Evolution

TWiM #275: The Myth of Clonality

Podcast and Annotation Information

  • Annotation by Aisha Hager, Karwitha Mamae, Mel Melendrez-Vallard, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #275 Podcast
  • Podcast transcript by Otter.ai and edited by Marvin Romo and Rebecca Seipelt-Thiemann: Access Podcast Transcripts
  • Papers Discussed:
    • Tomoiaga D, Bubnell J, Herndon L, Feinstein P. High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development. 2022. Sci Rep. 12(1):11515. doi: 10.1038/s41598-022-14598-9.
    • Kumar S, Schmitt C, Gorgette O, Marbouty M, Duchateau M, Giai Gianetto Q, Matondo M, Guigner JM, De Reuse H. 2022. Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori. mBio. 13(5):e0163322. doi: 10.1128/mbio.01633-22.

1. Paper Abstracts

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

The Most Interesting Things (according to students)

It is interesting that E. coli cells, despite what we are told, can harbor multiple plasmids at the same time.

“The concept of DNA transfer between bacteria was put forth by Griffith in 1928. During the dawn of molecular cloning of DNA in the 1980s, Hanahan described how the transformation of DNA  plasmids into bacteria would allow for cloning of DNA fragments. Through this foundational work, it is widely taught that a typical transformation produces clonal bacterial colonies. Using low concentrations of several plasmids that encode different fluorescent proteins, under the same selective antibiotic, we show that E. coli bacteria readily accept multiple plasmids, resulting in widespread a clonality and reveal a complex pattern of colony development. Co-transformation of plasmids occurs by either CaCl2 or by electroporation methods. A bacterium rod transformed with three plasmids—each expressing a high level of a unique fluorescent protein—and replated on agar, appears to reassign a random number of the three fluorescent plasmids to its daughter cell during cell division. The potential to simultaneously follow multiple lineages of clonally related bacteria in a bacteria colony would allow for mosaic analysis of gene function. We show that clonally related bacterium rods self-organize in a fractal growth pattern and can remain linked during colony development revealing a potential target against microbiota growth.” (Tomoiaga et al. 2022, no changes)

1.2. Main paper; discussion starts at 27:43 minutes

The Most Interesting Things (according to students)

Bacterial resistance is a major threat to humans. We never thought humans having bacterial resistance is bad. We thought it would be good since getting sick would be less threatening but then we remembered that we have good bacteria we need and can’t live without. We then understood that this was a problem. H. pylori can use a defense mechanism of expelling the treatment through its membrane vesicles and make it less effective.

“Bacterial antibiotic resistance is a major threat to human health. A combination of antibiotics with metals is among the proposed alternative treatments. Only one such combination is successfully used in clinics; it associates antibiotics with the metal bismuth to treat infections by Helicobacter pylori. This bacterial pathogen colonizes the human stomach and is associated with gastric cancer, killing 800,000 individuals yearly. The effect of bismuth in H. pylori treatment is not well understood in particular for sub-lethal doses such as those measured in the plasma of treated patients. We addressed this question and observed that bismuth induces the formation of homogeneously sized membrane vesicles (MVs) with unique protein cargo content enriched in bismuth-binding proteins, as shown by quantitative proteomics. Purified MVs of bismuth-exposed bacteria were strongly enriched in bismuth as measured by inductively coupled plasma optical emission spectrometry (ICP-OES), unlike bacterial cells from which they originate. Thus, our results revealed a novel function of MVs in bismuth detoxification, where secreted MVs act as tool to discard bismuth from the bacteria. Bismuth also induces the formation of intracellular polyphosphate granules that are associated with changes in nucleoid structure. Nucleoid compaction in response to bismuth was established by immunogold electron microscopy and refined by the first chromosome conformation capture (Hi-C) analysis of H. pylori. Our results reveal that even low doses of bismuth induce profound changes in H. pylori physiology and highlight a novel defense mechanism that involves MV-mediated bismuth extrusion from the bacteria and a probable local DNA protective response where polyphosphate granules are associated with nucleoid compaction.” (Kumar et al 2022, no changes)

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

Snippet Main
Vision and Change Topics
  • Structure and function (V&C_SF)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Impact of Microbes (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Evolution (V&C_E)
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 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • Fundamental Statement 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 25 (ASM_25): Microbes are used as models that provide fundamental knowledge about life processes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define plasmid.
  • Recall the evidence that identified that single bacteria can be transformed with and stably carry multiple plasmids.
 S L
  • When given an alternative model, predict how the results/outcomes of this experiment would have differed.
  • Defend why this work has important implications for researchers working with plasmids.
 S H
  • Identify why H. pylori infection is important to human health.
  • Recall the how H. pylori manage sub-lethal exposure to bismuth.
 M L
  • Predict the results of a hypothetical experiment proposed 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

  • Bacterial Transformation (9:45): This is a method for having bacteria take up DNA from their environment.  Some bacteria are naturally competent for transformation, but others are made competent for transformation using chemicals or electricity.  Here,  E. coli cells were transformed with genes encoding different fluorescent proteins: cerulean, Venus and Cherry to determine if colonies could maintain different plasmids and thus express the different fluorescent proteins at the same time.
  • Split Green Fluorescent Protein (Split GFP) (10:40 -11:30): This is a green fluorescent protein whose coding region has been split in half using molecular genetic techniques.  It only fluoresces when the coding regions for each half are expressed at the same time in the same cell. In this experiment, they used split GFP plasmids in a co-transformation to determine cells that had both plasmids because they fluoresced green.  Bacteria that had only one of the plasmids would not.
  • Electroporation (11:50): This is a method to induce competent cells to take up foreign DNA by using electrical pulses to create temporary membrane pores so molecules such as DNA can enter the bacterium.
  • Mosaic Analysis (16:30): Mosaic is when you have two or more kinds of something together.  Here, they are referring to two or more E. coli cells where the difference is in which plasmids they carry.  This was a proposed application, which would involve using essentially a pool of bacteria with different mutations in a single gene or combinations of different genes to study or select individuals of the pool with a particular phenotype.  They mention this is a way to determine whether a phenotype is autonomous (independent, such as a self-driving car) or non-autonomous (having a dependency, such as a human driving a car).

4.2. Main Paper

  • Quantitative Proteomics (33:38; 47:10): This is a method for identifying and quantifying proteins in a mixture, typically using mass spectrometry (MS).  The authors used MS to determine what proteins are in the vesicles. The membrane vesicles had 486 different proteins in treated vesicles, compared to 455 proteins in untreated vesicles. Most were identified as bismuth binding proteins.
  • Electron Microscopy (SEM/TEM) (41:10; 45:34; 49:34):  These are high resolution microscopy techniques that use electrons bent by magnetic fields generated by electromagnets to magnify images.  Here, it was used to analyze the response of bacteria to bismuth by characterizing the vesicles after sectioning the cells. The untreated samples had a wider size distribution of the membrane vesicles that are heterogeneous. The treated samples were of a more homogeneous size distribution.
  • Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) (41:20; 48:35): This is a method to identify and quantify the elements in a mixture.  Here it was used to determine the concentration of bismuth in the vesicles. Packaging bismuth into membrane vesicles allowed cells to escape toxicity by confining it.
  • Chromosome Conformation Capture (Hi-C)  (41:40; 53:45): It is a technique to study the 3-dimensional aspects of chromatin structure. This technique was used to observe the bismuth-induced differences in bacterial chromosome compaction.
  • Energy dispersive X-ray analysis (51:02): It is a technique used to determine the identity of elements in cellular structures. Here it is used to identify the composition of the electron dense regions that appear in the cytoplasm of cells exposed to bismuth.

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

5.1. Snippet Paper

  • Plasmids (3:32–4:53, 6:56; 8:30): E. coli cells can harbor multiple plasmids at the same time. Colonies were also found to be plasmid mosaics. Plasmid exclusion and fitness penalty were discussed. The podcasters noted that using three highly similar fluorescent proteins allowed the researchers to avoid effects due to differing fitness if different proteins had been used.  This essentially means that the similar proteins have a similar fitness cost to the bacteria, so no effects could be attributed to that.
  • Fluorescent Proteins and Bacterial Transformation (5:07; 6:12; 9:01–10:16): Plasmids carrying genes encoding fluorescent proteins cerulean, Venus and Cherry were used to transform E. coli cells to see if they would get colonies expressing all three fluorescence proteins at the same time.
  • Competent Cells (14:56): The ability to take up DNA is called competence and it can be quantified to ensure you have “good” cells.  For a sufficient level of competence, 1 ng of DNA should result in at least 106 colonies. It does not scale up with higher DNA mass, but they could take up more than one plasmid.
  • Congression (15:51): Congression is another name for co-transformation, or using multiple plasmids in your transformation mixture.  This was discussed in the context of Bacillus, which is naturally competent when grown in specific conditions. The podcasters discussed using one plasmid encoding a drug marker and one plasmid you can screen for (phenotype), such as failure to form spores.  They note the frequency of co-transformation/congression is 1:100 or 2:100 because what you are really selecting for is competent cells.

5.2. Main Paper

  • Antibiotic Resistance (28:44): Antibiotic resistance is the ability of a bacterium to survive exposure to an antibiotic substance. They discuss the use of metals and how they have used metals to control microbe growth and survival.
  • Antimicrobial Metals (31:10, 36:25):  Several metals such as copper or metal bismuth are antibacterial. The authors investigate the use of bismuth in treating H. pylori infections.  They note that metal based therapies in combination with antibiotics may be a way forward for treatment of these H. pylori infections to prevent them from transitioning to carcinoma.
  • Membrane Vesicles (32:52; 42:09; 43:56): The microbe’s response to bismuth exposure is to “bleb” off membrane vesicles that carry a cargo enriched in bismuth and bismuth binding proteins.
  • Nucleoid Region (34:45; 42:16; 51:56–53:27): The nucleoid region is an irregularly shaped region inside prokaryotic cells where genetic information is located. They applied bismuth to the cells and found that polyphosphate granules reorganize and protect the nucleoid from the bismuth-induced stress.
  • Mutation and Complementation (52:28): Polyphosphate kinase (PPK) helps polymerize the polyphosphate granules.  To see the effect of removing this gene, the researchers constructed a strain without this gene (a deletion mutant).  Bismuth exposure for this mutant strain showed no appearance of phosphorus rich electron dense regions (polyphosphate granules).  To make sure PPK was indeed responsible they added wilt-type ppk back to the mutant strain (complementation) and the defect was corrected; bismuth treatment induced polyphosphate granules.

6. Podcast Questions

  1. What is a plasmid?
    1. An organelle inside of a bacterial cell
    2. An extrachromosomal piece of DNA
    3. A part of a bacterial cell membrane
    4. A part of a bacterial chromosome
  2. What evidence did the authors and the podcasters present that shows a single bacteria cell can carry and stably maintain multiple plasmids?
    1. Fluorescent proteins of different colors encoded on different plasmids were visible in single bacterial cells.
    2. Selectable antibiotic resistance markers encoded on different plasmids produced cells resistant to all antibiotics.
    3. Nutritional biosynthetic enzymes encoded on different plasmids allowed cells to grow well on minimal media.
    4. A bioluminescent protein was encoded on one plasmid and a quencher was encoded on another plasmid.
  3. If the results had instead indicated that multiple plasmids could not be maintained in a single cell, how would the results have been different?
    1. Each cell would have plasmid recombination and produce fused “marker” proteins from the “mixed” plasmid.
    2. Each cell would have multiple plasmids and produce every “marker” protein encoded on each plasmid.
    3. Each cell would lose the plasmids and produce only the native proteins encoded in the genome.
    4. Each cell would have a single plasmid and produce the one “marker” protein encoded on that plasmid.
  4. How might the fact that a single bacteria or bacterial colony harbor multiple different plasmids affect a scientist’s work where they believe they are studying a single plasmid? For example, Kolodziejek et al. (2023) used plasmids to study a mutant phenotype in bubonic plague.
    1. If they are not careful in preventing contamination, they could introduce foreign plasmids which confound the results.
    2. If they are not careful in culturing bacteria and maintaining selective conditions, they could lose the plasmid entirely.
    3. If they are not careful in characterizing the plasmid and ensuring it is the only one, they could come to incorrect conclusions.
    4. If they are not careful in selecting the strain and confirming its genotype, they could misinterpret the genomic mutation.
  5. Why is controlling Helicobacter pylori infection as important to human health?
    1. Continued infection leads to stomach cancer.
    2. Continued infection lowers the immune response.
    3. Continued infection reduces gastric emptying.
    4. Continued infection increases acid reflux levels.
  6. The researchers used a number of techniques to identify how H. pylori manages to survive sub-lethal bismuth exposure. What did they find?
    1. Bacteria gather and store bismuth in the cell’s periplasm.
    2. Bacteria use a protein channel to pump bismuth out of the cell.
    3. Bacteria accumulate and secrete vesicles filled with bismuth.
    4. Bacteria precipitated bismuth into high density granules.
  7. The podcasters discuss where humans and bacteria might encounter bismuth naturally. They found that there is no real source other than metal smelting, which leads to a discussion of why bacteria react this way to bismuth.  Petra suggests that it is a general stress response. To test this, we could use the same methods and test nickel, antimony, arsenic, and/or copper.  If this effect is truly bismuth-specific and not a general stress response, what should we expect?
    1. Sub-lethal metal exposure will show metal precipitation into high density granules.
    2. Sub-lethal metal exposure should cause metal-filled vesicles to be secreted.
    3. Sub-lethal metal exposure should cause an increase in protein pump channels.
    4. Sub-lethal metal exposures will show different phenotypes based on the metal.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 3).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features and elements of experiment design in schematic and fluorescent microscopy images.
  • Evaluate data to identify evidence of successful transformation.
  • Analyze data to make conclusions about whether single colonies can carry multiple distinct plasmids.

Experimental Background (Tomoiaga et al., Figure 1)

Cloning, and maintaining and transferring genes on plasmids between strains and species are widely used methods in microbiology, molecular biology, genetics, and cell biology.  Historically, most scientists were taught that a single colony carried only a single plasmid once the transformed cell grew into a colony.  However, in the course of other studies, Tomoiaga et al (2022) found evidence that challenged this historical view of clonality.  To follow up on this, they constructed a set of nearly identical plasmids differing in the expression of a highly similar fluorescent protein (Cerulean, Venus, and mCherry) that can easily be distinguished by confocal microscopy. To determine whether single colonies could carry multiple distinct plasmids, they transformed chemically competent E. coli in two ways.  First, each single plasmid was used to transform cells that were then mixed and plated (panel A; results in panels B-E) or second, a mixture of all three plasmids was used to transform cells and the cells were plated (panel F; results in panels G-J).  The same colonies were then evaluated for expression of each fluorescent protein using confocal microscopy with excitation and emission filters set to show the specific fluorescent proteins (Cerulean = panels B & G, Venus = panels C & H, Cherry = panels D & I).

 

10-part figure showing plasmids separated (b through e) and mixed (g through j). A and F visualize the groupings with simple illustrations.
Figure 1: “Transformation with three plasmids separately or in mixed. (A) Three lactamase promoter (Lam→) constructs each expressing a different fluorescent protein (Ce-Cerulean, V-Venus or C-mCherry) were transformed separately into DH5⟨ E. coli. (B), (C), and (D) Colonies observed after 16 h of growth (all plasmids have the same resistance). Overlay in (E) shows no colony has double fluorescence using saturating laser excitation. (F) Three constructs co-transformed simultaneously. (G), (H), and (I) Colonies observed after 16 h of growth (all plasmids have the same resistance). (J) Four of the nine colonies in this visual field show coexpression (white arrows) using saturating laser excitation. Three of these colonies express all three fluorescent proteins and one only Cerulean and Venus (see colored arrows in (G, H, I).”(Tomoiaiga et al 2022, no changes)

 

7.1.2. Questions

  1. Match the fluorescent protein name with its visible color. [B = Blue; R = Red; Y = Yellow]
    1. ________ Cerulean
    2. ________ Venus
    3. ________ mCherry
  2. Panels A and F show the experimental design.  What is the essential difference in how the cells were transformed based on this?
    1. Fluorescent proteins were added individually to each aliquot in A; a fluorescent protein mixture was added to an aliquot in panel F.
    2. Individual aliquots were transformed with single plasmids then mixed in A; a mixture of plasmids were co-transformed in one aliquot  in F.
    3. Cells were infected with each of three fluorescent viruses in A; a mixture of fluorescent viruses were used to infect bacteria in panel F.
    4. Bacteria were transformed with single plasmids encoding each gene in A; bacteria were transformed with one plasmid carrying all three genes in F.
  3. What is the difference in the visualizations show in panels B-D?
    1. Each panel shows a different fluorescence for the same bacterial colonies.
    2. Each panel shows a different magnification for the same bacterial colonies.
    3. Each panel shows a different nutrient level for the same bacterial colonies.
    4. Each panel shows a different growth phase for the same bacterial colonies.
  4. What results indicate the transformation was successful for all three plasmids when transformed separately?
    1. We can see many colonies growing on the plate.
    2. We can see that there are some large colonies.
    3. We can see all plasmids were put into the tubes
    4. We can see fluorescent colonies of all three types.
  5. What results indicate the transformation was successful for all three plasmids when transformed in a mixture?
    1. We can see all plasmids were put into the tubes.
    2. We can see fluorescent colonies of all three types.
    3. We can see many colonies growing on the plate.
    4. We can see that there are some large colonies.
  6. In this experiment, the researchers wanted to determine whether single colonies could carry multiple distinct plasmids. What do their results indicate? What is your evidence?
    1. Yes, the pool of colonies show all fluorescent proteins (panels B-E; G-I).
    2. No, individual cells express only one kind of fluorescent protein (panels B-D).
    3. Yes, individual colonies show multiple fluorescent proteins (panels G-I).
    4. No; the pool of colonies show distinct fluorescent patterns (panels E, J).

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features and elements of experiment design in microscopy and elemental composition images.
  • Evaluate the data to identify evidence of equal/unequal distribution of elements in untreated and bismuth-treated cells.
  • Evaluate the data to make conclusions about the composition of the electron dense regions in bismuth-treated cells.

Experimental Background (Kumar et al., Figure 3)

Helicobacter pylori is a bacterium that can reside in the acidic environment of the human stomach where it can cause ulcers. If left untreated, prolonged infection can result in gastric cancer, which causes 800,000 deaths per year globally.  Treatments for H. pylori involve a combination of antibiotic and a metal called bismuth.  With the concern over a growing amount of antibiotic resistance worldwide, Kumar et al. (2022) were keen to investigate the effects of bismuth alone as a potential alternative treatment. To do this, they first needed to characterize the effects of bismuth on H. pylori. The two main effects were that: 1) bismuth-treated cells produced and secreted membrane vesicles containing bismuth and bismuth-binding proteins and 2) bismuth-treated cells formed electron dense structures.  To further characterize these structures, the authors performed scanning-transmission electron microscopy (STEM; left-most panels of A and B; shown in negative-contrast) and elemental composition analysis using energy dispersive X-ray (EDX) on bismuth-treated (panel B) and control, untreated (WT, panel A) bacteria.  The levels and distribution of phosphorus, calcium, nickel, oxygen, sulfur, magnesium, and bismuth were identified by EDX; the levels of phosphorus and bismuth are noted in the center and right-most panels of A and B, respectively.

Control and bismuth treated cells.
Figure 3: “Elemental mapping of bismuth-treated H. pylori cells. (A) Scanning-transmission electron micrographs (STEM) of untreated bacteria. The negative-contrast image of control bacterial sections is shown together with the EDX map for phosphorus and bismuth. (B) STEM negative-contrast image of H. pylori cells exposed to bismuth. Electron-dense regions are seen as bright white regions. EDX maps of phosphorus and bismuth are shown. Accumulation of phosphorus in the cytoplasm of these bismuth-treated bacteria is highlighted by yellow open circles and white arrows in the inset panels; this accumulation overlaps with the electron-dense regions.” (Kumar et al 2022, no changes)

 

7.2.2. Questions

  1. The control bacteria in this experiment are noted as _____ and are found in panel ______.
    1. Negative-contrast, A
    2. Control, B
    3. WT, A
    4. Bismuth treated, B
  2. The electron-dense regions of cells originally identified in H. pylori are confirmed in this experiment as well using STEM. STEM micrographs in these results are shown in negative-contrast. What color are the electron-dense regions in the negative-contrast images?
    1. Bright white regions
    2. Dark black/gray regions
    3. Gold speckled regions
    4. Purple speckled regions
  3. Based on the data, what is the elemental composition of the electron-dense regions? What is your evidence?
    1. Osmium; this metal is the most dense of all elements on Earth
    2. Bismuth; the dense regions show high levels of purple abundance
    3. Hydrogen; hydrogen is the the most abundant element on Earth
    4. Phosphorus; the dense regions show high gold-color abundance
  4. What can you conclude about the distribution and abundance of bismuth in bismuth-treated cells based on these data?
    1. Bismuth is abundant everywhere except in the nucleoid region of the cell.
    2. Bismuth is found at low levels, evenly distributed throughout the cell.
    3. Bismuth is found concentrated in vesicles inside the bacterial cell wall.
    4. Bismuth is found concentrated in electron-dense regions near the nucleoid.

8. Paper Information and Licensing

8.1. Snippet paper

  • Tomoiaga D, Bubnell J, Herndon L, Feinstein P. High rates of plasmid co-transformation in E. coli overturn the clonality myth and reveal colony development. 2022. Sci Rep. 13(5):e0163322. doi: 10.1038/s41598-022-14598-9.
  • 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 copyright information on the article’s website.

8.2. Main paper

  • Kumar S, Schmitt C, Gorgette O, Marbouty M, Duchateau M, Giai Gianetto Q, Matondo M, Guigner JM, De Reuse H. 2022. Bacterial Membrane Vesicles as a Novel Strategy for Extrusion of Antimicrobial Bismuth Drug in Helicobacter pylori. mBio. 12(1):11515. doi: 10.1128/mbio.01633-22
  • 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 copyright information on the article’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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