Evolution

TWiM #266: Bacteria That Can Record

Podcast and Annotation Information

  • Annotation by Rongze Ao, Charmaine Grace Eclevia, Sanjay Harinatha, Valerie Thao-Van La, Rebecca Seipelt-Thiemann, and Rachael Barry.
  • Podcast audio by TWiM: Listen to TWiM #266 Podcast
  • Podcast transcript by Otter.ai and edited by Harshita Sharma and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Wang F, Virginija Cvirkaite-Krupovic, Vos M, Beltran L, Kreutzberger MA, Winter J-M, Su Z, Li J, Schouten S, Mart Krupovic, et al. 2022. Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome. Cell. 185(8):1297-1307.e11. doi: 10.1016/j.cell.2022.02.019
    • Schmidt F, Zimmermann J, Tanna T, Farouni R, Conway T, Macpherson AJ, Platt RJ. 2022. Noninvasive assessment of gut function using transcriptional recording sentinel cells. Science. 376(6594):eabm6038. doi: 10.1126/science.abm6038.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:56 minutes

The Most Interesting Things (according to students)

  • The evolution of simple rod-like viruses to larger spindle viruses was fascinating because it shows microbes are constantly evolving in our bodies and the outside world. This is a testament to how viruses develop evolutionary adaptations to survive in more intense environments in order to be replicated amongst many different organisms and to the potential of our bodies to develop and evolve into more efficient systems.
  • The mere amino acid composition and conformation of proteins can confer unique abilities to organisms through higher-order structures, including the dislocation of the seven helical strands within capsids that allow the expansion from rod-like to spindle-shaped.

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 16:45 minutes

The Most Interesting Things (according to students)

  • By assessing how diet-induced microbiome changes affect health, this technology can be used to evaluate treatment effectiveness and potentially pave the way for personalized medicine.
  • In the co-culture experiment, E. coli can use the material produced by other bacteria. Such interactions show some bacteria can work together to thrive and adapt to changing conditions, ultimately supporting a balanced and resilient microbiome.
  • The use of modified CRISPR/Cas systems to analyze the gut microbiome is a significant discovery. With small modifications, this tool can be used to track microbe-microbe and microbe-host interactions, even allowing researchers to pinpoint where in the gut these interactions occur. This approach allows us to explore the human body at a microscopic level providing deeper insight into understanding the microbiome and potentially, other body systems.

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.

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

Snippet Main
Vision and Change Topics
  • Evolution  (V&C_E)
  • Structure and Function (V&C_SF)
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundament 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 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 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 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.
  • Fundamental Statement 23 (ASM_23): The health of the environment and all organisms (microbes, plants, humans, other animals) are closely linked and interdependent, as described by the One Health paradigm

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • List the two major categories of virus capsids and the various methods used to determine their structures.
  • Recall how the biochemistry of structural adaptation contributes to a spindle-shaped virus’ ability to withstand extreme environmental conditions.
 S L
  • Propose how the structural evolution of spindle-shaped viruses from rod-shaped ancestors is advantageous.
 S H
  • Outline the steps of engineering the Record-seq CRISPR system that records bacterial gene expression as the bacterium moves through the mouse gut.
 M L
  • Predict what types of genes would be recorded in the Record-seq plasmid of sentinel E. coli cells, given different gut environmental and/or dietary conditions.
 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

  • X-ray Crystallography (7:50–8:32): A technique used to determine the 3D structure of a crystal using an X-ray beam that creates a diffraction pattern. The pattern indicates the particular arrangements of atoms in the crystal. The structure of the capsids of archaea-infecting viruses was constructed, specifically showing the unique helical structure made up of seven strands that expand to accommodate viral genome packaging.

4.2. Main Paper

  • RNA Sequencing (28:38–38:00): This is a next generation sequencing technique that provides a snapshot of which genes are being actively expressed at a specific point in time by sequencing the RNA, via cDNA generation, collected from bacteria. This was used to compare gene expression in E. coli in response to different diets in mice.
  • Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) (18:54-28:01): A gene-editing tool that uses specialized proteins to cut DNA at specific sites to modify, remove, or add genetic material.
  • Record-Seq (18:03–18:22): Technique that records transcription by using a modified CRISPR and RNA as a template.
  • Competition Assay (30:00–34:00): These are assays that allow a direct comparison of the fitness of two strains, here the wild-type and mutant strains.  Strains lacking specific genes were grown in parallel with wild-type and the abundance quantified.  This was done to determine the importance of the “knockout” genes to fitness.
  • Co-Culture (37:15–39:04): Growing two or more bacterial species together to study their interaction. E. coli was co-cultured with Bacteroides thetaiotaomicron (a major gut inhabitant) to observe how E. coli gene expression changed in the presence of another organism.
  • DNA Barcoding/Multiplexing (39:02–39:31): Detection of specific species by small standardized pieces of DNA.  Researchers add sequence tags to bacteria strains which are unique sequence identifiers that enable researchers to distinguish between wild-type and mutant strains during experiments.
  • Gnotobiotic Mice (32:09–32:16; 38:26–38:37): Gnotobiotic mice are germ-free mice and have no other microbiome colonization other than the bacteria introduced by the researchers.  They are a good model for studying the impact of specific microorganisms in a controlled setting since they are raised in a sterile environment.

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

5.1. Snippet Paper

  • Viral Capsid Structures and Function (2:49–14:07): Viral capsids play a crucial role in protecting and packaging the viral genome. They often feature helical symmetry in rod-shaped viruses or icosahedral symmetry in icosahedral viruses.
  • Archaeal Membrane Lipids (15:06–15:30): Archaeal membranes can adapt and withstand extreme environments, unlike bacterial membranes. Therefore, this allows spindle-shaped archaeal viruses to infect archaea that live in extreme environments like low pH and high temperatures.

5.2. Main Paper

  • Gene Expression Regulation (27:54–43:08):  How gene expression changes based on different parameters. In this instance, looking at how dietary factors and pH changes influence gene expression in sentinel bacteria as they move through the gut.
  • Record Seq (18:54–28:50): Used for recording transcriptional data in sentinel cells. This specific system uses a reverse transcriptase that takes in the mRNA and converts it into DNA, which is then stored in the bacterial genome and later analyzed.
  • Microbiome (29:36–30:06): Refers to the composition of microbes in the gut. Certain shifts of microbial gene expression can occur under the influence of various diets.
  • Anaerobic vs Aerobic Respiration (35:00 – 43:27): Changes in terminal electron acceptors used in respiration by organisms in the gut environment.
  • Gnotobiotic Systems/Hosts (32:09–32:16; 38:26–38:37): Mouse models with no other microbiome colonization other than the bacteria introduced by the researchers. This allows them to isolate the effects of specific dietary conditions on the behavior and gene expression of the sentinel E. coli.
  • Symbiosis (37:15–39:04): Two or more organisms in a close relationship and often dependent on each other. In this study the scientists looked at the interaction between E.coli and Bacteroides thetaiotaomicron in co-culture represents a mutualistic relationship, in which different species interact and support each other metabolically.

6. Podcast Questions

  1. What are the two major categories of virus capsids?
    1. Rod-Shaped and Cone-Shaped
    2. Spherical and Complex
    3. Helical and Icosahedral
    4. Enveloped and Polyhedral
  2. What biochemical feature allows the 7-start helical structure of the capsid to withstand extreme environments?
    1. Hydrophobic residues are found on the inside of the structure.
    2. Covalent bonding allows a sturdy and stable structure to form.
    3. The capsid uses hydrogen bonding to form a large structure.
    4. The formation of beta barrels in the structure is protective of DNA.
  3. Which of the following would be an advantage of related to the structural adaptations observed for spindle-shaped viruses?
    1. They can evolve into smaller capsids that require fewer resources to produce.
    2. They are more easily engulfed by endocytosis or injected into the host cells.
    3. Their dynamic nature allows them to accommodate genomes of different sizes.
    4. They mimic host cell shapes, allowing them to avoid immune detection.
  4. What is the first step in the general process of performing Record-seq in mice?
    1. Collecting the fecal samples from the mice and analyzing their gut microbiome composition.
    2. Engineering the CRISPR array and incorporating that transcriptional recording gene in a plasmid.
    3. Administering sentinel E. coli to the mice orally and analyzing the abundance in fecal material.
    4. Administering molecular markers to mice by feeding them and then performing RNA sequencing.
  5. What/Which gene(s) would you expect to see represented in a Record-seq CRISPR array from a mouse that experienced an allergic reaction to a high-starch diet while colonized by the sentinel E. coli?
    1. Gluconate kinase and heat shock genes
    2. Glucose-6-phosphate and chaperone genes
    3. High-pH adaptation and heat tolerance genes
    4. Low-pH adaptation and pro-inflammatory genes

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Define cryo-EM and explain its importance in this study.
  • Identify key features in the microscopy and model diagrams.
  • Analyze the data to make conclusions about the 3-dimensional structure of the capsid.
  • Predict the structure of the capsid when carrying genomes of different sizes.

Experimental Background (Wang et al. Figure 1)

Sulfolobus monocaudavirus 1 (SMV1) is an archaeal virus known for infecting organisms that thrive in extreme environments. Spindle-shaped viruses like SMV1 are notable because they carry larger genomes and have unusual spindle and spindle-like shapes that are variable.  Understanding these adaptations could provide valuable information for understanding how these viruses survive in extreme conditions.  In this study, Wang et al. (2022) first used cryo-EM, a high-resolution imaging technique that uses flash-frozen samples to capture the three-dimensional structures, to obtain a high-resolution image of the virus (panel A).   They then used the data to reconstruct a model structure of the tail core (panel B) and tail surface (panel C).  Additionally, theoretical and data-driven ribbon models of a single major capsid protein (MCP) are shown with identification of asparagine 53, which is highly glycosylated, oriented to the outside of the three-dimensional structure, and suggested to play a role a protective role in extreme environments (panel D).

  • This article is not licensed for reuse. Thus, the abstract and figures cannot be copied here. Please see the article on the journal’s web page.
7.1.2. Questions
  1. Cryo-EM imaging was essential for studying the structure of SMV1 because it allowed scientists to:
    1. Visualize the biochemical pathways of viral replication.
    2. Determine the structure of the virion with high resolution.
    3. Observe viral infection in live host cells at it occurred.
    4. Study the lipid composition of the viral envelope.
  2. What role does the N-linked glycosylation of the MCP play in virus survival?
    1. It allows the virus to survive in more extreme conditions.
    2. It helps the virus to bind to its host more effectively.
    3. It allows the virus to bypass innate immune responses.
    4. It helps the virus to maintain a stable cylinder shape
  3. What best describes the theoretical symmetry and shape of the capsid based on the model data?
    1. It is a dodecahedron.
    2. It is icosahedral.
    3. It is a hollow tube.
    4. It is large sphere.
  4. The authors suggest that the MCP proteins slide against each other to accommodate the space necessary for the genome.  Based on this, which of the following structures would you expect to carry the genome with the largest number of base pairs?  What is your reasoning?

three caspid shape drawings: the first has a tall top and a shallow bottom, the latter is wide on both ends, and the latter is shallow on both ends.

    1. A, because it has the longest shape
    2. B, because it has the largest 3-dimensional size.
    3. C, because it has the most tightly compacted size.
    4. None of them, because shape doesn’t matter, stability matters.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in stacked bar charts, box plots, and bar charts.
  • Compare the abilities of Record-seq  and RNA sequencing to identify differentially expressed genes.
  • Analyze the data to make conclusions about the diet-based differences in differentially expressed genes using each technology.
  • Evaluate the data to draw conclusions about the diet-based effects on uxaC gene expression as quantified using each technology and across tissues.
  • Judge the relative success of specific E. coli strains in a competitive colonization assay.

Experimental Background (Schmidt et al., Figure 3ACD)

The gut microbiome is a dynamic system of microbial communities that are important for maintaining health and its disruption (dysbiosis) is known to be involved in a number of diseases including autoimmune diseases.  Studying the dynamic and interactive nature of these communities has been a challenge.  In this study, Schmidt et al. (2022) evaluate a system for capturing bacterial gene expression changes as bacteria make their way through the gut environment.  The technology, which they call Record-seq is a plasmid-based, modified reverse-transcriptase CRISPR system, they engineered and used to transform E. coli “sentinel” bacteria.  In this technology, bacterial RNAs will be reverse-transcribed and the resulting DNA stored in the sentinel’s plasmids, which can be sequenced.  If shown to be effective, this would be an improved and noninvasive way to survey progressive gene expression changes in the gut as compared to the current methodology of RNA sequencing.  RNA sequencing provides a snapshot of gene expression. However, it can be invasive and must be performed across a number of locations and time-points to observe trends.  To compare these techniques, the authors designed a study to examine the effects of different diets on the gut microbiome.  They fed mice traditional mouse chow or a starchy diet and then either introduced sentinel cells or sampled gut tissues (cecum, proximal colon, distal colon, and feces). The authors analyzed and compared sequencing data from their Record-seq sentinel bacteria acquired from feces and data from RNA sequencing of the gut samplings. Differentially expressed genes (DEGs) were identified when mice were fed a starchy diet compared to mouse chow for both technologies and then compared (panel A).  The researchers next wanted to characterize a specific gene’s expression that they identified using both technologies, uxaC, which is involved in hexuronate catabolism.  To do this, they normalized and compared gene expression counts from both technologies (panel C*).  To further validate the finding that uxaC was important, the researchers inoculated germ-free mice with a 1:1 mixture of wild-type E. coli and E. coli with a deleted uxaC gene (ΔuxaC).  Fecal counts of both wild-type and E. coli ΔuxaC  were quantified at multiple time points and the competitive index calculated as a ratio (panel D).

  • This article is not licensed for reuse under Creative Commons. Thus, the abstract and figures cannot be copied here. Please see the article on the journal’s web page.

* For help on boxplots, you can go here: Visualizing the Box and Whisker Plot or Atlassian Box Plot Guide.

7.2.2. Questions

  1. About 45% of differentially expressed genes were found in both the fecal transcriptome and Record-seq data for mice fed chow (panel A).  A further 54% were found only in Record-seq.  Of the 54%, a large number of genes were found to be differentially expressed in particular intestinal regions.  What proportion of the Record-seq “only” DEG were found as DEG only in the proximal intestine?
    1. 10%
    2. 33%
    3. 54%
    4. 56%
  2. Which of the following statements shows the “best use” of each technology, based on the data presented in panel A? [pick all that apply]
    1. Record-seq appears to be better at detecting how gene expression changes occur along the gastrointestinal tract.
    2. Record-seq appears to be better at detecting the total of all gene expression changes across the gastrointestinal tract.
    3. RNA sequencing appears to be better at detecting all genes expressed in fecal samples, but not in individual tissues.
    4. RNA sequencing appears to be better at detecting relative and more subtle differences across time and tissues.
  3. The gene expression for gene uxaC was quantified for both technologies and along the gut axis (panel C). The data are displayed by box plots, which help display statistical measures that bar plots do not show.  Since the legend and paper do not indicate certain features, match the box plot feature with its typical description. [1 = mean; 2 = median; 3 = outlier; 4 = interquartile range; 5 = individual measures; 6= full data range excluding outliers]
    1. ______ Box
    2. ______ Horizontal line in box
    3. ______ Whiskers
    4. ______ Symbols, such as circles
  4. Which region of the intestine shows the greatest difference in uxaC gene expression when chow and starchy diets are compared, and which technology(ies) show(s) this? [pick all that apply]
    1. cecum; Record-seq
    2. proximal colon; Record-seq
    3. distal colon; Record-seq
    4. feces; Record-seq
    5. cecum; RNA-seq
    6. proximal colon; RNA-seq
    7. distal colon; RNA-seq
    8. feces; RNA-seq
  5. Based on competitive colonization experiments (panel D), what can you conclude about relative success of colonization for wild-type E. coli and the uxaC mutant (ΔuxaC) E. coli?
    1. The uxaC mutant E. coli have a competitive fitness disadvantage compared to wild-type strains under starch-rich diets.
    2. Wild-type strains generally show lower competitive fitness than uxaC mutant E. coli under starch diet conditions.
    3. Both strains exhibit similar competitive fitness, regardless of dietary conditions, as evidenced by equivalent bar heights.
    4. The uxaC mutant E. coli show higher competitive fitness across all diets compared to the wild-type strains.

8. Paper Information and Licensing

8.1. Snippet paper

  • Wang F, Virginija Cvirkaite-Krupovic, Vos M, Beltran L, Kreutzberger MA, Winter J-M, Su Z, Li J, Schouten S, Mart Krupovic, et al. 2022. Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome. Cell. 185(8):1297-1307.e11. doi: 10.1016/j.cell.2022.02.019
  • 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 on the journal’s web page.

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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