Information Flow and Genetics

TWiM #192: A Qtip for Phages

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper discussion starts at 4:39 minutes

The Most Interesting Things (according to students)

  • The study of Armillaria gallica C1 is at least 2,500 years old, weighs 400,000 kg, and spans 75 hectares.
  • Armillaria gallica C1 may have advanced DNA repair mechanisms or growth strategies that prevent mutations from accumulating.

Anderson et al (2018) is not licensed for Creative Commons use, so the abstract cannot be copied here. Please see the article on the journal’s web page.

1.2. Main paper discussion starts at 21:41 minutes

The Most Interesting Things (according to students)

  • VP882 represents the first known case of a phage using a host-produced autoinducer to regulate its life cycle.
  • VP882 can time its lysis-lysogeny decision based on host population density.

Silpe et al. (2019) is not licensed for Creative Commons use, so the abstract cannot be copied here. Please see the article on the journal’s web page.

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

 Snippet Main

Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)

ASM Fundamental Statements
  • Fundamental Statement 8 (ASM_18): Genetic variation can influence microbial structures and their functions.
  • 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 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 25 (ASM_25):Microbes are used as models that provide fundamental knowledge about life processes.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve the quality of life.

3.  Potential Learning Objectives for the Podcast

The student will be able to:

Paper1

Order2
  • Identify how the mutations found in C1 are an indicator of genome stability.
  • Recall the unique process of mushroom reproduction, including the kinds of mating. filaments required and the unique cells formed after cell fusion.

S

L
  • Hypothesize how having different mutation rates would affect evolutionary studies.

S

H
  • Identify the normal function of the VqmA quorum sensing pathway in Vibrio cholerae.
  • Recall ways to induce and repress lytic and lysogenic reproduction.
  • Identify the main finding of the main paper’s discussion.

M

L
  • Hypothesize the advantages of the VP882 quorum-sensing system as a candidate for phage-based therapies.

M

H

1 Papers: Snippet (S) or Main (M)

2Learning 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

  • Sample (7:43–12:25): A sample is a small part or quantity that is studied to generalize results to a whole or population.  Here, researchers collected 248 samples of Armillaria across a large forest area and recorded precise GPS coordinates to map spatial distribution and track individual fungal clones.
  • Culturing and DNA Extraction (7:43–12:25): Culturing is a method of growing a species, usually in the laboratory.  DNA extraction is a method for isolating the genomic material (or plasmids) for further study.  Here, rhizomorphs were surface-sterilized, cultured on malt extract agar, and the mycelium was grown and harvested. DNA was then extracted using a CTAB (cetyltrimethylammonium bromide) chemical method for purity.
  • Somatic Compatibility Testing (7:43–12:25): This is a growth assay to determine genetically similar and different fungi of the same species.  Fungal samples were grown together in lab cultures to see if they would merge (indicating the same genetic individual) or form a rejection zone (indicating different individuals). This method helped confirm the identity of the big C1 clone.
  • Illumina Sequencing (12:25–14:47): Illumina sequencing is a next generation type of sequencing based on reagents and instrumentation by the company called Illumina.  HiSeq Illumina sequencing was by paired-end with 155 bp reads at the Centre for Applied Genomics at the Hospital for Sick Children, Toronto. A total of 15 strains were Illumina-sequenced, but only 14 were included in the search for variants.
  • Bioinformatics (12:25–14:47): Bioinformatics is the study of biology using computers.  Here, computational tools were used to map DNA reads to a reference genome and identify genetic changes (mutations) in the form of single nucleotide polymorphisms (SNPs). Only highly confident SNPs were used to assess mutation accumulation.

4.2. Main Paper

  • Cloning Techniques (34:49–35:50): These are molecular biology techniques to isolate, amplify, and study genetic material, usually by ligating DNA into plasmids.  Genes like vqmAᵖʰᵃᵍᵉ  and qtip were cloned under arabinose- or tetracycline-inducible promoters for controlled expression in bacteria.
  • Growth and Lysis Assays (34:49–36:39): Like bioassay, these are assays to help researchers learn more about the specific biology process by monitoring it.  Here, lysis was measured by monitoring culture turbidity (optical density). Induction of VqmAᵖʰᵃᵍᵉ caused rapid bacterial cell death, visible as a drop in optical density at 600 nm.
  • Phage Curing (34:49–36:39): You an use specific methods to remove phage from a bacterium.  Here, they removed the prophage from host cells (cured) to show that VqmAᵖʰᵃᵍᵉ-induced lysis requires phage presence. Lysis was abolished in phage-free strains.
  • Bioassay (36:39–37:45): Bioassays are assays for detecting a particular compound or process or to identify the role of the compound in a process.  Here, DPO (3,5-dimethylpyrazin-2-ol), the quorum-sensing signal, was depleted by omitting threonine, and later re-added to confirm that DPO is required to trigger phage lysis.
  • In vitro Tn5 (IVT) Mutagenesis (38:54–41:30): This is a mutagenesis strategy using transposons. Here, it was used to randomly disrupt phage genes, revealing that gp62 is essential for triggering lysis. Mutants lacking functional gp62 failed to lyse, but lysis was restored when gp62 was reintroduced.
  • Electrophoretic Mobility Shift Assays (EMSA) (47:32–48:13): This is a method to determine whether a protein binds nucleic acid in a sequence specific way.  It was used to test whether VqmAᵖʰᵃᵍᵉ binds directly to specific DNA sequences like the qtip promoter.
  • Co-Immunoprecipitation (Co-IP) (49:30–50:40): This is a protein method to identify interacting proteins using an antibody to at least one of the pair.  Here, Co-IP was used to demonstrate direct physical binding between Qtip and cI repressor proteins, confirming their interaction in the lysis regulation pathway.
  • Confocal Microscopy (49:30–50:40): This is a high resolution fluorescent microscopy technique that you can use to generate 3-dimensional models.  Here, it was used to visualize the subcellular localization of the cI repressor. Qtip caused cI to aggregate at the cell poles, inactivating its repressor function and promoting lysis.

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

5.1. Snippet Paper

  • Mutations: Spontaneous Mutations (12:25–16:02): Random changes in the genome are extremely rare in Armillaria gallica. The mutation rates are extraordinarily low, suggesting high genomic stability for this fungus, over thousands of years.

5.2. Main Paper

  • Quorum Sensing (23:33–24:51): Quorum sensing is bacterial communication using chemical signals (autoinducers) to coordinate behavior. In Vibrio, the autoinducer DPO triggers the expression of genes via vqmA. A phage uses this host signal to decide when to lyse the host cell.
  • Horizontal Gene Transfer (General) (26:01–26:15): Lysogeny represents horizontal gene transfer, as phage DNA can integrate into host genomes, transferring new traits such as toxins (i.e. cholera toxin).
  • Viral Reproduction: Lytic (26:01–27:09): The lytic cycle is discussed when phages lyse bacteria after sensing stress via DNA damage or quorum sensing.
  • Viral Reproduction: Lysogeny (27:09–29:31): The lysogenic cycle is when phage DNA integrates into the host without immediate lysis. Phage VP882 can remain episomal in Vibrio cells.
  • SOS Response (27:52–28:16): A bacterial stress response to DNA damage; induces prophages to exit lysogeny. The host cell’s SOS response, triggered by DNA damage, causes cleavage of the C1 repressor protein, initiating the lytic cycle in lambda and related phages.
  • Operon vs Regulon (38:54–41:36): A set of genes regulated together by a single promoter, such as phage lysis genes. The lysis genes are organized as an operon regulated by transcriptional terminators and anti-terminators like Q protein.
  • Positive Control (Activators) (41:36–42:31): Gene expression is promoted by activators. Q protein functions like an activator by allowing transcription of downstream lysis genes when the repressor is inactivated.
  • Negative Control (Repressors) (41:36–51:15): C1 repressor is a classic example of negative control, preventing transcription of lytic genes until a signal, such as DNA damage or quorum sensing, disables it.
  • Regulation of Gene Expression (General Mechanisms) (41:36–51:15): Discussed thoroughly via the regulation of phage gene expression by repressors (C1), anti-terminators (Q protein), and novel regulators (Q-tip).
  • Phage Therapy (56:20–57:46): Engineered phages are proposed as targeted antimicrobial “kill switches” based on quorum-sensing control mechanisms.

6. Podcast Questions

  1. What mutations were found in the C1 individual of Armillaria gallica, and what do they suggest about genome stability?
    1. High-frequency mutations, suggesting a genome prone to instability.
    2. Few mutations over time, indicating strong genome stability mechanisms.
    3. Random mutations across the non coding regions with no clear pattern of stability.
    4. A high mutation rate throughout the genome similar to rapidly evolving bacteria.
  2. When two Armillaria hyphae fuse and the nuclei do not fuse, the cell is considered __________.
    1. diploid
    2. haplospore
    3. dikaryon
    4. haplotype
  3. Typically, we look at evolutionary studies using DNA or amino acid sequence similarities and differences. Let’s say we were able to use samples from fossilized materials for members of family A and members of family B.  If family A has a standard mutation rate and family B has a lower mutation rate, what would we likely see?
    1. Family A would likely have evolved faster than Family B due to having more mutations available for natural selection.
    2. Family A would likely have evolved more slowly than Family B due to having more mutations hampering DNA replication.
    3. Family A and Family B would likely have evolved at the same rate because mutation rate has little to do with evolution.
    4. Family A would likely have evolved to have fewer species than Family B due to the higher lethal mutation rate.
  4. The podcasters note a number of ways the lytic-lysogenic decision is controlled. Match the condition with its phage lytic-lysogenic state. (1 = lytic; 2 = lysogenic)
    1. _________ presence of C1
    2. _________ absence of C1
    3. _________ mitomycin C exposure
  5. What is the normal outcome of activating VqmA quorum sensing circuit for V. cholerae?
    1. Biofilm formation in the presence of external calcium.
    2. Repression of genes necessary for biofilm formation.
    3. Upregulation of genes needed for biofilm formation.
    4. Suspension of cell division until calcium levels recover.
  6. What was the major finding of the main paper?
    1. Vibriophage VP882 uses a bacterial quorum sensor to enhance bacterial survival in hypoxia.
    2. Vibrio cholerae promoters are the key determinant of the lysis-lysogeny decision, not via quorum sensing.
    3. Vibrio cholerae virulence islands encode proteins that can regulate lysis-lysogeny decision of any phage.
    4. Vibriophage VP882 uses a bacterial quorum sensor to regulate the lytic-lysogenic switch.
  7. What advantages of the VP882 quorum-sensing system make it a potential candidate for phage-based therapies?
    1. It allows the phage to integrate bacterial communication signals, making lysis activation more precise.
    2. VP882 randomly infects and lyses bacteria regardless of species, so it would be effective for many infections.
    3. The quorum-sensing system makes bacteria carrying VP882 resistant to antibiotics, improving its survival.
    4. VP882 uses a number of quorum-sensing molecules in natural environments, so it can respond to many conditions.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 2) 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 a phylogenetic tree and spatial diagram.
  • Analyze the data and make conclusions about the closest sample pairs in space and genetic similarity.
  • Analyze the data and make conclusions about the relationship of genetic similarity and sampling location.
  • Use the results to make a reasonable prediction for how the fungus spreads.
Experimental Background (Anderson et al., Figure 2)

Over 40 years ago a very large underground fungus spanning over 37 hectares was discovered in the Upper Peninsula of Michigan in the United States.  Researchers found this fungus was Armillaria gallica and they designated it as individual C1.  In this study, Anderson et al. (2018) re-visited this fungus to examine it more closely using more modern genetic analyses.  They took 248 samples across the coverage area (now 75 hectares) for their study.  To get a better idea of the genetic stability and variation across this large organism, which they estimated to be 2500 years old, they isolated DNA, performed next generation sequencing, and then performed phylogenetics analysis for fourteen isolates representing different areas of the sampling locations.  The data are displayed as a phylogenetic tree (upper area) with the spatial organization of fungal sampling represented in the lower area.

  • Anderson et al (2018) is not licensed for Creative Commons use, so, the figure cannot be copied here. Please see the article at the journal’s web page.

7.1.2. Questions

  1. What does the scale bar labeled “10 changes” indicate in the phylogenetic tree?
    1. The number of fungal strains collected in the study
    2. The number of mutations found in each strain
    3. The number of years since the last mutation occurred
    4. The scale for relating mutation number to branch length
  2. In the Armillaria gallica spatial distribution sampling diagram, which of sampling site pairs below are closest to each other?
    1. 159:179
    2. 194:170
    3. 179:201
    4. 119:176
  3. In the Armillaria gallica phylogenetic tree, which of isolates of those listed below most genetically similar to each other?
    1. 174:142
    2. 174:170
    3. 174:175
    4. 174:196
  4. What is the relationship between the spatial distribution and genetic variants in Armillaria gallica individual C1 based on these data?
    1. It indicates that different genetic variants are randomly distributed across the area.
    2. It shows that more similar isolates map to closer geographic locations within the fungus.
    3. It demonstrates that all fungal isolates have no mutations and identical genomes
    4. It shows mutations are clustered in specific areas of the genome and the rest is stable.
  5. Do these data support fungal growth as a continuous growth outward in space or formation of spores that become airborne? What is your reasoning?
    1. formation of spores that become airborne; genetically similar isolates were found nearer each other.
    2. continuous growth outward in space; genetically similar isolates were found in no spatial organization.
    3. formation of spores that become airborne; genetically similar isolates were found distant to each other.
    4. continuous growth outward in space; genetically similar isolates were found nearer each other.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in bar graphs, electromobility shift assay results, and experimental design for the data presented.
  • Analyze the electrophoretic mobility shift assay (EMSA) data to make conclusions about DNA-binding preferences of VqmAᵖʰᵃᵍᵉ  and VqmAVc​.
  • Analyze the reporter gene assay results to make conclusions about which promoter, if any, is preferentially activated by VqmAᵖʰᵃᵍᵉ and VqmAVc.
  • Defend how these data support or refute the hypothesis that VP882 hijacks bacterial quorum sensing to control its lysis-lysogeny decision.
Experimental Background (Silpe and Bassler, Figure 5)

Bacteriophages (phages) are thought to contribute significantly to bacterial evolution and a major model in this area is Vibrio cholerae, which owes several of its virulence factor gene acquisitions to phage infection, including CTX which is a major cholera virulence factor for human disease. In this study, Siple and Bassler (2019) investigate a vibriophage that appears to be responsive to a bacterial quorum sensing pathway.  Both phage and bacteria respond to quorum sensing molecule 3,5-dimethylpyrazin-2-ol (DPO).  DPO binds to its bacterial receptor VqmA (VqmAVc) and represses bacterial biofilm formation and virulence gene expression. DPO also binds to a phage-encoded version (VqmAphage) and induces lysis.

To learn more about the interplay between these systems and the mechanism each uses, Siple and Bassler (2019) first wanted to know whether VqmAVc and VqmAphage were interchangeable.  To do this, they constructed a reporter gene that is responsive to VqmAVc (PvqmR–mKate2), which produces fluorescence when activated.  They then engineered a bacterial strain lacking vqmAVc (ΔvqmAVc).  They then added empty vector as a negative control, a plasmid expressing VqmAVc, or a plasmid expressing VqmAphage and assayed for reporter gene activation by quantifying fluorescence (panel A).  They next wanted to investigate the ability of each protein to bind relevant promoter DNAs.  The bacterial promoter they chose regulates expression of bacterial gene vqmR (PvqmR) and the phage promoter they chose regulates expression of phage gene qtip (Pqtip).  They mixed these promoter DNAs and purified proteins VqmAVc and VqmAphage to perform electromobility shift assays (EMSA*; panel B). In this experiment 8x and 1x refer to the amounts of purified protein used in the binding assay.  They next tested for activation of the representative promoters in vivo (E. coli) using reporter gene systems that produce bioluminescence when activated (Pqtip-lux and PvqmR-lux). If the proteins binding to promoters increased transcription, the sample would emit bioluminescence.  In these E. coli strains, they added plasmids engineered to overexpress vqmAVc or vqmAphage in the presence of arabinose. They quantified the amount of bioluminescence for the different promoter-protein combinations in the presence of arabinose (panel C).  In the last experiment for this figure, they investigated the phage lysis effects attributed to activating the quorum pathway in V. cholera.  The strain they used carries the lysogenized vibriophage VP882 and lacks its native vqmA (ΔvqmAVc).  Each protein (VqmAVc or VqmAphage) is again engineered to be overexpressed only in response to arabinose.  They counted the remaining bacteria by measuring optical density of bacterial cultures in the presence and absence of arabinose (panel D).

  • Silpe and Bassler (2019) is not licensed for Creative Commons use, so, the figure cannot be copied here. Please see the article at the journal’s web page.
  • For help with EMSA, see Suresh SK. 2024 Beginner’s guide to investigating protein: DNA interactions using electrophoretic mobility shift assays (EMSAs). Biochem (Lond) 46 (5): 8–11. doi: https://doi.org/10.1042/bio_2023_125

7.2.2. Questions

  1. Which of the following is true regarding the DNA-binding of VqmAPhage and VqmAVc as observed in the electrophoretic mobility shift assay (EMSA)?
    1. VqmAPhage and VqmAVC bind to the same DNA sequences with identical affinities.
    2. VqmAPhage shows stronger binding to the promoter of PvqmR, while VqmAVC shows stronger binding to Pqtip.
    3. VqmAPhage and VqmAVc exhibit distinct DNA-binding preferences, suggesting different regulatory roles.
    4. Both VqmAPhage and VqmAVc bind to any DNA sequence without preference.
  2. Which promoter is preferentially activated by VqmAPhage based on the luciferase reporter assay?
    1. PvqmR
    2. Pqtip
    3. Both promoters equally
    4. Neither promoter
  3. How does the differential gene regulation by VqmAPhage and VqmAVc support the hypothesis that VP882 hijacks bacterial quorum sensing to control its lysis-lysogeny decision?
    1. The differences in gene regulation suggest that VqmAPhage and VqmAVc modulate quorum sensing to favor lysis under high cell density and lysogeny under low cell density.
    2. The differential regulation of promoters supports the idea that VP882 uses quorum sensing to control bacterial survival, but not its lysis-lysogeny decision.
    3. The gene regulation mechanisms are unrelated to quorum sensing and do not impact the lysis-lysogeny decision.
    4. The regulation of Pqtip promoter is the key determinant of VP882’s lysis-lysogeny decision, regardless of quorum sensing.
  4. Which experiment would be most appropriate to determine whether similar quorum-sensing-based phage regulation mechanisms exist in other bacteriophages?
    1. Perform a luciferase reporter assay on different bacteriophages to measure their activation of quorum-sensing-related promoters.
    2. Perform a genome sequencing study to identify quorum-sensing genes in all bacteriophages.
    3. Use CRISPR-Cas9 to edit quorum-sensing genes in a bacteriophage and observe the effects on bacterial lysis.
    4. Investigate whether different bacteriophages use the same DNA-binding motifs as VqmAPhage and VqmAVc.

8. Paper Information and Licensing

8.1. Snippet paper

  • Anderson JB, Bruhn JN, Kasimer D, Wang H, Rodrigue N, Smith ML. 2018. Clonal evolution and genome stability in a 2500-year-old fungal individual. Proc. R. Soc. B 285: 20182233. http://dx.doi.org/10.1098/rspb.2018.2233.
  • 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.

8.2. Main paper

  • Silpe JE, Bassler BL. 2019. A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision. Cell. 176(1-2):268-280.e13. https://doi.org/10.1016/j.cell.2018.10.059
  • 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.

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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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