Structure and Function

TWiM #270: Magnets and Salt Improve Plastics Production by Archaea

Podcast and Annotation Information
  • Annotation by  Jenna Frizzell, Sandra Hanna, Anna Kate Kinnear, Rebecca Seipelt-Thiemann, Roger Greenwell
  • Podcast audio by TWiM: Listen to TWiM #270 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Chen S, Cui YW, Huang MQ. 2022. Coupling Magnetic Field and Salinity Upshock To Improve Polyhydroxyalkanoate Productivity by Haloferax mediterranei Feeding on Molasses Wastewater. Appl Environ Microbiol. 88(13):e0030522. doi: https://doi.org/10.1128/aem.00305-22 .
    • Tal N, Millman A, Stokar-Avihail A, Fedorenko T, Leavitt A, Melamed S, Yirmiya E, Avraham C, Brandis A, Mehlman T, Amitai G, Sorek R. 2022. Bacteria deplete deoxynucleotides to defend against bacteriophage infection. Nat Microbiol. 7(8):1200-1209. doi: https://doi.org/10.1038/s41564-022-01158-0

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:30 minutes

The Most Interesting Things (according to students) 

  • The function of microorganisms in distinctive industrial processes, such as biofilm stability or nutrient recycling.
  • Conversations could link microbial discoveries to uses in medicine, such as new antibiotics or probiotic treatments.

This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.

1.2. Main paper; discussion starts at 27:00 minutes

The Most Interesting Things (according to students)

  • The ways in which bacteria acquire resistance to antibiotics and the molecular foundations of these protective strategies. Subjects such as efflux pumps and enzyme-driven inactivation.
  • Emphasizing the dynamic relationships between microbial communities and their hosts, which encompasses immune evasion strategies and the alteration of host biology by both commensal and pathogenic microbes.

This article is not licensed for Creative Commons use; see the article copyright information. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 12 (ASM_12). Bacteria and Archaea exhibit extensive diversity, including nitrogen fixation, methane production, and anoxygenic photosynthesis, many of which are unique to these two domains.
  • Fundamental Statement 21(ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 26(ASM_26):Humans leverage microbes and their products to address problems and improve quality of life.
  • 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 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define halophile.
  • Define aseptic technique and identify a reason why the researchers did not use it.
  • Recall the strategies used to increase polyhydroxyalkanoate (PHA) production in these microbes.
 S L
  • Predict the effect of different environments on PHA production based on the relationships of growth, stress, and PHA production.
 S H
  • Identify different bacterial defenses against phages.
  • Recall the function of the enzymes in this study or discussed on the podcast
 M L
  • Hypothesize phage resistance mechanisms to defend against cytidine deaminase.
 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

  • Growth Conditions (5:00): Microbes have different growth conditions that can make them more difficult or easier to grow in the lab or for certain applications.  The bacteria in this study are halophiles that grow best in high salt conditions making them easier to grow in pure culture without having to use aseptic technique.

4.2. Main Paper

  • Recombinant DNA (30:23–31:31): Recombinant DNA technologies use molecular techniques to modify DNA, introduce it into cells, and enable gene expression in the new cell. The researchers used these to introduce the gene encoding cytosine deaminase gene into  MG1655 E. coli  to determine whether it confers defense against phages.
  • Mass Spectrometry (38:28–40:00): Mass spectrometry is a method to identify and quantify molecules using their mass to charge ratio.  Using mass spectrometry, the relative levels of dNTPs in control and MG1655 E. coli were determined pre- and post-infection with phage in order to understand the function of the defensive genes of interest.

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

5.1. Snippet Paper

  • Halotolerance & Osmotic Pressure (4:47–6:10; 14:16 -16:07): Increasing the salinity in the growth conditions of Haloferax mediterranei (an extreme halophile), the internal concentration of PHA (a green-plastics precursor) significantly increases.
  • Volumetric Productivity & Fermentation (7:00–10:45): This involves a concept important for applied microbiology areas where you balance investment (cost) and return (products).  Here, the researchers were trying to increase polyhydroxyalkanoate (PHA) and reduce cost.
  • Electron Carriers (10:11–13:14): Subjecting microbes to a low-level magnetic field facilitates redox shuffling.
  • Horizontal Gene Transfer (HGT) (10:46–11:47): Processes that involve DNA transfers that are not from cell division, such as conjugation, transformation, or transduction.
  • Microbial Evolution (11:48–19:17): Analyzing the movement and adaptation of genetic components in reaction to environmental challenges. The emphasis on microbial ecology and new genetic methods underscored the practical and creative elements of the research presented.
  • Mobile Genetic Elements (19:19–21:58): Mechanisms illustrating how components such as plasmids or transposons enhance microbial diversity and resistance to antibiotics. Emphasizing the significance of microbial resistance strategies in addressing global health issues.

5.2. Main Paper

  • Viral Reproduction: Lytic (33:41–35:47): In response to lytic T7 phage infection, E. coli carrying the cytidine deaminase gene rapidly convert their population of cDTP to uDNP thus inhibiting the phage from DNA replication and reproduction.
  • Host-Microbe Interactions (40:31–45:18): Study of the symbiotic or pathogenic associations between microorganisms and their hosts.
  • Sigma Factors (42:42–44:00): Gene 5.7 in T7 encodes a protein that shuts down transcription of a sigma transcription factor within the host cell, which is responsible for the initiation of mRNA production. Mutations in this gene allow for partial resistance in T7 phage.
  • Microbial Metabolism and Biochemical Pathways (45:26–47:51): Examining the metabolic adaptations or pathways that microbes employ to thrive and grow in particular environments.
  • Ecological and Functional Roles of Microbes (47:53–48:47): Investigating microbial diversity and their roles in larger ecological frameworks, including nutrient cycling or disease interactions.

6. Podcast Questions

  1. What is a halophile?
    1. A species that is able to grow in high salt environments.
    2. A species that requires a partner bacterium species to grow.
    3. A species that does not require water for fermentation.
    4. A species that can endure high radiation stress conditions.
  2. The podcasters make a point that the researchers did not need to use aseptic technique in this study. Why did the researchers not use it?
    1. The researchers used media containing ten different antibiotics.
    2. The researchers used adherent strains not affected by washing.
    3. The microbes grow on/in differential media that other bacteria don’t.
    4. The microbes produce compounds toxic to other microbial species.
  3. What strategies did researchers use to optimize PHA production in Haloferax mediterranei?
    1. Magnetic fields and non-halophilic microbes to enhance fermentation.
    2. Osmotic lysis for cell recovery and high salt concentrations as stressors.
    3. Low salt concentration and recycled water for biomass improvement.
    4. Sterile media and reducing waste stream use to optimize carbon sources.
  4. Given the strategies and results of the PHA production experiments, what would be a reasonable condition to try to enhance PHA production and why?
    1. Aeration increased PHA production, so they could bubble oxygen through the media.
    2. Adding xylose sugar increased production, so they could try adding other sugar.
    3. Magnetizing the metal flasks increased PHA production, so try rare metal magnets.
    4. Stressing the microbes increased PHA production, so they might try high heat stress.
  5. The podcasters mention and/or discuss a number of ways that bacteria defend against phages.  Which are bacterial defense systems/enzymes? [pick all that apply]
    1. CRISPR/Cas
    2. Restriction-modification systems
    3. Uridine deaminase
    4. Cytidine deaminase
  6. Based on the podcast discussion, which role does deoxyguanidine triphosphatase play in bacterial defense against phage infection?
    1. It removes the base, guanine, from deoxyguanosine triphosphate to intermittently pause phage replication.
    2. It converts deoxyguanosine triphosphate to deoxyguanosine monophosphate to prevent phage replication.
    3. It dephosphorylates deoxyguanosine triphosphate to deoxyguanosine, preventing phage DNA replication.
    4. It removes the deoxyribose sugar from deoxyguanosine to induce lethal mutations into the phage genome.
  7. Based on the podcast discussion, what is the function of cytidine deaminase?
    1. Inhibits RNA polymerase subunit a.
    2. Converts cytidine to uridine.
    3. Is a cofactor for DNA polymerase.
    4. Removes amino groups from proteins.
  8. If you were to isolate phage resistant to cytidine deaminase, what might you find? [pick all that apply]
    1. Phage encode an enzyme converting uridine to cytidine.
    2. Phage with no cytidine bases in their genome.
    3. Phage with abasic nucleotides in their genomes.
    4. Phage encode an enzyme converting DNA to RNA.

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 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify important features in a line graph.
  • Identify the dependent and independent variables in an experiment.
  • Make conclusions about the optimal conditions for Haloferax mediterranei biomass accumulation, PHA production, and volumetric productivity based on data provided.
  • Design an experiment to test how salinity level affects the expression of enzymes in PHA synthesis.
Experimental Background (Chen et al., Figure 1)

In an effort to produce environmentally friendly alternatives to petrochemical plastics that take years to degrade in the environment, scientists have identified a biodegradable polymer called polyhydroxyalkanoates (PHAs).  However, the cost to produce them is still very high.  Chen et al. (2022) investigated ways to increase PHA production in a cost-effective way using the halophile Archaea Haloferax mediterranei.  To identify what salt concentration was the best growing condition for both biomass accumulation and PHA production, the researchers grew the microbe in three different salt concentrations and measured dry cell weight (panel a), what percent of the dry weight was PHA (panel b).  These measures allowed the researchers to then quantify the efficiency of PHA production as volumetric productivity, which is the rate at which PHA is produced per culture volume (panel c).  This measure is important for ensuring low cost, high production methods needed in applied microbiology. For panel c, a denotes statistical significant at P , 0.05 and b denotes no difference.

7.1.2. Questions

  1. What feature is noted by the red line in the graph in panel a?
    1. PHA production of the microbe when grown in 300 g/L NaCl.
    2. Logarithmic growth of the microbe in medium with 100 g/L NaCl.
    3. Growth of the microbe over time in medium with 200 g/L NaCl.
    4. Accumulation of dry cell mass for microbes grown in 200 g/L NaCl.
  2. For the experiment showing PHA production (panel b), the dependent variable is ______ and the independent variable is _______.
    1. % PHA of cell content; salt concentration
    2. Dry cell mass; % PHA of cell content
    3. Salt concentration; volumetric productivity
    4. Volumetric productivity; dry cell mass
  3. What is the optimal salinity concentration and culture time for the biomass accumulation (panel a) of Haloferax mediterranei?  What is your evidence?
    1. 100 g/L NaCl; 70 hours; cell mass peaks most stably here
    2. 200 g/L NaCl; 50 hours; cell mass peaks the earliest here
    3. 300 g/L NaCl; 70 hours; cell mass peaks the latest here
    4. 300 g/L NaCl; 40 hours; cell mass is stable and high here
  4. What is the optimal salinity concentration and culture time for the PHA production (panel b) of Haloferax mediterranei? What is your evidence?
    1. 100 g/L NaCl; 50 hours; % PHA peaks the earliest here
    2. 200 g/L NaCl; 70 hours; % PHA is the most stable here
    3. 200 g/L NaCl; 50 hours; % PHA is stable and high here
    4. 300 g/L NaCl; 70 hours; % PHA reaches the highest here
  5. Volumetric production (panel c) takes into account biomass accumulation(cell mass; panel a), product produced (% PHA of cell; panel b), time, and volume for production so it is a good way to quantify efficiency.  Which salinity concentration supports the best volumetric production for PHA?  What is your evidence?
    1. The volumetric production for 100 g/L and 300 g/L were equally small and statistically significantly different than 200 g/L.
    2. The volumetric production for 100 g/L was lowest and statistically significantly different than 200 g/L and 300 g/L.
    3. The volumetric production for 200 g/L was highest and statistically significantly different than 100 g/L and 300 g/L.
    4. The volumetric production for 300 g/L was highest and statistically significantly different than 100 g/L and 200 g/L.
  6. Using both the dry cell mass (panel a) and % PHA (panel b) results, what relationship do you see that could aid the researchers in choosing a next step for their experiments?
    1. Increasing the salt concentration increased the relative PHA production so the researchers should increase the salt concentrations in the medium above 300 g/L and incubation time.
    2. The microbes grew best, as determined by dry cell mass, in medium with 200 g/L NaCl, so the researchers should use enhanced aeration techniques to get more oxygen to the microbes.
    3. While microbes grown in 300 g/L NaCl produce relatively more PHA, their growth is slowed.  It would be good for the researchers to identify ways to enhance growth in the 300 g/L medium.
    4. The microbes had the most consistent % PHA levels at 70 hours of culture incubation, so the researchers should culture the microbes for 70 hours but use lower salt concentrations in the medium.
  7. Which experimental approach would best determine how salinity levels affect the expression of key enzymes involved in PHA synthesis in Haloferax mediterranei?
    1. Measure the intracellular PHA content at different salinities using gas chromatography.
    2. Use spectrophotometry to measure light scattering across different salinities and pH levels.
    3. Monitor the growth curve of Haloferax mediterranei at optimal and nonoptimal salinities.
    4. Perform a proteomic analysis to quantify enzyme levels under varying salinity conditions.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify relevant features in schematics, bar graphs, and/or heatmaps.
  • Evaluate the data to determine whether or not cytidine deaminase functions in phage defense, and if so, against which phages.
  • Analyze how cytidine deaminase impacts T7 phage DNA replication.
  • Describe the relationship of different nucleotide pools when E. coli do or do not express cytidine deaminase.
  • Hypothesize phage resistance mechanisms to defend against cytidine deaminase.
Experimental Background (Tal et al., Figure 1)

Defenses against viral infection are important for both eukaryotic and prokaryotic cells.  Virally-infected human cells use a mechanism to deplete nucleotide pools so the virus cannot replicate its genome.  In this study, Tal et al. (2022) attempt to identify similar defense mechanisms in bacteria.  They began by looking for genes located in or near known genomic defense “hot spots” in bacterial genomes and identified a putative defense gene, cytidine deaminase (panel a).  They next investigated whether expression of this gene in E. coli could affect phage infection and T7 phage DNA replication.  These data on plaque formation using a variety of bacteriophage, with and without cytidine deaminase are displayed as a heat map (panel b), while T7 phage DNA replication is displayed as a bar chart (panel g).  Next, the researchers investigated whether mutations in specific cytidine deaminase functions could affect phage success/bacterial defense (panel c).  To determine whether nucleotide pools were affected the researchers quantified levels of relevant nucleotides from bacteria expressing cytidine deaminase or not using liquid chromatography-mass spectrometry (panels d-f).

7.2.2. Questions

  1. In the schematic diagram for cytidine deaminase (panel a), the researchers identify the names and locations of two domains.  What are they?
    1. Kinase; deaminase
    2. E. coli AW1.7;  576 aa
    3. Deaminase; E. coli AW1.7
    4. WP_045268677.1; Kinase
  2. What does the color key in panel b indicate?
    1. The more intense red, the bigger the phage plaques were.
    2. The more intense red, the faster the bacteriophage replicated.
    3. The more intense red, the more bacteria defense was observed.
    4. The more intense red, the greater the number of phage plaques.
  3. Which phage is most sensitive to cytidine deaminase encoded by AW1.7? What is your evidence?
    1. SECphi4, SECphi6, T7; they produce only smaller plaques.
    2. T6 and T7; they have a 103 fold reduction in plaque number.
    3. SECphi27, SECphi17, T5; they have no reduction in plaque size.
    4. T5, SECphi6, SECphi17; they have no fold change in plaque size.
  4. Based on the phage T7 phage infection of bacteria expressing different mutant cytosine deaminase proteins, which domain(s) of cytidine deaminase is/are critical for phage infection?
    1. Deaminase
    2. Kinase
    3. Both
    4. Neither
  5. What effect does the expression of cytidine deaminase have on phage DNA replication (panel g)?
    1. Phage DNA replication is accelerated.
    2. Phage DNA replication is unaffected.
    3. Phage DNA replication is stopped.
    4. Phage DNA replication is impaired.
  6. Based on the functions of cytidine deaminase, the researchers hypothesized that cytidine deaminase converts dCTP to dUMP.  They performed mass spectroscopy to test this hypothesis (panel e-f).  Do these data support, refute?
    1. Support.  As dUMP increases, dCTP decreases, and vice versa.
    2. Support.  As dUMP increases, dCTP increases, and vice versa.
    3. Refute. As dUMP increases, dCTP peaks and returns to baseline.
    4. Refute. As dUMP increases, dCTP remains stable and high.
  7. If you were to isolate phage resistant to cytidine deaminase, what might you find? [pick all that apply]
    1. Phage encode an enzyme converting uridine to cytidine.
    2. Phage with no cytidine bases in their genome.
    3. Phage with abasic nucleotides in their genomes.
    4. Phage encode an enzyme converting DNA to RNA.

8. Paper Information and Licensing

8.1. Snippet paper

  • Chen S, Cui YW, Huang MQ. 2022. Coupling Magnetic Field and Salinity Upshock To Improve Polyhydroxyalkanoate Productivity by Haloferax mediterranei Feeding on Molasses Wastewater. Appl Environ Microbiol. 88(13):e0030522. https://doi.org/10.1128/aem.00305-22 .
  • This article is not licensed for Creative Commons use; see https://journals.asm.org/doi/10.1128/aem.00305-22. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.

8.2. Main paper

  • Tal N, Millman A, Stokar-Avihail A, Fedorenko T, Leavitt A, Melamed S, Yirmiya E, Avraham C, Brandis A, Mehlman T, Amitai G, Sorek R. 2022. Bacteria deplete deoxynucleotides to defend against bacteriophage infection. Nat Microbiol. 7(8):1200-1209. https://doi.org/10.1038/s41564-022-01158-0
  • This article is not licensed for Creative Commons use; see https://www.nature.com/articles/s41564-022-01158-0#rightslink. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.

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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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