TWiM #191: By the Pulp of Their Teeth

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:01 minutes

Rascovan et al (2018) is not under Creative Commons licensing. Hence, abstract cannot be copied. Thus, the abstract cannot be copied here. Please see the article on the journal’s web page.

1.2. Main paper; discussion starts at 29:30 minutes

” It has been hypothesized that mitochondria evolved from a bacterial ancestor that initially became established in an archaeal host cell as an endosymbiont. Here we model this first stage of mitochondrial evolution by engineering endosymbiosis between Escherichia coli and Saccharomyces cerevisiae. An ADP/ATP translocase-expressing E. coli provided ATP to a respiration-deficient cox2 yeast mutant and enabled growth of a yeast–E. coli chimera on a nonfermentable carbon source. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings, and GFP-expressing E. coli endosymbionts could be observed in the yeast by fluorescence microscopy and X-ray tomography. This readily manipulated system should allow experimental delineation of host–endosymbiont adaptations that occurred during evolution of the current, highly reduced mitochondrial genome.” (Mehta et al. 2017, no changes).

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

3.  Potential Learning Objectives for the Podcast

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

• Single Nucleotide Variant (SNV) Analysis: (18:02–18:51): This is a method for identifying genetic variants by comparing an isolated (or historical) genome to a reference genome.  Here, single base substitutions in the isolated ancient DNA were analyzed and compared to known strains of Yersinia pestis to construct a phylogenetic tree.
• Molecular Clock Analysis: (20:08–20:52): Using this method, the timescale of evolutionary events, such as when two life forms may have diverged, can be estimated by looking at changes in DNA sequences to determine ancient mutation rates.
• Admixture Analysis: (21:47–22:55): This is a mathematical analysis that involves examining genetic differences in mixed populations to learn more about the composition of their ancestral populations.

4.2. Main Paper

• Construction of Petite Mutants (31:14–32:15): Using specific yeast protocols, the researchers generated mutant yeast that essentially lacked functional mitochondria.  These are called petite mutants.
• Genetic Modification (32:16–32:56; 35:36–35:56; 42:02–42:57): Methods such as gene insertion via plasmids, as mentioned in the paper, and CRISPR can be used to genetically modify organisms to express desired phenotypes. An example is the researchers here making E. coli deficient in thiamin
• Spheroplast Fusion (33:17–34:13): This is a method to produce yeast without a cell wall or remove the cell wall so that they will take up materials. Here, the researchers use this method, along with polyethylene glycol to make the yeast more likely to take up E. coli cells.
• Fluorescent Microscopy (35:58–36:07): This is a type of microscopy that is used to follow or locate a fluorescent protein.  Usually a protein of interest is fused to a fluorescent protein.  Here, tagging the E. coli cells with fluorescent green proteins (GFP) allowed them to be located under a fluorescent microscope.
• X-ray Tomography (36:07–36:58): By directing X-rays at an object from multiple orientations, the interior of solid objects can be visualized in a non-destructive manner. This technique allowed for the imaging of the intact yeast cells in slices, sort of like a CT scan.

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

5.1. Snippet Paper

• Genomics (10:13–10:32, 15:45–18:49): The sequencing and analysis of the genomes of different Yersinia pestis strains to study the evolution of the species over thousands of years by examining genetic differences between strains both ancient and modern.
• Base Substitution Mutations (17:31–18:51): Single nucleotide variations (SNV), created from individual base substitution mutations accumulating in populations over many generations, can be used in the analysis of evolutionary histories and the construction of phylogenies.
• Iron acquisition, Active Transport (18:52–19:34): Iron acquisition, either through the use of small iron-binding molecules called siderophores or active transporters (energy using), such as those coded for by Ton genes, is essential to the pathogenicity of Yersinia pestis. Mutations were found to have appeared in these genes over time during SNV analysis
• Epidemiology (25:10–26:27): Factors such as widespread malnourishment, stress, and high population density, to which the Trypillia population was thought to be exposed to, around the time of the emergence of ancestors that eventually developed into plague, are known to favor the spread of plague and lead to epidemics.

5.2. Main Paper

• Endosymbiosis (30:13–31:40; 36:14–37:09; 42:57–44:22): To study endosymbiosis, the scientists engineered a model of yeast cells and E. coli to be reliant on each other, wherein E. coli was residing inside the yeast cells in a symbiotic relationship.
• Fermentation Model (31:45–32:12): The defective yeast, or petite mutants, contained no mitochondria, so they could not perform cellular respiration and had to rely on fermentation. They could not grow on glycerol, as it is a non-fermentable carbon source.
• Lysosomes (34:42–35:20): Eukaryotic cells use enzyme-containing organelles called lysosomes to break down unwanted substances, including intracellular bacteria. Some intracellular bacteria , like Chlamydia, have evolved mechanisms to escape lysosomal degradation.
• Molecular Cloning (35:36–35:56): Molecular cloning is the insertion and amplification of foreign DNA in a host organism. Snare-inhibiting protein genes were transferred from chlamydia to the E. coli here is an example of this process.
• Toxicity of Oxygen (44:33–45:16): As the oxygen concentration increased on earth, due to organisms performing photosynthesis, organisms had to evolve mechanisms to cope with its potentially toxic effects. Acquiring mitochondria through endosymbiosis helped them safely utilize oxygen because the machinery to fight toxic effects of oxygen is the same as that used for UV damage response.

6. Podcast Questions

1. Why was it possible for scientists to extract Yersinia pestis from the teeth of ancient human samples?
   1. Live Yersinia pestis could be recovered from the teeth because the pulp of a tooth is completely protected.
   2. Yersinia pestis could be extracted from the thick biofilms present on the teeth of ancient humans.
   3. If the concentration of bacteria is sufficiently high in the bloodstream, bacteria can get into the pulp of the teeth.
   4. Yersinia pestis is a cavity-causing bacteria, and its DNA could be extracted from cavities in ancient human samples.
2. What results were used to infer phylogenetic relationships of the strains of Yersinia?
   1. Single base substitutions in the Yersinia genomes
   2. The appearance of the organisms under a transmission electron microscope
   3. The presence or absence of various exoenzymes secreted by the organisms
   4. Genetic analysis of virulence-associated plasmids found within the organisms
3.  What is the molecular clock technique used for in this study?
   1. Determining the whole genome sequences of Yersinia.
   2. Determining the changes in genetic sequences of the gene under study.
   3. Determine in history when the different strains or species diverged from each other.
   4. Determining the rate of mutation of the bacterial DNA under study.
4. There are two theories of how Yersinia moved: migration and trade. Which of the following supports the trade model?
   1. The same Yersinia strains were found in all the human remains sampled.
   2. All the human remains were genetically blended and carried the same strain of Yersinia.
   3. The human remains showed genetic diversity but the Yersinia strains were identical.
   4. The different Yersinia lineages were in distinct regions without evidence of major population movements.
5. In the main paper, the mutant yeast cells had lost the function of which organelle?
   1. Nucleus
   2. Mitochondria
   3. Golgi apparatus
   4. Centrosomes

6. What ability did the researchers need to provide the E. coli to allow them to survive inside the yeast cells?

   1. The ability to avoid immune system detection
   2. The ability to survive a different tonicity
   3. The ability to ferment sugars into alcohol
   4. The ability to avoid lysosomal degradation

7. What was the primary objective that scientists were trying to accomplish in the main paper? Were they successful? What is your evidence?

   1. Understand the evolutionary steps that led to the development of the first bacterial cells; yes; bacteria and yeast co-evolved in vitro.
   2. Reconstruct the endosymbiosis events that led to the rise of modern eukaryotic cells; yes; bacteria persisted and the yeast survived.
   3. Engineer Rickettsia to serve as a gene therapy vector; yes; bacteria acquired new beneficial phenotypes not present in nature.
   4. Create a new pathogen capable of surviving and replicating inside of cells; yes; the yeast acquired E. coli‘s outer membrane defenses.

7. Figure Reading Exercises

The following are two figure reading exercises, both from Main Paper (Figures 2 and 3).

7.1. First Figure Reading Exercise

Experimental Background (Mehta et al., Figure 2)

According to endosymbiotic theory, mitochondria were once free-living prokaryotes that entered into and remained inside a host cell as endosymbionts. Mehta et al. (2018) were interested in the early stages of mitochondrial evolution that aided this event. To model this evolutionary process, they decided to build  a comparable system using a strain of Escherichia coli engineered to produce and export ATP as the endosymbiont and a yeast lacking mitochondrial ATP synthesis as the host.  First, they generated E. coli cells where  the thiamin biosynthetic gene (thiC) was replaced with a cassette encoding both green fluorescent protein (gfp)  and kanamycin resistance (kanR) [strain E. coli ΔthiC::gfp-kanR] and then also transformed this strain with a plasmid encoding an inducible ADP/ATP translocase that was only produced in the the presence of arabinose sugar (pAM94) [strain E. coli ΔthiC::gfp-kanR(pAM94)].  To test whether the engineered E. coli strains could take up ATP, the strains were incubated with radioactive ATP (noted as [γ-35S]ATP; panel A; uptake).  To measure whether the engineered strains could release ATP, the radioactivity-loaded cells were then incubated with ADP, AMP, or potassium phosphate and the radioactivity retained in  the cells  was quantified (panel A).  To confirm whether the differences in retained radioactivity were in fact due to ATP release, they also measured released ATP for the E. coli ΔthiC::gfp-kanR(pAM94) strain with and without arabinose (the inducer of translocase expression), as well as with and without ADP (panel B).

7.1.2. Questions

1. Which E. coli strain has the plasmid that encodes the ATP translocase gene and what induces translocase expression?
   1. ΔthiC::gfp-kanR; ATP
   2. ΔthiC::gfp-kanR(pAM94); arabinose
   3. ΔthiC::gfp-kanR; arabinose
   4. ΔthiC::gfp-kanR(pAM94); ATP

2. What ability did the E. coli gain following transformation with the pAM94 plasmid when grown in the appropriate inducing medium?

   1. Metabolize AMP using organic phosphate
   2. Produce ATP via cellular respiration
   3. Grow on arabinose containing medium
   4. Transport ATP in and out of the cell

3. What is the dependent variable of the assay shown in panel B?

   1. The ability to ferment arabinose
   2. The amount of ATP taken up
   3. The amount of ATP released
   4. The level of resistance to kanamycin
4. Which results show whether ATP uptake is dependent on the ATP translocase?
   1. Panel A, first pair of bars (ATP uptake) –  without the pAM94 plasmid, the uptake is very small (blue bar), but with it, the uptake is high (red bar).
   2. Panel B, first and second bars–without the pAM94 plasmid, the uptake is very small (second bar), but with it, the uptake is high (first bar).
   3. Panel A, the first, 4th, and 5th pairs of bars–without the pAM94 plasmid, the uptake is very small (blue bar), but with it, the uptake is high (red bar).
   4. Panel B, first and 3rd bars–without the arabinose, the uptake is very small (the third bar), but with it, the uptake is high (the first bar).
5. Which incubation environment (shown in panel B) led to the greatest release of ATP from E. coli ΔthiC::gfp-kanR (pAM94)?
   1. [γ-35S]ATP (+ or–arabinose)
   2. ADP + arabinose
   3. AMP–arabinose
   4. Pi + arabinose

6. In the context of the larger project, why was it important that the E. coli have the ability to translocate ATP?

   1. To provide the E. coli cells with energy to produce the thiamin
   2. To allow the E. coli cells to grow on a nonfermentable medium
   3. To allow them to provide ATP to mitochondrial deficient yeast cells
   4. To reduce the ADP concentration in the extracellular space

7.2. Second Figure Reading Exercise

Experimental Background (Mehta et al., Figure 3)

According to endosymbiotic theory, mitochondria were once free-living prokaryotes that entered into and remained inside a host cell as endosymbionts. Mehta et al. (2018) were interested in the early stages of mitochondrial evolution that aided this event. To model this evolutionary process, they decided to build  a comparable system.  For the host, they used a yeast lacking mitochondrial ATP synthesis (NB97).  These yeast are unable to grow on a non-fermentable carbon source, such as glycerol medium.  For the endosymbiont they engineered Escherichia coli to produce and export ATP.  They first found that the E. coli were unable to be maintained in the yeast.  To understand why this was not working as expected they dug into the literature for intracellular pathogens.  They located evidence that pathogenic bacteria use SNARE-like proteins to avoid degradation in the lysosomes, so they also added genes encoding three different SNARE-like proteins from Chlamydia to produce three new E. coli strains.  To enable the endosymbiotic relationship between the bacteria and yeast, the bacteria were also engineered to be auxotrophic for thiamin and/or NAD, which could be supplied by the yeast host.  One E. coli strain  is unable to produce its own thiamin [strain ID ΔthiC], a second E. coli strain is unable to produce its own NAD  [strain ID is ΔnadA]; and the final E. coli strain is unable to produce its own thiamin and NAD [strain ID is ΔthiCΔnadA].  Now, they believed they had a yeast dependent on E. coli for its ATP and an E. coli dependent on the yeast host for thiamin and/or NAD, so they performed the experiments to test this.  To test the persistence of the E. coli-yeast endosymbiosis, they grew the yeast with no E. coli as well as each chimera on glycerol medium, replating the plain yeast and chimeras four times (rounds 2-4 are shown in panel A).  Additionally, they used quantitative PCR to quantify the relative abundance of the E. coli and yeast genomes  in the chimera of NB97 and E. coli ΔthiC  (below left-most panel A).  To compare colony survival, they plated  equal cell numbers of the chimera of NB97 and E. coli ΔnadA on rich medium (YPD) and selective glycerol medium with carbenicillin, which together eliminates non-endosymbiont yeast and bacteria (panel B). Finally, to investigate whether both genomes were present in the putative chimeras, they isolated DNA from 10 random chimera colonies and used polymerase chain reaction (PCR) with agarose gel electrophoresis to detect bacterial-derived gfp and yeast-derived MatA DNA fragments (panel C).

7.2.2. Questions

1. Which strain(s) of E. coli is/are auxotrophic?
   1. E. coli–ΔthiC
   2. E. coli- ΔnadA
   3. E. coli- ΔthiCΔnadA
   4. all of the above
2. Why were yeast that did not have an E. coli strain unable to replicate?
   1. They lacked the ability to produce ATP to meet their energy needs
   2. The E. coli inside them acted as pathogens and killed the yeast cells
   3. The medium they were inoculated onto contained fungicidal compounds.
   4. Trick question. The control yeast did replicate on the glycerol medium.

3. What does the number of E. coli genomes per yeast genome at the end of re-plating round 4 indicate?

   1. More E. coli cells were initially transferred into the yeast cell than survived.
   2. The E. coli were able to replicate independently in the cytoplasm of yeast.
   3. E. coli are being degraded by host cell lysosomes, releasing their DNA.
   4. The E. coli infected the yeast and became intracellular pathogens.
4. Which results show the researchers successfully mimicked endosymbiosis? (Check all that apply)
   1. The presence of gfp and MatA DNA in the same DNA sample.
   2. The growth of colonies on selective glycerol medium with carbenicillin.
   3. The ratio of E. coli and yeast genomes determined by qPCR in strain 2.
   4. The growth of colonies on rich medium (YPD) medium for NB97 –E. coli- ΔnadA.

5. How did the media used in this experiment simulate evolutionary pressures that led to the development of mitochondria in eukaryotic cells?

   1. The media induced mutations in the yeast and bacteria cells, leading to independent co-evolutionary changes for each.
   2. It created an environment that is dangerous to the bacteria cells, forcing them to rely on the yeast cells for protection
   3. It created a more strict environment (non-fermantative) that forced resource dependency between the yeast and intracellular bacteria cells
   4. It allowed the yeast and bacteria cells to function independently of one another, but survive due to a symbiotic relationship.

8. Paper Information and Licensing

8.1. Snippet paper

• Rascovan N, Sjögren KG, Kristiansen K, Nielsen R, Willerslev E, Desnues C, Rasmussen S. 2018. Emergence and Spread of Basal Lineages of Yersinia pestis during the Neolithic Decline. Cell. 176(1), 295-305. https://doi.org/10.1016/j.cell.2018.11.005
• This article is not licensed for Creative Commons use; see the article on the journal’s website.

8.2. Main paper

• Mehta AP, Supekova L, Chen JH, Pestonjamasp K, Webster P, Ko Y, Henderson SC, McDermott G, Supek F, Schultz PG. 2018. Engineering yeast endosymbionts as a step toward the evolution of mitochondria. PNAS. 115(46), 11796-11801. https://doi.org/10.1073/pnas.1813143115
• This article is licensed for Creative Commons using CC-BY-NC, under the standard PNAS license for non-commercial, educational purposes. See the article’s copyright information for more information.