Evolution

TWiM #269: Bacterial but Not Microbial

Podcast and Annotation Information

  • Annotation by Danielle Desmarteau, Quinlin Dixon-Lim, Emily Nguyen, Jonelle Mattiacio, Pete Chandrangsu, and Rebecca Seipelt-Thiemann.
  • Podcast audio by TWiM: Listen to TWiM #269 Podcast
  • Podcast transcript by Otter.ai and edited by Danielle Desmarteau, Quinlin Dixon-Lim, Emily Nguyen, and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Volland JM, Gonzalez-Rizzo S, Gros O, Tyml T, Ivanova N, Schulz F, Goudeau D, Elisabeth NH, Nath N, Udwary D, et al. 2022. A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles. Science. 376(6600):1453-1458. doi: 10.1126/science.abb3634.
    • Ongenae V, Mabrouk AS, Crooijmans M, Rozen D, Briegel A, Claessen D. 2022. Reversible bacteriophage resistance by shedding the bacterial cell wall. Open Biol. 12(6):210379. doi: 10.1098/rsob.210379.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 6:45 minutes

The Most Interesting Things (according to students)

  • Thiomargarita magnifica exemplifies how prokaryotes can adapt to have a larger cell size previously only considered to exist in eukaryotes.
  • Potential biotechnology applications of the material that this microbe is made out of could actually give us a new medical grade catheter that wouldn’t grow bacteria.
  • This article is not licensed for Creative Commons use; see https://www.science.org/doi/10.1126/science.abb3634.  Thus, the abstract and figures cannot be copied here.

1.2. Main paper; discussion starts at 33:01 minutes

The Most Interesting Things (according to students)

Bacteria can survive without a cell wall given the correct osmoprotective environment.

“Phages are highly abundant in the environment and pose a major threat for bacteria. Therefore, bacteria have evolved sophisticated defense systems to withstand phage attacks. Here, we describe a previously unknown mechanism by which mono- and diderm bacteria survive infection with diverse lytic phages. Phage exposure leads to a rapid and near-complete conversion of walled cells to a cell-wall-deficient state, which remains viable in osmoprotective conditions and can revert to the walled state. While shedding the cell wall dramatically reduces the number of progeny phages produced by the host, it does not always preclude phage infection. Altogether, these results show that the formation of cell-wall-deficient cells prevents complete eradication of the bacterial population and suggest that cell wall deficiency may potentially limit the efficacy of phage therapy, especially in highly osmotic environments or when used together with antibiotics that target the cell wall.” (Ongenae et al. 2022)

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

 Snippet Main

Vision and Change Topics
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)

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 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • 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 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify how this bacterium’s features challenge our view of prokaryotes.
  • Recall the evolutionary adaptations of Thiomargarita magnifica.

S

L
  • Propose a method to identify culture conditions for this mega-bacterium.

S

H
  • Order the steps of virus-induced lysis by phage infection.
  • Describe how peptidoglycan contributes to the form and function of the cell wall.
  • Recall the mechanism of cell wall shedding to create cell wall deficient bacteria (L-form; CWD).

M

L
  • Design an experiment based on a hypothesis from the podcast discussion.

M

H

1Papers: 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

  • Ribosomal DNA (rDNA) Sequencing (12:40–12:56): This next generation sequencing (NGS) technology involves analyzing ribosomal genes (rDNA) to identify and quantify species and archaea in complex samples, as well as categorize its evolutionary relationship to other species. The authors compared it to whole genome sequencing, which was used in this study to show that the microbe, Thiomargarita magnifica, is related to other mega-bacterium with some features that are eukaryotic-like.

4.2. Main Paper

  • Viable Count (45:14–48:56): There are a number of methods to determine living cells from dead cells, including staining methods and having cells form colonies on agar plates.  This is usually done because living and dead cells can look alike in microscopy and also both scatter light in optical density measurements.  The authors determined viability of the bacterial cells after phage infection by analyzing colony forming units (CFU).
  • Microscopy (44:17–45:13; 53:21–55:14): The authors used a variety of microscope techniques including electron microscopy and phase contrast to visualize viable cell wall-deficient (CWD) bacteria cells. Electron microscopy is a technique that uses a beam of electrons to illuminate a specimen and create a high resolution image of extremely small objects like microbes.

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

5.1. Snippet Paper

  • Membrane Bound Vesicles (13:15–13:33; 21:18–26:00): Thiomargarita magnifica are found to have genomes distributed all along the periphery of the cell in pepins, or membrane vesicles, that allows the cells to synthesize proteins right where they need them.
  • Polyploidy (13:59–15:28): While most bacteria are monoploid (having a single chromosome copy), Thiomargarita was found to be a polyploid, meaning they have multiple copies of their genome.
  • Cell Membrane (16:48–19:17) Thiomargarita has an invaginated cell membrane that helps increase the surface area to volume ratio but interestingly has nothing growing on it.
  • Proton Motive Force (PMF) (21:58–23:59): An electrochemical gradient across the bacterial membrane that drives essential cellular processes. Thiomargarita has PMF distributed along the entire length of the bacterium.

5.2. Main Paper

  • Viral Lytic cycle (33:36–37:46: 57:50–59:07): Phage is able to mount a lytic cycle against microbes, lysing and killing the infected cell and releasing newly assembled virus particles.
  • Peptidoglycan Structure (35:36–37:12, 48:52–50:04): Peptidoglycan is a polymer found in the cell walls of most bacteria, providing structural support and helping the cell withstand osmotic pressure. In this paper the authors show that upon exposure to the phage, the microbe undergoes a behavioral change, where they rapidly and nearly completely convert themselves from walled cells to a cell wall deficient state
  • Osmoprotective Environment (39:30–40:37): An environment where bacteria can maintain stable internal osmotic pressures, even when facing changes in the external environment. This is crucial for bacterial survival, as fluctuations in osmolarity can disrupt cellular processes and potentially lead to cell damage.
  • L-form (46:10–46:59): A unique form of bacteria lacking a rigid cell wall; also known as cell wall-deficient bacteria (CWD) in the paper.

6. Podcast Questions

  1. Which statement(s) about prokaryotic cells is/are challenged by the discovery and characterization of mega-bacterium Thiomargarita magnifica? [pick all that apply]
    1. All prokaryotes have two cell membranes.
    2. All prokaryotes have circular DNA, not linear DNA.
    3. All prokaryotes lack membrane-bound organelles.
    4. All prokaryotes are unicellular, not multicellular.
    5. All prokaryotes have high surface-volume ratios.
  2. A major adaptation of Thiomargarita magnifica is its large central vacuole. What is the main benefit of this adaptation?
    1. It stores oxygen and oxygen-containing molecules for later use.
    2. It eliminates the need for a proton motive force in cellular respiration.
    3. It reduces the active cytoplasmic volume, enhancing diffusion.
    4. It enables the cell to survive in extremely acidic environments.
  3. The podcasters mention that this mega-bacterium cannot yet be grown in culture.  Which of the following is a next best step in trying to establish culture conditions for this bacterium that was isolated from a marine (salt-water), sulfur-rich environment (a sunken leaf)?
    1. Identify the salt composition and concentrations in the water and then supplement standard liquid broth with these concentrations.
    2. Take water from the original location, use chemical and physical separation techniques to make fractions, then try to grow them in each.
    3. Use a variation of the disk diffusion assay where a water sample is applied to the disk and bacteria are spread onto the plate.
    4. Use mass spectrometry to identify compounds present in the sulfur-rich, salt water environment they were found in.
  4. What is the correct order of steps for a lytic phage attack?
    1. genome incorporation into host, replication, assembly, lysis
    2. attachment, penetration, genome incorporation into host
    3. lysis, replication, penetration, assembly, attachment
    4. attachment, penetration, replication, assembly, lysis
  5. What role does peptidoglycan play in the structure and function of bacterial cells?
    1. This molecule provides structure and protection against osmotic pressure.
    2. This lipid serves as the primary component of the outer cell membrane.
    3. This protein is a channel that transports nutrients across the membrane.
    4. This polysaccharide is used to generates cell energy through glycolysis.
  6. Which of the following best describes how cell wall deficient bacteria come to have no cell walls following stress or phage attack?
    1. By decreasing availability of surface bound signal receptors
    2. By secreting enzymes that break down peptidoglycan layer
    3. By expressing and secreting spacer proteins to burst the wall
    4. By incorporating phage DNA into their cell membranes
  7. The podcasters identify that the ability to become cell wall-less is a likely an adaptation that has costs and benefits, so the ability to control the switch back and forth is most adaptive. How could you identify the regulator of this switch?
    1. Use fluorescent microscopy to identify the phage entry receptor on the bacteria.
    2. Sequence the genome and look for quorum sensor pathways involved in cell wall.
    3. Isolate mutants that fail to switch back to having a cell wall and identify the genes.
    4. Perform mass spectrometry on cell lysates for normal and cell wall-less cells.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1AB;D-G) and one from the main paper (Figure 1A-E;H).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in schematics and microscopy images.
  • Calculate the relative size differences between species.
  • Analyze the data and make conclusions about the unicellularity/multicellularity of the species.
  • Analyze the data and make conclusions about the species’ structural features.

Experimental Background (Volland et al., Figure 1AB;D-G)

Bacteria are widely known to be microscopic organisms, but one recently-discovered species of bacteria has sparked curiosity as to whether larger bacteria exist. In Volland et al (2022) the authors investigate and characterize a novel species, Candidatus Thiomargarita magnifica.  The researchers first show the size relationship of this species to bacteria and small eukaryotic species (panel A).  To gain additional insights into the species structure, they next use light microscopy to detail the upper half of a cell (panel B main area) and closely examine a broken basal region (panel B inset area).  To examine the extent and continuity of this cell’s membrane, red fluorescent stain was used to identify membranes, which were visualized using confocal scanning laser microscopy (CLSM; panel D). Transmission electron microscopy was then used to gain a more fine detail view of the membrane with its constrained cytoplasm due to a large central vacuole (panel E).  Additional structures, such as sulfur granules and pepins, were also identified using even higher magnification (panel F and G)

7.1.2. Questions

  1. The organisms identified in panel A are shown in blue and green.  What does the color indicated?
    1. Blue are eukaryotes and green are prokaryotes.
    2. Blue are algae and green are prokaryotes.
    3. Blue are multicellular and green are unicellular.
    4. Blue are Gram-positive and green are Gram-negative.
  2. How much bigger is Ca. Thiomargarita magnifica than the average E. coli?
    1. 10x bigger
    2. 50x bigger
    3. 100x bigger
    4. 1000x bigger
  3. What does the red fluorescence in panel D indicate and what does the staining pattern suggest?
    1. Vacuoles; there is one very long vacuole for nearly the whole length of the structure.
    2. Membranes; there is a very long cell rather than many different cells stuck together.
    3. Sulfur containing compounds; the cell uses sulfur compounds for cellular respiration.
    4. Peptidoglycan; there is a rigid structure than enables the cell to maintain its surface area.
  4. Light microscopy of the broken basal area indicated a tube-like structure (panel B) which is further detailed in the electron microscopy images (panel E).  What takes up this tube-like space and what effect does this feature have on the species’ internal structure?
    1. nothing; this tube-like structure holds gases for flotation
    2. sulfur granules; the cellular respiration machinery is blocked
    3. pepins; the structures are consolidated at the membrane
    4. a vacuole; the cytoplasm is squished into a small area

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in microscopy images, bar graphs, and experimental design for these experiments.
  • Evaluate the data to make conclusions about the impact of phage LA7 exposure on morphological and structural features of cells.
  • Analyze the data to make conclusions about the impact of medium choice on cell survival and phage LA7 exposure.
  • Analyze the data to make conclusions about the impact of medium choice on phage success.
  • Predict the impact of medium choice on the experimental system.

Experimental Background (Ongenae et al., Figure 1a-e;h)

Phages, also known as bacteriophages, are a type of virus that exclusively infect bacteria.  Understanding how phages disrupt bacterial communities is important for both ecological microbiology and potential therapeutic applications, such as phage therapy to combat antibiotic-resistant bacteria. Lytic phages, specifically, are a type of phage that hijack bacterial DNA replication mechanisms to reproduce their own viral DNA. The bacterial cell then dies via cell lysis and releases new phage into the environment. As a result of this threat, many bacterial strains have adaptations to avoid or prevent this type of viral infection. In this study, Ongenae et al (2022) characterize a new mechanism of bacteriophage resistance based on their previous work with cell wall deficient cells (CWD) and stress in osmoprotective environments.  They knew Streptomyces strain MBT86 produced CWD cells when stressed and reasoned that bacteriophage exposure might induce stress and thus, also, CWD.  To test this, they grew Streptomyces strain MBT86 in an osmoprotective medium (LBP) in the absence (panel a; upper panels of panel c) and presence of phage LA7 (panel b and lower panels of panel c).  Light microscopy (panels a and b) was used to compare morphology and fluorescent microscopy (panel c) was used to compare DNA content (SYTO-9) and membranes (FM5-95) of both treatments, and peptidoglycan (WGA Oregon) of phage-treated cells (panel d).  They next were interested in the phage-related effects in traditional medium, so they compared the growth/survival (optical density) of phage-treated and non-phage treated cells in traditional (DNB) and osmoprotective medium (LPB) (panel e).  To determine the ability of cells to recover, they next plated phage-treated MBT86 cells on DNB or phage-treated CWD cells on LPB to compare the colonies produced (panels e and f).  Finally, to determine how successful LA7 phage reproduction was in each condition, growth media were serial diluted in plaque assays to quantify the number of plaque forming units (PFU) (panel h).

Several labeled images including A through d, microscopy images, E (a bar chart), and F-G (agar plates), showing results with and without phage. Results with phage are more distributed and spread out.
Figure 1.

“Formation of CWD cells after phage infection in the actinomycete MBT86. (a) Morphology of MBT86 after 48 h in LPB medium. Scale bar represent 100 µm. (b) Morphology of MBT86 in LPB medium 24 h after phage LA7 infection. (c) Morphology of MBT86 with and without phage LA7 24 h after infection. Cells were stained with the DNA dye SYTO-9 (green) and FM5-95 (red) to dye membranes. Intensity of fluorescence was adjusted for visualization purposes. Scale bars represent 20 µm. (d) After 24 h of phage LA7 infection, the resulting CWD cells of MBT86 were stained with the peptidoglycan stain WGA Oregon. Some cells already start to rebuild their peptidoglycan layer. Scale bar represent 10 µm. (e) OD600 measurement 24 h after phage LA7 infection in DNB medium and LPB medium. The experiment was performed in triplicates with standard deviation presented as error bars. In LPB medium after phage infection, the OD600 was still 0.285 ± 0.05. (f) Regrowth of MBT86 after phage infection in DNB medium. Note that small mycelial fragments grew back into colonies. (g) Regrowth of MBT86 CWD cells after phage infection in LPB medium. Note that these colonies have developmental defects, as seen by a mixture of white and grey colonies. (h) Plaque assay showing PFUs of LA7 on DNB and osmoprotective LPB medium. Images were taken 24 h after LA7 infection.” (Ongenae et al. 2022)

7.2.2. Questions

  1. Good experimental design includes controls.  What control condition was included in the microscopy experiments (panels a-d)? What was the purpose of using this as a control
    1. MBT86 cells grown in/on osmoprotective media without any phage; It is used to identify colony morphology changes due to phage exposure.
    2. MBT86 cells grown in/on standard media without any phage; It is used as a comparison to show morphology changes due to medium type.
    3. MBT86 cell wall deficient cells grown without phage; It is used as a comparison to show enhanced mycelia growth due to phage exposure.
    4. MBT86 cell wall deficient cells grown with phage; It is used as a baseline for quantifying symbiotic growth of the phage and MBT86 cells.
  2. What morphology difference do you see when you compare MBT86 cells exposed to phage (panel b) and not exposed to phage?
    1. Cells not exposed to phage are lysed, while cells exposed to phage are healthy and spherical.
    2. Cells not exposed to phage form mycelia, while cells exposed to phage lost their cell walls.
    3. Cells not exposed to phage form secretory structures, while cells exposed to phage do not.
    4. Cells not exposed to phage are motile and travel, while cells exposed to phage are sessile.
  3. The authors use fluorescent dyes to visualize the location and quantity of different molecules (panels c-d).  Match the fluorescent dye and color with the molecule it stains. [Note: not all are used] Dyes: S = SYTO-9; F = FM5-95; W = WGA Oregon ; Colors: Y= yellow; R = Red; G = Green
    1. _____ lipids
    2. _____ DNA
    3. _____ proteins
    4. _____ peptidoglycan
  4. Based on the fluorescent microscopy results in panel (c), what can you conclude about the impact of phage LA7 exposure on MBT86 cells? What is your evidence?
    1. Phage LA7 promotes extracellular matrix production; staining shows DNA and membranes remain intact.
    2. Phage LA7 induces cell lysis; cell structure is degraded as shown by loss of DNA and membrane staining.
    3. Phage LA7 enhances filamentous growth; consistent mycelia morphology is shown in the staining.
    4. Phage LA7 induces cell wall loss; cell structure is intact as evidenced by DNA and membrane staining.
  5. Based on the fluorescent microscopy results in panel (d), what can you conclude about the capability of phage-exposed MBT86 cells to recover from phage-induced stress?  What is your evidence?
    1. Green fluorescence indicates the bacteria is capable of rebuilding its cell wall.
    2. Green fluorescence indicates the bacteria is not capable of rebuilding its cell wall.
    3. The cells are dead, as indicated by the absence of any green fluorescent signal.
    4. The cells lack both a cell wall and a cell membrane, as indicated by diffuse fluorescence.
  6. Optical density (OD) is used in this experiment to quantify cell growth and survival (panel e).  What is actually being measured by OD?
    1. The concentration of extracellular polysaccharides in the medium.
    2. The number of viable phage particles remaining after infection.
    3. The light scattering of bacterial cells in the liquid medium.
    4. The intensity of fluorescence emitted by stained bacterial DNA.
  7. Cell growth/survival is quantified for phage-infected and non-infected cells in standard and osmoprotective media (panel e).  What notation in this panel indicates the comparison being made is statistically significant?
    1. bar height
    2. whiskers
    3. asterisks
    4. nothing
  8. Cell growth/survival is quantified for phage-infected and non-infected cells in standard and osmoprotective media (panel e). Based on the optical density results in panel (d), in which medium are cells protected from phage-induced lysis? What is your evidence?
    1. DNB; There is a larger difference between DNB samples than LPB samples.
    2. LPB; There are some surviving cells in LBP with phage, but not DNB with phage.
    3. DNB; There are more surviving cells in DNB medium than LPB medium.
    4. LPB; LPB is listed as osmoprotective, but DNB is not listed as osmoprotective.
  9. One of the questions that these authors had was whether cell wall deficient cells were protected from phage-induced lysis. To do this the researchers tested the success of phage replication in standard (DNB) and osmoprotective media using plaque assays (panel h).  Does the formation of cell wall deficient cells impact phage replication success?  What is your evidence?
    1. yes; There were more phage in LPB than DNB.
    2. no; There were equal amounts of phage in each.
    3. yes; There were more phage in DNB than LPB.
    4. no; There were no phage isolated in either media.
  10. Based on the results of the growth/survival experiments (panel e), what would the researchers have likely seen if had used standard medium instead of osmoprotective medium for their initial experiments (panels a-d)?
    1. cell lysis and death
    2. Increased survival
    3. more cell wall deficient cells
    4. biofilm formation

8. Paper Information and Licensing

8.1. Snippet paper

  • Volland JM, Gonzalez-Rizzo S, Gros O, Tyml T, Ivanova N, Schulz F, Goudeau D, Elisabeth NH, Nath N, Udwary D, et al. 2022. A centimeter-long bacterium with DNA contained in metabolically active, membrane-bound organelles. Science. 376(6600):1453-1458. doi: 10.1126/science.abb3634
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.

8.2. Main paper

  • Ongenae V, Mabrouk AS, Crooijmans M, Rozen D, Briegel A, Claessen D. 2022. Reversible bacteriophage resistance by shedding the bacterial cell wall. Open Biol. 12(6):210379. doi: 10.1098/rsob.210379
  • 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 on the journal’s website.

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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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