Information Flow and Genetics

TWiM #235: Green Algae and Fatty Acids

Podcast and Annotation Information
  • Annotation by Isabel Bartlett, Kwaku Bonsu, Adam Helfenbein, Rebecca Seipelt-Thiemann, and Roger Greenwell
  • Podcast audio by TWiM: Listen to TWiM #235 Podcast
  • Podcast transcript by Otter.ai and edited by Grace Helle and Harshita Sharma: Access Podcast Transcripts
  • Papers Discussed:
    • Moniruzzaman M, Weinheimer AR, Martinez-Gutierrez CA, Aylward FO. 2020. Widespread endogenization of giant viruses shapes genomes of green algae. Nature. 588(7836):141-145. doi: 10.1038/s41586-020-2924-2.
    • Ellermann M, Jimenez AG, Pifer R, Ruiz N, Sperandio V. 2021. The Canonical Long-Chain Fatty Acid Sensing Machinery Processes Arachidonic Acid To Inhibit Virulence in Enterohemorrhagic Escherichia coli. mBio. 12(1):e03247-20. doi: 10.1128/mBio.03247-20.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 4:02 minutes

The Most Interesting Things (according to students)

  • Nucleocytoplasmic large DNA viruses integrate part of their genomes into green algae in a process known as endogenization. In some green algae species, up to 10% of the host genome consists of this viral DNA. It is interesting how this DNA was integrated into and makes up a large portion of the algae’s genome, allowing new genes and proteins that function in the new host.
  • Similarly to how viral DNA is integrated into green algae’s genome, conferring new functional proteins, the same thing has happened in humans. As provided as an example in this podcast, a protein called syncytin is required for the formation of a placenta. This protein is amongst many in the human genome which diverge from former retroviral proteins.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract and figures cannot be copied here.

1.2. Main paper; discussion starts at 17:40 minutes

The Most Interesting Things (according to students)

  • Arachidonic acid was identified as an antimicrobial, what is of interest to me was the mechanism behind that antimicrobial activity.  Arachidonic acid is sensed by the FadR transcriptional regulator and the FadL transporter which then lead to a reduction in the expression of virulence genes, reducing the ability of these bacteria to adhere to host cells.
  • Additionally, the findings of this paper are interesting because it identifies a potentially new target for antimicrobials or antibiotics. As the fatty acid sensing machinery was important for virulence, targeting this machinery may serve as a potential new drug/antimicrobial target.

“The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.” (Ellermann et al. 2021)

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Metabolic Pathways (V&C_MP)
  • Information Flow and Genetics (V&C_IFG)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • 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 9 (ASM_9):The replication of viruses is determined by their unique structures, DNA or RNA genomes, and the cells they infect.
  • Fundamental Statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.
  • 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.
  • Fundamental Statement 16 (ASM_16):Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 29 (ASM_29): The extent of microbial damage can be minimized by host-derived and external factors, including the microbiome, antibiotics, and immunity.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the main purpose of this study.
  • Recall a human gene, protein, or process that originated from a viral genome.
  • Identify the conclusions of the podcasters’ discussion on fitness advantages related to this topic.
  • Organize the order of steps in retroviral genome integration.
 S L
  • Evaluate a hypothetical experiment to determine whether viral DNA elements are active.
 S H
  • Recall the effect of arachidonic acid on E. coli.
  • Identify the regulator of arachidonic acid’s effect on E. coli.
 M L
  • Predict the experimental results for alternative hypotheses.
 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

  • Computational Biology/ Bioinformatics (7:20–8:15): Computational biology are methods using computers to inform biological data.  These techniques were used to discover the viral DNA in green algae.

4.2. Main Paper

  • Reporter Gene Assay (36:30–37:40): A reporter gene is a system used to monitor gene expression using a fluorescent protein. This assay was used to measure the expression of the espA gene, which is part of the LEE pathogenicity island, in response to certain fatty acids.
  • Secretion Assay (38:30–39:41): This is an assay to detect secretion of specific proteins.  EspA and EspB were measured to determine how arachidonic acid affects the secretion of virulence factors.
  • Growth and Viability Assay (42:50–44:46): The researchers used growth and viability assays to determine whether the antivirulence effect of arachidonic acid is due to a bacteriostatic or bactericidal mechanism, which it is neither.
  • Confocal Microscopy (50:30–52:35): Confocal microscopy is a type of high resolution fluorescent microscopy that can generate 3D images.  It was used to visualize the formation of attaching effacing lesions on epithelial cells.

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

5.1. Snippet Paper

  • Genomics (4:18–18:17): Genome sequencing, identification of viral elements, gene identification, and the genetic impact on host genomes were explored to provide a comprehensive understanding of how the giant viruses that affect green algae contribute to genetic diversity and evolution.
  • Viral Reproduction, Lysogeny (5:43–8:18): These nucleocytoplasmic large DNA viruses integrate part of their genomes into green algae, a trait characteristic of lysogenic viruses.

5.2. Main Paper

  • Novel Antimicrobials (24:00–24:30): The paper highlights how arachidonic acid has a potent inhibitory effect on pathogens through the disruption of membrane integrity.
  • Toxicity of Oxygen (24:00–24:30): Arachidonic acid inhibits the growth of pathogens through an oxidative stress pathway.
  • Virulence Factors (28:13–30:22): Virulence factors such as the LEE pathogenicity island, effector proteins, and type three secretion systems are integral in establishing infection, causing disease, and evading the host immune response
  • Mechanisms of Pathogenesis (28:13–30:22): The use of the aforementioned virulence factors plays an important role in how infection by EHEC E. coli happens.
  • Protein Secretion (28:13–30:22):  EspA and EspB were measured to determine how arachidonic acid affects the secretion of virulence factors. The type three secretion system is a key virulence mechanism for certain bacteria.

6. Podcast Questions

  1. What is the main purpose of the study of the paper on algal genomes?
    1. Discovering that the human genome is 7% retrovirus DNA
    2. Exploring known viral genes in green algae genomes
    3. Exploring the size of viral genomes in eukaryotic cells
    4. Retroviral DNA in humans is responsible for virus immunity
  2. Based on the paper discussion, what type of fitness does having retroviral DNA in eukaryotic genomes provide?
    1. Retroviral DNA is not functional in algal cells, so there is no fitness advantage.
    2. Retroviral DNA makes viral defense proteins, so there is a fitness advantage.
    3. Algae can excise viral DNA but it requires significant ATP, so fitness is reduced.
    4. This event occurs but its advantages or disadvantages require further study.
  3. Which of the following genes, proteins, or processes was originally from a virus? [pick all that apply]
    1. Dystrophin
    2. Syncytin
    3. Axon guidance
    4. Placenta formation
  4. What is the order of the integration of a retrovirus into a genome? [1 = first, 5 = last]
    1. _____ Viral DNA is transcribed
    2. _____ RNA enters the cell
    3. _____ Viral RNA is packaged
    4. _____ RNA is reverse transcribed into DNA
    5. _____ DNA integrates into the host genome
  5. The podcasters note that the study was entirely computational and that more studies need to be done to determine if the viral-derived segments are functional.  Which results would indicate the viral elements have some function?
    1. Products of virtual elements can be amplified using polymerase chain reaction.
    2. Products of virtual elements can be detected using restriction enzyme digestion.
    3. Products of virtual elements can be detected using western blot analysis.
    4. Products of virtual elements can be detected using Southern blot analysis.
  6. What is the effect of host-derived arachidonic acid on E. coli?
    1. Enhances virulence
    2. Inhibits virulence
    3. Inhibits growth
    4. Enhances growth
  7. Which protein regulates the arachidonic acid-induced changes to transcription of the locus of enterocyte effacement (LEE) pathogenicity island?
    1. FadL
    2. EspB
    3. FadR
    4. EspA
  8. In the discussion, Michael points out that fatty acid metabolism is not required for this arachidonic acid effect on LEE operons because the bacteria with a mutant gene in the first step of the metabolism has the same pattern of expression as wild-type when exposed to arachidonic acid (reduction in LEE-encoded operon expression).  What would the results have shown if fatty acid metabolism had been required for this effect?
    1. The strain with the mutant gene would have less LEE-encoded operon expression when treated with arachidonic acid compared to the vehicle.
    2. The strain with the mutant gene would have the same amount of LEE-encoded operon expression regardless of arachidonic acid or solvent treatment.
    3. The strain with the mutant gene had less LEE-encoded operon expression than the wild-type strain when treated with the vehicle solvent.
    4. The strain with the mutant gene had the same amount of LEE-encoded operon expression as the wild-type treated with arachidonic acid.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1 and 3).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in schematics, bar charts, and western blots.
  • Identify key aspects of experimental design, such as positive and negative controls in the experiments.
  • Analyze the data and make conclusions about the impact of fatty acid exposure on LEE operon and specific gene expression.
  • Predict the virulence of bacteria based on conclusions of these experiments.
Experimental Background (Ellerman et al., Figure 1A-E)

Enterohemorrhagic Escherichia coli is a highly pathogenic strain of E. coli that produces Shiga toxin.  It causes severe intestinal damage that results in bloody diarrhea, dehydration, and possibly kidney distress.  Typical infection occurs after eating undercooked contaminated meat, but can result from ingesting any contaminated food.  In the gut, bacteria such as these encounter metabolites produced by the host and other bacteria that can alter their virulence phenotype.  In this study Ellerman et al. (2021) more fully explore how exposure to different long-chain fatty acids affects the regulatory functions of a virulence regulator they had previously identified, FadR.  Of particular interest was the effect on the regulation of a pathogenicity island called the locus of enterocyte effacement (LEE; panel A) which encodes a number of pathogenesis-related proteins including a needle-like molecular machine used to attach and inject bacterial factors into a host cell.  The bacterial operons are activated in this order: LEE1 (ler; red), LEE2 (escC; blue), LEE3 (escV; green), LEE4 (espA; purple), and LEE5 (tir; orange).

The researchers treated bacterial cells with vehicle (the solvent used to dissolve the fatty acid), palmitic acid (panels B and C), or arachidonic acid (panels D and E).  They measured the activation of specific LEE-encoded operons by quantifying gene expression of specific genes in the operon at the RNA level (panels B and D) and at the protein level (panels C and E).  RNA was quantified using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and is displayed as the relative amount of RNA compared to the vehicle (panels B and D). Protein level was analyzed using western blot analysis of specific secreted proteins encoded in each operon, which was then quantified using densitometry.  The level was calculated as “fold change” in level using the amount of operon-encoded protein compared to the loading control (LC); both an example blot and the quantified results are shown (panels C and E).

Schematic, bar charts, and western blots.Figure 1. “LCFAs inhibit the LEE pathogenicity island in EHEC. (A) Schematic of Ler regulation of the LEE pathogenicity island in EHEC. (B to E) EHEC was grown microaerobically under LEE-inducing conditions in the presence of 8 µM palmitic acid (PA), 8 µM arachidonic acid (AA), or the vehicle control (V). (B) Relative expression of the LEE-carried gene espA in EHEC as assessed by targeted qRT-PCR. (C) EHEC secretion of the LEE effector EspA at late log phase as assessed by Western blotting (right) and densitometry (left). (D) Relative expression of representative genes from each of the 5 LEE operons in EHEC as assessed by targeted qRT-PCR. (E) EHEC secretion of EspA and EspB at late log phase as assessed by Western blotting (right) and densitometry (left). …. LC, loading control. All data are represented as the mean ± SEM from at least 3 independent experiments. P values were determined by Student’s unpaired t test (B and D) Mann-Whitney test (C and E), one-way ANOVA (F),…. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.” (Ellermann et al. 2021, cropped to include data and text from panels A-E only )

7.1.2. Questions

  1. How many operons does the  locus of enterocyte effacement (LEE) contain?
    1. 2
    2. 3
    3. 4
    4. 5
  2. What do the asterisks above the bar graphs indicate?
    1. Statistical significance
    2. Median differences
    3. Largest sample sizes
    4. Controls were used
  3. The amount of espA RNA is quantified in the presence of particular compounds (panel B).  In this experiment, ______ is  the positive control and _______ is the negative control.
    1. Vehicle; no negative control
    2. Palmitic acid; no negative control
    3. No positive control; vehicle
    4. No positive control; palmitic acid
  4. To identify the effects of palmitic acid on LEE operon gene expression, the researchers quantified the levels of espA RNA (panel B) and secreted EspA protein (panel C) when palmitic acid was present (PA) or absent (V).  What can you conclude about the effects of PA on this virulence-related gene?
    1. The espA RNA is reduced when palmitic acid is present, but EspA protein is increased.
    2. The espA RNA and EspA protein are both increased when palmitic acid is present.
    3. The espA RNA is increased when palmitic acid is present, but EspA protein is reduced.
    4. The espA RNA and EspA protein are both decreased when palmitic acid is present.
  5. To identify the effects of arachidonic acid on LEE operon gene expression, the researchers quantified the levels of RNA  from one gene in each operon (panel D) and secreted EspA and EspB protein (panel E) when arachidonic acid was present (AA) or absent (V).  What can you conclude about the effects of AA on the LEE-encoded operons? [pick all that apply]
    1. AA exposure has no impact on the secretion of EspA protein but increases EspB protein.
    2. The levels of all operon RNAs are reduced when AA is present compared to vehicle.
    3. The levels of all operon proteins are reduced when AA is present compared to vehicle.
    4. EspA and EspB proteins are secreted less in the presence of AA compared to vehicle.
  6. The levels of secreted EspA are shown in panels C and E for palmitic acid and arachidonic acid, respectively.  If you were to quantify the virulence of these bacteria, which would you expect to be more virulent? What is your evidence?
    1. PA-treated; PA is more effective in curbing EspA secretion compared to AA.
    2. AA-treated; AA is the only fatty acid that reduces EspA and EspB secretion.
    3. AA-treated; AA is more effective in reducing EspA secretion compared to PA.
    4. PA-treated; PA decreases the fold change in EspA compared to the control.
    5. Neither; A combination of PA and AA together decreases secretion of EspA.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in schematics, bar charts, and western blots.
  • Identify positive and negative controls in the experiments.
  • Compare EspA secretion and EspB secretion
  • Analyze the data and make conclusions about the role of each fad gene in the arachidonic acid-induced changes to LEE operon expression.
  • Predict the virulence of bacteria based on the data and with hypothetical changes to the experiment.
Experimental Background (Ellermann et al., Figure 3)

Enterohemorrhagic Escherichia coli is a highly pathogenic strain of E. coli that produces shiga toxin.  It causes severe intestinal damage that results in bloody diarrhea, dehydration, and possibly kidney distress.  Typical infection occurs after eating undercooked contaminated meat, but can result from ingesting any contaminated food.  In the gut, bacteria such as these encounter metabolites produced by the host and other bacteria that can alter their virulence phenotype.  In this study Ellerman et al. (2021) had previously determined that exposure to arachidonic acid reduced expression of operons in a pathogenicity island called the locus of enterocyte effacement (LEE).  They also had identified a virulence regulator, FadR, which was known to be involved in long-chain fatty acid (LCFA) sensing (panel A).  The researchers were interested in determining whether there was a relationship among FadR, arachidonic acid (AA), and LEE-encoded virulence.  To do this, they constructed bacteria with a mutant version of each of three different LCFA sensing pathways genes (fadL, fadR, fadE) and measured the effects of arachidonic acid exposure on LEE-encoded operons by quantifying secreted EspA and EspB proteins, which are encoded in one of the LEE operons.  Mutant deletion alleles are noted by ΔfadL (panel B), ΔfadR (panel C), and ΔfadE (panel E).  WT denotes wild-type bacteria.  Protein level was analyzed using western blot analysis of specific secreted proteins, which was then quantified using densitometry.  The level was calculated as “fold change” in level using the amount of specific protein compared to the loading control (LC); both an example blot and the quantified results are shown.

Schematic, bar chars, and western blots.
Figure 3: “Inhibition of the LEE by arachidonic acid is dependent on FadR. (A) Schematic of canonical long-chain fatty acid (LCFA) sensing in Escherichia coli. (B to D) EHEC was grown microaerobically under LEE-inducing conditions in the presence of 8 µM arachidonic acid (AA) or the vehicle control (V). (B) Secretion of the LEE effectors EspA and EspB at late log phase by EHEC WT or ΔfadL strain as assessed by Western blotting (left) and densitometry (right). (C) Secretion of EspA and EspB by EHEC WT or ΔfadR strain as assessed by Western blotting (left) and densitometry (right). (D) Secretion of EspA and EspB by EHEC WT or ΔfadE strain as assessed by Western blotting (right) and densitometry (left). LC, loading control. All data are represented as the mean ± SEM from at least 3 biological replicates. P values were determined by Kruskal-Wallis test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.” (Ellermann et al. 2021)

7.2.2. Questions

  1. A loading control (LC) is used in all western blot analysis experiments.  What is the purpose of this control? [pick all that apply]
    1. The loading control allows researchers to ensure that the proteins loaded onto the gel were not degraded.
    2. The loading control allows researchers to identify artifacts in membrane transfer during western blot analysis.
    3. The loading control allows researchers to determine the levels of RNA compared to levels of protein.
    4. The loading control allows researchers to fairly compare/normalize the protein of interest across samples.
  2. WT is the designation the researchers use for wild-type, non-mutant E. coli. What does ΔfadL denote?
    1. It is a strain that can’t import short-chain fatty acids.
    2. It is a strain of E. coli that is missing the fadL gene.
    3. It is a strain of E. coli that overexpresses FadL protein.
    4. It is a strain of E. coli that produces arachidonic acid.
  3. What does the bar height in the bar graphs indicate?
    1. Statistical significance
    2. Standard deviation
    3. Mean/average
    4. Standard error
  4. The amount of secreted EspB protein in the presence of vehicle (V) and arachidonic acid (A) is displayed in the western blot in panel D.  In this experiment, ______ is  the positive control and _______ is the negative control.
    1. Vehicle; no negative control
    2. Arachindonic acid; FadE
    3. FadE; arachidonic acid
    4. No positive control; vehicle
  5. Based on the schematic of canonical LCFA sensing (panel A).  Which protein is responsible for permitting long-chain fatty acids to cross the inner bacterial membrane?
    1. FabB
    2. FadD
    3. FadE
    4. FadL
  6. To identify the role of different LCFA sensing proteins in arachidonic acid-induced effects on LEE operon gene expression, the researchers quantified the levels of secreted EspA and EspB proteins in wild-type (WT) and a bacterium deficient in FadR (ΔfadR) when arachidonic acid was present (AA) or absent (V).  What can you conclude about the effects of AA on these virulence-related proteins in the FadR-deficient bacteria?
    1. Secreted EspA is reduced when arachidonic acid is present, but EspB protein is increased.
    2. Secreted EspA  and EspB protein are both increased when arachidonic acid is present.
    3. Secreted EspB is reduced when arachidonic acid is present, but EspA protein is increased.
    4. Secreted EspA and EspB protein are unchanged by the presence of arachidonic acid.
  7. The levels of secreted EspA are shown in panels B, C, and D for bacteria with mutations in genes fadL, fadR, and fadE, respectively. Wild-type is shown in each panel (WT).  If you were to quantify the virulence of these four strains of bacteria, which would you expect to be more virulent? What is your evidence?
    1. ΔfadE; this is the only strain that has reduced Esp secretion with arachidonic acid exposure.
    2. ΔfadL; a mutation in this gene blocks EspA and EspB secretion even with arachidonic acid.
    3. ΔfadR; a mutation in this gene enhances EspA secretion when arachidonic acid is present.
    4. ΔfadL or ΔfadR; these bacterial strains are both unaffected by arachidonic acid exposure
    5. Wild-type; this strain will be the most virulent because it is wild-type and has no mutations.

8. Paper Information and Licensing

8.1. Snippet paper

  • Moniruzzaman M, Weinheimer AR, Martinez-Gutierrez CA, Aylward FO. 2020. Widespread endogenization of giant viruses shapes genomes of green algae. Nature. 588(7836):141-145. doi: 10.1038/s41586-020-2924-2. https://doi.org/10.1038/s41586-020-2924-2
  • 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

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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