Evolution

TWiM #213: Fugitive Emissions

Podcast and Annotation Information

  • Annotation by Julian Luna, Eline Mast, Rebecca Seipelt-Thiemann, and Curtis Henderson
  • Podcast audio by TWiM: Listen to TWiM #213 Podcast
  • Podcast transcript by Otter.ai and edited by Grace Helle and Harshita Sharma: Access Podcast Transcripts
  • Papers Discussed:
    • Warnes SL, Little ZR, Keevil CW. 2015. Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials. mBio. 6(6):e01697-15. doi: 10.1128/mbio.01697-15
    • van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, Williamson BN, Tamin A, Harcourt JL, Thornburg NJ, Gerber SI, Lloyd-Smith JO, de Wit E, Munster VJ. 2020. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. N Engl J Med. 382(16):1564-1567. doi: 10.1056/NEJMc2004973
    • Bublitz DC, Chadwick GL, Magyar JS, Sandoz KM, Brooks DM, Mesnage S, Ladinsky MS, Garber AI, Bjorkman PJ, Orphan VJ, McCutcheon JP. 2019. Peptidoglycan Production by an Insect-Bacterial Mosaic. Cell. 179(3):703-712.e7. https://doi.org/10.1016/j.cell.2019.08.054

1. Paper Abstracts

1.1. Snippet papers; discussions start at 1:35 and 15:41 minutes

The Most Interesting Things (according to students)

  • SARS-CoV-2 spreading relies on humidity so places with more humid climates should be alert.
  • Viral particles get degraded when coming into contact with copper.
Snippet 1 abstract

“The evolution of new and reemerging historic virulent strains of respiratory viruses from animal reservoirs is a significant threat to human health. Inefficient human-to-human transmission of zoonotic strains may initially limit the spread of transmission, but an infection may be contracted by touching contaminated surfaces. Enveloped viruses are often susceptible to environmental stresses, but the human coronaviruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently caused increasing concern of contact transmission during outbreaks. We report here that pathogenic human coronavirus 229E remained infectious in a human lung cell culture model following at least 5 days of persistence on a range of common nonbiocidal surface materials, including polytetrafluoroethylene (Teflon; PTFE), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. We have shown previously that noroviruses are destroyed on copper alloy surfaces. In this new study, human coronavirus 229E was rapidly inactivated on a range of copper alloys (within a few minutes for simulated fingertip contamination) and Cu/Zn brasses were very effective at lower copper concentration. Exposure to copper destroyed the viral genomes and irreversibly affected virus morphology, including disintegration of envelope and dispersal of surface spikes. Cu(I) and Cu(II) moieties were responsible for the inactivation, which was enhanced by reactive oxygen species generation on alloy surfaces, resulting in even faster inactivation than was seen with nonenveloped viruses on copper. Consequently, copper alloy surfaces could be employed in communal areas and at any mass gatherings to help reduce transmission of respiratory viruses from contaminated surfaces and protect the public health.” (Warnes, et al. 2015)

Snippet 2 abstract

This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract cannot be copied here. Please see the article at the journal’s web page.

1.2. Main paper; discussion starts at 27:49 minutes

The Most Interesting Things (according to students)

  • Endosymbiosis can occur between more than two organisms.
  • Mass spectroscopy can be used to find peptidoglycan in an organism.

“Peptidoglycan is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to peptidoglycan biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a peptidoglycan layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.” (Bublitz et al. 2019)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • 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 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • 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 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall conditions for the virus to remain infectious.
  • Explain copper’s effect on SARS-CoV-2.
 S L
  • Predict the meaning of virus surveillance test results.
 S H
  • Define endosymbiosis.
  • Recall the evidence that horizontal gene transfer occurred between the insect and bacteria.
 M L
  • Propose an experiment to identify evidence of horizontal gene transfer from Tremblaya to mealybugs.
 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 Forming Units (PFU) (6:39–15:50): Infectious particles can be measured to test their transmissibility across different surfaces and, depending on the infectious particle, whether they can cause infection on certain surfaces.
  • Electron Microscopy (EM) (14:51–15:50): A high resolution type of microscopy that generates an image by shooting a beam of electrons to study small particles including viral particles.
  • Polymerase Chain Reaction (PCR) (17:00–17:09): A molecular technique used to amplify short segments of DNA.  This test can be used to test whether a patient has contracted SARS-CoV-2.

4.2. Main Paper

  • Mass Spectrometry (MS) (38:56–39:31): A technique that quantifies and identifies compounds by measuring their mass-to-charge ratio (m/z) to determine the amount of specific compounds in a sample.
  • Fluorescent In Situ Hybridization (FISH) (39:31–41:25): This technique is used to localize specific DNA or RNA sequences within cells or tissues by using a labeled probe to bind to complementary sequences so they can visually be seen.
  • Immunohistochemistry (IHC) (44:18–45:11): A technique that uses antibodies to locate specific proteins in tissues or cells.

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

5.1. Snippet Papers

  • Measuring Virulence (6:31–8:59): About 10,000 SARS-CoV-2 infectious particles need to be present for infection to occur. Otherwise, the infectivity of SARS-CoV-2 begins to decrease after a few days of staying on a surface.
  • Virulence Factors (1:35–5:08): SARS-CoV-2 spreads more in less humid environments because the aerosol droplets can remain floating in the air longer before they hit the ground. Meanwhile in more humid environments, the viral droplets fuse with the humidity droplets where gravity makes them fall faster to the ground.
  • Quantifying Virus (6:31–8:29): Viral particles (in this case SARS-CoV-2) can be measured based on the amount of infectious particles present, not just by the amount of viral mRNA present.
  • Specificity (8:59–11:52, 13:47–17:08): Certain surfaces will sustain SARS-CoV-2 longer than other surfaces. When comparing stainless steel (a common surface for healthcare workers) to copper, stainless still didn’t do much to stop the spread of SARS-CoV-2. Meanwhile, copper was able to inactivate the virus particles much quicker.
  • Surveillance, Prevalence, Incidence (17:28–17:42): One study in China shows how the floor is a hotspot for SARS-CoV-2 particles.
  • Transition Metal Effects on Pathogens  (11:52–15:50): Transition metals like copper and zinc works best against SARS-CoV-2. This happens because copper liberates free radicals, where the copper one and the copper two interact with oxygen, and make hydrogen peroxide singlet oxygen hydroxy ions. These hydroxy ions are what inactivate the virus. The EM snapshot shows how copper surfaces degrade the viral nucleic acids when they both come into contact.

5.2. Main Paper

  • Building Peptidoglycan (32:26–37:45): Normally, the immune system recognizes peptidoglycan as a foreign substance and releases cytokines against it. Through horizontal gene transfer, the Moranella endosymbiont and other bacteria have passed genes important in making peptidoglycan the mealybug genome. In other words, the bacteria have a way to synthesize peptidoglycan without encoding all the genes.
  • Horizontal Gene Transfer (29:03–31:36): Horizontal gene transfer occurs when genes are passed from one organism to another as opposed from parent to progeny. This helps evolution occur which is seen with the chloroplasts and mitochondria genes that were absorbed by eukaryotes. Some of their genes were passed onto the eukaryotic genome while others were lost. The podcast talks about how the mealybug genome has some peptidoglycan synthesizing genes for its endosymbionts. Through horizontal gene transfer, the Moranella can synthesize peptidoglycan.
  • Endosymbiosis Theory (30:09–:32): Endosymbiosis occurs whenever an organism lives inside a host for a while and shares genes through horizontal gene transfer. The most common example is how the mitochondria and chloroplast became part of the eukaryotic cell. The podcast talks about the mealybug sharing genes with Moranella and Tremblaya bacteria. Both Moranella and Tremblaya genomes have been reduced compared to a free-living species.
  • Gram Negative Cell Envelope (35:48–37:45): Three classes of reactions based on where the enzyme location happens to make peptidoglycan. First is the cytoplasmic synthesis of the precursor of peptidoglycan. The second set of reactions involve an inner membrane associated with enzymes that flip this peptidoglycan precursor into the periplasm of the bacterium. And then the third set involves crosslinking of precursors into the growing peptidoglycan matrix and modifications.
  • Chloroplasts and Mitochondria (29:23–30:08): Both the chloroplasts and mitochondria are examples of endosymbiosis within eukaryotes. They were originally bacteria that lost some of their genes when they got engulfed by the eukaryotic cells. However some of those genes were kept and through horizontal gene transfer got integrated into the nuclear genome.
  • Antimicrobials and Their Targets (32:26–33:08, 41:09–42:22): Most antimicrobials target peptidoglycan since it’s a structure unique to bacteria. When eukaryotes detect peptidoglycan, the immune system begins making cytokines to fight the foreign bacteria. This is why many antimicrobials target the peptidoglycan synthesis pathway. More specifically, antimicrobials will inhibit new peptidoglycan from being added to a growing peptidoglycan chain. This makes antimicrobials useful for experimental use to see structural changes in the membranes of the endosymbiotic bacteria.

6. Podcast Questions

  1. Which of the following affect(s) a viral particle’s ability to remain infectious? [pick all that apply]
    1. The ability to persist on a particular surface
    2. The match between virus and host receptor
    3. The thickness of the virus’ protein capsule
    4. The type of nucleic acid present in the virus
  2. What was the relationship between copper and virus inactivation?
    1. The higher the copper in the metal alloy, the better the virus could infect.
    2. The higher the copper in the metal alloy, the more quickly the virus inactivated.
    3. The higher the copper in the metal alloy, the better the virus was protected.
    4. The higher the copper in the metal alloy, the more quickly the virus inactivated.
  3. The podcasters discuss different virus surveillance tests, including plaque forming unit (PFU) assays and polymerase chain reaction (PCR) tests.  If a particular sample showed a negative result in PFU assay and a positive result in PCR test, what could this indicate?
    1. The virus  or its RNA was present and also able to infect cells.
    2. The virus or its RNA was not present and also unable to infect.
    3. The results conflict, so this must be considered a false result.
    4. The virus RNA was present, but no infective virus was present.
  4. Endosymbiosis is when_______________.
    1. One organism phagocytoses another organism.
    2. Two organisms have identical internal structures.
    3. One organism lives within another organism.
    4. Two organisms live in the same ecological habitat.
  5. What is the main evidence that mealybugs acquired some genes from Moranella by horizontal gene transfer?
    1. The genome of Moranella is very small due to extreme gene loss, so it is unable to make many biosynthetic enzymes for its own processes.
    2. The mealybug produces peptidoglycan for itself as well as Moranella, which was determined by using nitrogen-15 labeled D-alanine precursors.
    3. Moranella provides a natural immunity that also protects the mealybug, so this gives both a selective advantage when living in nature.
    4. Moranella, not mealybugs, use peptidoglycan. Some peptidoglycan synthesis genes are found in the mealybug, not the Moranella genome.
  6. Tremblaya is the other endosymbiont of the mealybug.  How could you find evidence of horizontal transfer from this species to the mealybug?
    1. Examine how the Tremblaya’s peptidoglycan layer is formed using N-15 labeled D-alanine. Just like for the Moranella experiment you would see N-15 layers.
    2. Examine which biological pathways are incomplete in the small genome of Tremblaya and look in the mealybug genome for genes encoding those enzymes.
    3. Compare the sequence of the Tremblaya genome to the sequence of the mealybug genome and identify any genes present only in one of the species.
    4. Make cell lysates of Tremblaya only cells and mealybug only cells, then use a fluorescently tagged antibody to identify proteins that are present in both.

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 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of line graphs.
  • Identify experimental design features, including controls in these experiments.
  • Analyze viral inactivation data to draw conclusions about antiviral effectiveness of different surfaces and inoculum phases.

Experimental Background (Warnes et al. Figure 2)

Methods of combating infectious diseases that are spread by surface contact include waiting for the pathogens to decay, use of specific cleaners to inactivate the pathogens, or use of specific surfaces to speed up inactivation processes.  Previous research had suggested that copper might have an antiviral effect.  So, to test which copper-containing surfaces might be least favorable for stability of infectious particles of the coronavirus family of viruses, Warnes et al. (2015) sprayed HCoV-229E virus (wet-droplet) onto two types of metal alloys containing copper: brass (panels A and B) and copper nickels (panel C), as well as non-copper metals (steel, zinc, or nickel), and 100% copper.  They then recovered the virus at different time points and quantified the infectivity of the recovered virus using a plaque forming assay using human lung cells in culture.  These data are reported as plaque forming units (PFU). Next, to determine whether drying affected viral inactivation on three different surfaces (copper, brass, and steel), they dried the inoculum immediately and performed the same recovery and infectivity tests (panel D).

Four line graphs of pfu per coupon by contact time, showing change on different surface types.
Figure 2. “Rapid inactivation of human coronavirus occurs on brass and copper nickel surfaces. Approximately 103 PFU HuCoV-229E (20 µl infected-cell lysate) was applied to 1-cm2 coupons of a range of brasses (A and B [early time points only]), copper nickels (C), and control metal surfaces that did not contain copper (stainless steel, zinc, and nickel). Virus was removed at various time points and assayed for infectivity as described in the text. Coronavirus was inactivated in ≤40 min on brasses and 120 min on copper nickels containing less than 70% copper. Analysis of the initial 30 min of contact between virus and brasses (Fig. 2B) reveals an initial lag followed by rapid inactivation. Stainless steel and nickel did not demonstrate any antiviral activity, although mild antiviral activity was observed on zinc (this was significant only at 60 min [P = 0.046]). (D) The same inoculum was applied as 1 µl/cm2, was dried immediately to simulate fingertip touch contamination, and was found to have inactivated the virus approximately 8 times faster. Error bars represent ± SEM, and data are from the results of multiple experiments.” (Warnes et al. 2015)

7.1.2. Questions

  1. Viral inactivation is shown for brass alloys in both panel A and panel B.  What is different between these panels?
    1. They show the different metal alloys.
    2. The time scale is different for them.
    3. The alloys have different copper %.
    4. The show a replicated experiment.
  2. Which control(s) in any of the panels show(s) the amount of virus inactivation we should expect in the absence of any copper?
    1. Zinc; black circles
    2. Steel; blue triangles
    3. Nickel; orange diamonds
    4. Cartridge brass; red square
  3. Based on the data in panel B, what is the relationship of copper content and virus inactivation?
    1. As copper % increases, virus inactivation increases.
    2. As copper % increases, virus inactivation decreases.
    3. As copper % increases, there is no effect on the virus.
    4. As copper % increases, the virus increases stability.
  4. Based on the data in panel D, which is/are good antiviral surface(s)?
    1. Steel
    2. Copper
    3. Cartridge brass
    4. All are good.
  5. The effect of dry versus wet inoculum can be investigated by comparing some of the data in panel B (wet) to the data in panel D (dry).  What effect does the phase of the inoculum have on the time needed to inactivate the virus?
    1. Drying increases viral inactivation time on copper surfaces.
    2. Drying increases viral inactivation time on steel surfaces.
    3. Drying decreases viral inactivation time on steel surfaces.
    4. Drying decreases viral inactivation time on copper surfaces.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify features in the diagrams, including the origin of genes, current location of genes, and cellular location of encoded enzymes in the pathway.
  • Analyze the diagram to evaluate evidence of horizontal gene transfer.
  • Predict how biosynthetic pathways might be “completed” given other circumstances.
  • Hypothesize benefits for these types of endosymbiotic events, as well as what they mean for the evolutionary history of a species.

Experimental Background (Bublitz et al., Figure 2)

Planococcus citri (mealybugs) are small sap-eating insects with a powdery appearance that colonize plants. These insects live with a double endosymbiont inside them (panel A).  One bacterium (Candidatus Moranella endobia; a gammaproteobacterium; kidney bean shape) lives inside another (Candidatus Tremblaya princeps; a betaproteobacterium; golden rectangle shape) that lives in a specialized organ (the bacteriocyte; blue rectangle shape) inside the mealybug.  In this study, Bublitz et al. (2019) explore the distribution of genes among the three genomes that are involved in the synthesis of peptidoglycan, which is only part of gram negative bacterial cell walls (panel B).

A schematic model of a bacteriocyte (A) and a visualization of its membranes as a flowchart (B).
Figure 1. “A Complete PG Biosynthesis Pathway Is Predicted by Genomics. (A) Schematic representation of a single P. citri bacteriocyte (blue), where Moranella cells and their two lipid bilayers (green) reside inside of triple-membrane-bound Tremblaya cells (yellow). (B) Adapted from Typas et al., 2011. A pictorial representation of the genes present and expressed on the genomes of P. citri (blue represents native eukaryotic genes, brown represents HGTs of Gammaproteobacteria origin, orange represents HGTs of Bacteroidetes origin, and yellow represents Alphaproteobacteria origin) and Moranella (green) that are involved in PG production. The locations of the small colored cell schematics labeled with gene names are based on the predicted location of the protein product of that gene.” (Bublitz et al. 2019)

7.2.2. Questions

  1. All the genes located in the mealybug genome are noted as a cartoon bugs (colored oval with little spikes; panel B).  Match each gene type with its current genome location,  originating species by horizontal gene transfer (HGT), and the encoded protein’s location. There will be three answers for each shape/color, one from each column.
Shape/color  Genome Location Horizontal Gene Transfer From Protein Location
_____ Green Kidney bean M. Moranella 1. Alphaproteobacteria A. Moranella cytoplasm
_____ Brown bug P. Mealybug 2. Bacteroidetes B. Moranella inner membrane
_____ Orange bug 3. Gammaproteobacteria C. Moranella outer membrane
_____ Yellow bug 4. Not applicable D. Moranella periplasm
_____ Blue bug
  1. Including HGT events, how many species contributed enzymes for the cytoplasmic portion of peptidoglycan synthesis for Moranella?
    1. One
    2. Two
    3. Three
    4. Four
  2. We know that mealybug cells do not use peptidoglycan for their cells.  Based on this information, what in the diagram is the evidence that there were horizontal gene transfer events from the
    1. Peptidoglycan synthesis occurs.
    2. GlmSMU are mealybug genes.
    3. The HGT notation tells us.
    4. Both D- and L-alanine are used.
  3. Given the intracellular location of the Moranella, within Tremblaya, what is the most likely reason for the retention of a peptidoglycan biosynthesis pathway?
    1. It suggests Moranella is fully prepared to migrate to extracellular locations
    2. Peptidoglycan production could allow Moranella to escape from Tremblaya.
    3. The host requires Moranella to produce peptidoglycan for its immune system.
    4. Peptidoglycan biosynthesis may be essential for maintaining its structure.
  4. If a gene that is required for synthesis of another Moranella biosynthetic pathway is missing in the genome of Moranella, what is the best explanation for how the pathway could continue to function?
    1. The biosynthetic pathway is not necessary for intracellular bacterial.
    2. Tremblaya or the insect host could provide the missing gene product.
    3. Moranella could follow a different, alternative  synthesis pathway.
    4. The gene is silenced, but still present, so it could be reactivated.
  5. What does the presence of insect-encoded genes from multiple, other bacterial origins tell us about its evolutionary history?
    1. Mealybugs evolved independently of bacteria besides their endosymbionts.
    2. Mealybugs acquired most of their genome from their bacterial endosymbionts.
    3. Mealybugs show evidence of extensive horizontal gene transfer from bacteria.
    4. Mealybugs have acquired mutations that made their genes similar to bacteria.

8. Paper Information and Licensing

8.1. Snippet papers

  • Warnes SL, Little ZR, Keevil CW. 2015. Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials. mBio. 6(6):e01697-15. doi: 10.1128/mBio.01697-15.
  • This article is licensed for Creative Commons use using CC BY-NC-SA 3.0, which allows non-commercial re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.
  • van Doremalen N, Bushmaker T, Morris DH, Holbrook MG, Gamble A, Williamson BN, Tamin A, Harcourt JL, Thornburg NJ, Gerber SI, Lloyd-Smith JO, de Wit E, Munster VJ. 2020. Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. N Engl J Med. 382(16):1564-1567. https://www.nejm.org/doi/full/10.1056/NEJMc2004973
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the figures cannot be copied here. Please see the article at the journal’s web page.

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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