Metabolic Pathways

TWiM #134: Lipids That Live Forever

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 4:16 minutes

The Most Interesting Things (according to students)

  • The idea that bacteria can be engineered to release therapeutic agents directly at disease sites is fascinating.
  • It shows how synthetic biology can harness natural processes for medical applications. The use of microfluidic devices to characterize the engineered bacteria and track their dynamics in real-time is an impressive technological advancement.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

1.2. Main paper; discussion starts at 31:38 minutes

The Most Interesting Things (according to students)

  • The discovery that hopanoids act as membrane stabilizers and are crucial for stress tolerance in certain bacterial cells highlight their significant role in bacterial survival under extreme conditions.
  • The potential of hopanoids as general environmental stress biomarkers and their involvement in nitrogen storage provides valuable insights into their evolutionary importance and geological significance.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.2.

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

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
  • Impact of Microorganisms (V&C_IM)
  • Metabolic Pathways (V&C_MP)
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 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 15 (ASM_15): Most microbial life is currently unculturable and therefore both cultivation dependent and cultivation-independent techniques are used to identify microbial populations and their potential metabolic pathways.
  • 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
  • Identify the goal of bacterial engineering in the snippet discussion’s paper.
  • Recall the techniques used for a specific purpose in the experiments.
  • Recall how the quorum engineered lysis circuit (SLC) synchronizes bacterial lysis.
 S L
  • Hypothesize potential applications of this bacterial engineering system.
 S H
  • Define hopanoid.
  • Identify the function of hopanoids.
  • Recall the feature of hopanoids that allows them to be used as environmental stress biomarkers in ancient sedimentary rocks.
 M L
  • Propose a way to engineer Nostoc for a practical, hopanoid-related application.
 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

  • Microfluidics (5:00): These are methods that use small amounts of reagents and allow for fine control.  Here the bacteria and cells were grown in microfluidic chambers that allow for tight control for nutrient flow and temperature, allowing for observation of cell behavior under these special conditions. This allowed for seeing how synthesis lysis circuits can trigger cell death.
  • Restriction Enzymes and Cloning (10:00): These are molecular methods for engineering DNA.  Restriction enzymes cut DNA at specific sequences (a restriction digest) and are used engineer plasmids. Cloning allowed for new DNA to be inserted into the plasmid and then replicated in host organisms.

4.2. Main Paper

  • Bacterial and Plant Growth Conditions (33:57): Bacteria and plants have specific growth requirements.  Here, two strains of the bacteria were used, one that can perform symbiosis with the plants and another that could not. They were grown in Allen and Aaron media with nitrate, ammonium, and MOPS and even Akinetes were induced in cultures. There were also temperature-stressed cultures at 13 and 40 degrees Celsius.
  • Strain Construction (39:03): These are biotechnology engineering methods to construct strains with specific features/genes.  Here, the researchers mutated several genes and introduced the plasmids into the bacteria through conjugation. This enabled the bacteria to produce the proteins of the genes that were introduced.  Plasmid selection used neomycin and ampicillin.

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

5.1. Snippet Paper

  • Synthetic Lysis Circuit with Feedback Regulation (4:16): They engineered a bacteria-synchronized lysis circuit which consists of a promoter with an activator (positive feedback) and a lysis gene (negative feedback).
  • Quorum Lysis (5:00): This is used to slowly create the signaling molecule AHL to a threshold level and then rapidly lyse the bacteria population to their contents. This process is continuously conducted.

5.2. Main Paper

  • Hopanoid Function in Cyanobacterial Membrane Stability (32:17): The production of hopanes is linked to rigidifying cell membranes, stress tolerance, plant microbe symbiosis, and nutrient storage in cyanobacteria Hopanoids.
  • Symbiotic Adaptation of Nostoc punctiforme (35:36): It can be studied in Nostoc punctiforme which is a diazotrophic symbiotic cyanobacterium that lives in different types of habitats.

6. Podcast Questions

  1. Which techniques was used to monitor bacterial cell lysis and accurately measure the number of S. typhimurium cells?
    1. Visibility-controlled bacterial growth chambers
    2. Optical density using a spectrophotometer
    3. Recombinant DNA technology and cloning
    4. Microfluidics and fluorescence microscopy
  2. What type of bacterial engineering is primarily discussed in the podcast snippet?
    1. Genetic modification for antibiotic resistance
    2. Engineering bacteria to produce biofuels
    3. Programming bacteria with a synchronized lysis circuit
    4. Using bacteria to degrade plastics in the environment
  3. How is bacterial lysis triggered in the engineered quorum lysis system?
    1. When a critical concentration of signaling molecules (AHL) is reached
    2. When the host’s immune system releases a specific host molecule
    3. When nutrients of a particular type are depleted in the environment
    4. When the local environmental temperature exceeds a certain threshold
  4. Which of the following is a potential application of engineered bacteria using synchronized lysis circuits (SLCs)?
    1. Producing more natural food flavor enhancers
    2. Degrading chemical spills in marine environments
    3. Delivering chemotherapy agents to tumor metastases
    4. Detecting air quality using biomarkers in industrial zones
  5. What are hopanoids?
    1. They are cholesterol-like molecules found in membranes.
    2. They are cytosolic proteins that enhance translation.
    3. They are membrane proteins that protect bacterial at high pH.
    4. They are quorum sensor molecules used for communication.
  6. What is the primary function of hopanoids when bacteria are under high-temperature stress?
    1. They inhibit protein synthesis to reduce heat-induced damage
    2. They increase the permeability of the membrane to water
    3. They disrupt the lipid bilayer to allow faster nutrient uptake
    4. They stabilize the membrane by maintaining fluidity and integrity
  7. Geologists and paleontologists use hopanoids in ancient rocks as biomarkers for bacterial activity.  What feature of hopanoids makes this possible?
    1. They were very plentiful in extinct bacterial species.
    2. They do not decompose like other molecules.
    3. They were used in structures that were dehydrated.
    4. They were only generated during times of stress.
  8. The podcasters discuss the finding that hopanoid-deficient Nostoc are unable to make granules that store carbon and nitrogen, which are granules typically found in a stress-induced state called the akinete. If you wanted to engineer Nostoc as a nitrogen accumulation “factory,” what might be a reasonable way to do this?
    1. Engineer Nostoc to produce hopanoids constitutively, that is all the time.
    2. Engineer Nostoc to produce hopanoid methylases only during osmotic shock.
    3. Engineer Nostoc to produce akinete cell structures rather than reproductive cells.
    4. Engineer Nostoc to produce hopanoid methylases constitutively, that is all the time.

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify the function of the genes within the synthetic genetic circuit.
  • Describe the temporal sequence of events in a synchronized lysis cycle, from bacterial growth to quorum sensing and eventual lysis.
  • Explain how the quorum-sensing mechanism triggers synchronized lysis in the synchronized lysis circuit (SLC) system.
  • Interpret fluorescence data to correlate reporter gene expression with functional events such as lysis in synthetic gene circuits.
  • Evaluate how clinically-relevant variables influence the periodic behavior, and therefore success, of synchronized lysis.
Experimental Background (Din et al., Figure 1)

Din et al. (2016) explore the possibility that engineered bacteria can be used for therapeutic purposes.  They engineered a a bacterium, Salmonella typhimurium, with a Synchronized Lysis Circuit (SLC, panel a) with the aim of regulating bacterial population levels and enabling controlled drug delivery via cell lysis. The SLC system combines positive feedback (via LuxI/AHL autoinduction) and negative feedback (via the bacteriophage lysis gene ϕX174 E), allowing bacteria to coordinate lysis based on quorum sensing through the signaling molecule AHL (N-acyl homoserine lactone; panel b). Briefly, as AHL accumulates, it is expected to trigger a population-wide lysis event, releasing cellular contents and thus killing most of the bacteria. The process then restarts with surviving bacteria producing AHL anew—creating a repeating “integrate-and-fire” cycle.  Din, et al (2016) tested the SLC system by tracking the lysis dynamics using microscopy (panel b, microscopy images) and a microfluidic device (panel c).   Fluorescence (superfolder green fluorescent protein; sfGFP) was used as a reporter for gene expression and as a proxy for population behavior (panel b microscopy images and panel c).  The effect of temperature (red data) and flow inside the microfluidic device (blue data) on the system was also investigated (panel d).

  • The figure cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1. A version of this article is available on PubMed at: https://pubmed.ncbi.nlm.nih.gov/27437587/

7.1.2. Questions

  1. What triggers the synchronized lysis in the synchronized lysis circuit (SLC)  system?
    1. Decrease in cell population to a specific number
    2. Increase in environmental temperature to 30C
    3. Accumulation of AHL to a critical concentration
    4. External chemical inducers provided by host cells
  2. In the genetic circuit (panel a), which gene is responsible for triggering cell lysis?
    1. sfGFP
    2. hylE
    3. luxI
    4. φX174E
  3. What role does the LuxR protein play in the system (panel a)?
    1. It is the AHL-dependent activator of all circuit genes
    2. It blocks the luxI promoter to stop and start transcription
    3. It is the enzyme that synthesizes the therapeutic drug
    4. It is a fluorescent reporter gene for quantifying cell integrity
  4. What is the sequence of stages leading to synchronized lysis (panel b)?
    1. Growth → Synchronized lysis → Quorum threshold
    2. Synchronized lysis → Growth → Quorum threshold
    3. Growth → Quorum threshold → Synchronized lysis
    4. Quorum threshold → Growth → Synchronized lysis
  5. What does the fluorescence signal over time indicate (panel c)?
    1. Constant fluorescent gene expression over time
    2. Coordinated and repeated cell growth and lysis
    3. Accumulation of AHL from cell host tissues
    4. Quorum-related cell death due to nutrient depletion
  6. Since this system is meant to be used in animal tissues, the researchers wanted to know the effects of varying temperature (clinical low to high body temperature; 36C to 40C) and perfusion (flow of fluids). What conclusions can you make about the success/limitations of the lysis circuit at different “body” temperatures and flow rates?
    1. Lysis was coordinated at low temperatures and all flow rates, so this would be effective for animal tissues but not with fever.
    2. Lysis was coordinated at high flow rates and all temperatures, so this would be effective for animal tissues with sufficient nutrients.
    3. Lysis was coordinated at high temperatures and all flow rates, so this would be effective for animal tissues only with fever.
    4. Repeated and coordinated lysis was successful for all temperatures and flow rates, so this would likely work in animal tissues.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of experimental design noted in schematics and figure resources.
  • Evaluate the data to make conclusions about activation of the lysis circuit.
  • Analyze the data to make conclusions about the relationship of circuit activation and cell viability.
  • Analyze the data to make inferences about what experimental aspects are responsible for changes in cell viability.
  • Evaluate the data to draw conclusions about the impact of seeding volume on experimental outcomes.
Experimental Background (Din et al., Figure 3)

Din et al. (2016) explore the possibility that engineered bacteria can be used for therapeutic purposes.  They engineered a a bacterium, Salmonella typhimurium, with a Synchronized Lysis Circuit (SLC) with the aim of regulating bacterial population levels and enabling controlled drug delivery via bacterial cell lysis. In the SLC system bacterial lysis is triggered via an engineered quorum sensing mechanism when the bacterial population reaches a certain level.  When the quorum level is reached gene expression for the lysis molecule (ϕX174E), drug molecule (HylE), and superfolder green fluorescent protein (sfGFP) are all induced.  To provide proof of principle regarding its use of delivering toxic products (HylE) to cancer cells, the researchers co-cultured SLC-engineered S. typhimurium bacteria with human cervical cancer cells (HeLa). Using a microfluidic setup (panel a), the researchers visualized HeLa cell survival as the engineered bacteria reached quorum levels and lysed (panel b).  Bacteria viability was quantified by their engineered green fluorescent protein expression (blue data; panels b and c) and HeLa cell viability was also quantified (black data; panels b and c). Din et al. (2016) also tested whether HylE release or bacterial lysis was responsible for the effect by quantifying HeLa cell viability after exposure to supernatants made from various bacterial strains (see figure legend; panel d). The researchers also examined whether timing of population growth (panel e) and lysis (panel e) could be controlled by adjusting the initial bacterial seeding density.

  • The figure cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

7.2.2. Questions

  1. The authors note that the microfluidic device was modified from their original experiments.  What is the purpose of their modification for this experiment?
    1. To increase the bacterial volume and growth rate
    2. To create maximum flow for cell nutrient acquisition
    3. To allow cell adherence and therapeutic diffusion
    4. To enhance the oxygenation, promoting aerobic respiration
  2. Match the experiment feature with its description.
Feature Description
a. _______ HylE 1. The cervical cancer cell
b. _______ HeLa 2. The reporter gene for bacteria cell integrity
c. _______ Salmonella 3. The therapeutic drug molecule
d. _______ SLC 4. The engineered bacteria species
e. _______ GFP 5. The engineered lysis circuit
  1. What does the increase in fluorescent signal over the 120 minute experiment indicate (panel c)?
    1. Cell division and viability of HeLa cells
    2. Decreasing viability of HeLa cells
    3. Accumulation of therapeutic toxin
    4. Quorum activation of GFP expression
  2. What does the cell viability data over the 120 minute experiment indicate (black data in panel c)?
    1. Cell division and viability of HeLa cells increase as bacteria decrease.
    2. The viability of HeLa cells decreases as the bacteria numbers increase.
    3. Toxin accumulates then decreases causing a resurgence in cell number
    4. Quorum activation does not occur, which is shown by a steady cell number.
  3. An important question is whether the impact on cell viability was due to general bacterial lysis molecules or specifically to release of HylE from lysed bacteria.  To test this the researchers exposed HeLa cells to lysates of different bacterial strains expressing: 1) the SLC only, 2) HylE all the time, 3) SLC containing hylE, or 4) no plasmid (panel d).  Which bacterial strain lysate exposure results in the lowest HeLa cell viability? What does these results indicate?
    1. Strain with SLC + HlyE (Strain 4); HeLa cell death is due to lysis-related release of HylE.
    2. Strain with SLC only (Strain 5); HeLa cell death is due to the presence of the lysis circuit.
    3. Strain with constitutive hlyE only (Strain 6); HeLa cell death is due to production of HylE.
    4. Strain with no plasmid (Strain 7); HeLa cell death is occurring in a non-specific way.
  4. The effect of initial bacterial seeding density on HeLa cell viability was examined by using different volumes of the engineered bacteria (0 uL to 10 uL) to seed the microfluidic device?  What can you conclude based on the data presented (panel e)?
    1. Higher seeding density delays fluorescence and complete HeLa cell death
    2. Lower seeding density results in faster growth but slower toxin release
    3. Higher seeding density leads to earlier complete HeLa cell death due
    4. Seeding density has no impact on cell death timing or fluorescence
  5. The dynamics of SLC circuit activation (firing rate) and HeLa cell viability were examined and the data presented in panel f.   What is relationship between firing rate and toxin exposure time?
    1.  As firing rate increased exposure time increased.
    2. As firing rate increased exposure time decreased.
    3. Exposure time is independent of firing rate.
    4. There is a linear relationship between these.
  6.  The cumulative exposure magnitude across different bacterial seed volumes was investigated and the results presented in the inset of panel f.  What can you conclude about the relationship of magnitude exposure and initial bacterial seeding volume?
    1. It decreases with increasing seed
    2. It increases exponentially with seed
    3. It remains relatively constant
    4. It varies unpredictably

8. Paper Information and Licensing

8.1. Snippet paper

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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