survival in oxidative stress conditions.
Experimental Background (Green et al., Figure 1)

Acinetobacter baumannii is a multi-drug resistant bacterium that is the cause of hospital acquired pnuemonia and septicimia.  It poses additional significant risks in healthcare settings due to its persistence on dry surfaces and resist typical disinfectant measures. In this study, Green et al. (2022) investigate how desiccation affects A. baumannii survival (panels A), organ colonization of infected mice (panels C and D), and bacterial survival under conditions of oxidative stress (panel E). Additionally, survival of desiccation for different clinical isolates was also examined (panel B).  Organ colonization was measured by quantifying living bacteria from tissue lysates and is represented at colony forming units (CFU).  Oxidative stress, which is a hallmark of immune cell killing mechanisms, was induced by hydrogen peroxide exposure (H2O2; panel E).

  • This article is not licensed for Creative Commons use, so the abstract and figures cannot be copied here. Please see the article on the journal’s web page.

7.2.2. Questions

  1. Which of the following best describes the trend in A. baumannii survival over time under desiccation conditions (panel A)?
    1. A. baumannii shows an immediate decrease in survival, reaching 0% within a few days.
    2. A. baumannii maintains high survival for a period of time before gradually declining.
    3. A. baumannii survival declines and stabilizes at 50% for the remainder of the observation period.
    4. A. baumannii survival steadily increases over time, showing enhanced tolerance to desiccation.
  2. How many days were the bacterial species and A. baumanii strains treated to desiccation conditions before testing survival (panel B)?
    1. 7 days
    2. 14 days
    3. 28 days
    4. 35 days
  3. What do the bar heights and individual symbols indicate for panels B and D?
    1. median
    2. standard deviation
    3. standard error
    4. mean
  4. How does A. baumannii’s survival following desiccation compare to other bacterial species’ survival following desiccation?
    1. A. baumannii has a lower desiccation tolerance compared to other bacterial species tested.
    2. A. baumannii shows similar desiccation tolerance to E. coli and K. pneumoniae.
    3. A. baumannii has a markedly higher desiccation tolerance than other bacterial species.
    4. All bacterial species tested show equivalent tolerance to desiccation conditions.
  5. Often clinical isolates and laboratory strains can have quite different response to environmental conditions, which is tested here in panel B.  What can you conclude about the survival following desiccation for the clinical isolates of A. baumannii’s?
    1. Clinical isolates are all at least as tolerant to desiccation as the lab strain.
    2. Clinical isolates are twice as tolerant to desiccation as the lab strain.
    3. Clinical isolates are much less tolerant to desiccation compared to the lab strain.
    4. Clinical isolates are variably tolerant to desiccation compared to the lab strain.
  6. Which organ(s) show(s) higher colonization for infection with desiccated A. baumannii compared to infection with non-desiccated (control) A. baumannii (panel C)? [pick all that apply]
    1. Lung
    2. Liver
    3. Spleen
    4. Heart
  7. Which organ colonization is affected the most by A. baumannii dessication (panel C)?
    1. Lung
    2. Liver
    3. Spleen
    4. Heart
  8. How does desiccation impact A. baumannii survival of oxidative stress, as induced by hydrogen peroxide (H₂O₂)?
    1. Desiccated A. baumannii shows reduced survival compared to control at all H₂O₂ concentrations.
    2. Desiccated A. baumannii has increased resistance to H₂O₂ at high concentrations compared to control.
    3. Both desiccated and control A. baumannii have equivalent survival rates at all H₂O₂ concentrations.
    4. Control A. baumannii displays higher resistance to H₂O₂ than desiccated samples at all tested concentrations.

8. Paper Information and Licensing

8.1. Snippet paper

  • Rousset F, Depardieu F, Miele S, Dowding J, Laval AL, Lieberman E., … & Bikard D. 2022. Phages and their satellites encode hotspots of antiviral systems. Cell Host & Microbe. 30(5): 740-753. doi: 10.1016/j.chom.2022.02.018.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.

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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