Metabolic Pathways

TWiM #243: Beef and Bacillus

Podcast and Annotation Information
  • Annotation by Sakiem Winston, Madison Wolford, Rebecca Seipelt-Thiemann, and Angela Wilson
  • Podcast audio by TWiM: Listen to TWiM #243 Podcast
  • Podcast transcript by Otter.ai and edited by Isabelle Norris and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Yang X, Wang H, Hrycauk S, Klassen MD. 2021. Effects of Peroxyacetic Acid Spray and Storage Temperature on the Microbiota and Sensory Properties of Vacuum-Packed Subprimal Cuts of Meat. Appl Environ Microbiol. 87(11):e03143-20. doi: 10.1128/AEM.03143-20
    • Khanna K, Lopez-Garrido J, Sugie J, Pogliano K, Villa E. 2021. Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation. Elife. 10:e62204. doi: 10.7554/eLife.62204.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:45 minutes

The Most Interesting Things (according to students)

  • A single almond takes a gallon of water to grow.
  • They tested a variety of temperatures and still didn’t cook the ribeyes.

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 29:24 minutes

The Most Interesting Things (according to students)

  • Bacillus subtilis uses a different form of division for sporulation that’s not at the center of the cell.
  • The part about thinning the cells using ion beams so they could use cryo-ET. We didn’t expect physical tools like that to be used on bacterial samples

“The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.” (Khanna et al. 2021)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • 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 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • 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.
  • 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.
  • 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 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall experimental conditions used in the meat spoilage study.
  • Describe the conclusions of the meat spoilage study.
 S L
  • Predict how changes in the experiment will affect the results.
 S H
  • Describe how Bacillus subtilis cell division differs for vegetative growth and sporulation.
  • Match the proteins involved in Bacillus subtilis cell division with their functions.
  • Identify the technique improvement that made this new imaging possible.
 M L
  • Predict how engineered changes in protein localization would affect septum location.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described

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

  • 16S Amplicon Sequencing (13:14–24:32): This is a technique where the 16S rDNA gene is amplified and used to identify its originating species using a database of known sequences.  Here, the researchers used it to identify which bacteria were present and how the composition changed in the meat treated with the compound and stored in different conditions and times.
  • Sensory Assessment (13:14–24:32): This is the technique of smelling the item to detect spoilage.  The researchers used people with experience and training in detecting odors of the meat that was treated and stored in different ways.  They used a Likert scale of 1-5.

4.2. Main Paper

  • Cryo-electron Tomography (Cryo-ET) (32:40–36:53): This is a 3D imaging technique used to visualize internal structures of bacteria. It allowed the researchers to compare the structure of the septa during vegetative growth and sporulation.
  • Cryo-focused Ion Beam (FIB) Milling (36:48–37:10): This is a method that physically thins the thick bacterial cells with a focused ion beam so they could be imaged using cryo-ET.

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

5.1. Snippet Paper

  • Microbiome, Spoilage, and Food-borne Illness (06:28–11:54; 14:30–22:32): The podcasters discuss how bacteria contribute to and protect from specific food-borne illnesses, mostly E. coli O157H7 and E. coli that produce Shiga toxin.
  • Microbial Load (7:00–10:00): The podcasters discuss where the bacteria involved in spoilage originate in meat processing including the term microbial load, which is how many bacteria are present.  The goal is to reduce the microbial load because all cattle carry pathogens.

5.2. Main Paper

  • Asymmetric Cell Division (29:24–31:23): Bacillus subtilis divides asymmetrically during sporulation, forming a smaller forespore and a larger mother cell. This division occurs near one pole of the cell, unlike the mid-cell binary fission used during vegetative growth
  • Sporulation Septum vs. Vegetative Septum (31:23–32:50): The septum formed during sporulation is thinner than the one formed during vegetative growth. The division machinery also localizes differently, with FtsZ and SpoIIE playing key roles.

6. Podcast Questions

  1. How many days was the meat stored in the meat spoilage experiment?
    1. 0, 2, 4
    2. 10, 30, 90
    3. 34, 104, 180
    4. 94, 180, 240
    5. 1,2,3
  2. Why did the researchers use the storage temperatures of 4C, 2C, and -1C?
    1. These are common consumer meat storage temperatures.
    2. These are temperatures recommended by the meat industry.
    3. These are temperatures known to influence pH level changes.
    4. These are temperatures that favor anaerobic fermentation.
  3. How did peroxyacetic acid (PAA) treatment affect the pH of packaged meat as the storage length increased?
    1. Increased the pH
    2. Decreased the pH
    3. Remained the same
    4. Was highly variable
  4. Which bacterium was found in the highest amounts in the meat samples with the highest spoilage?
    1. Serratia
    2. E. coli
    3. Yersinia
    4. Clostridium
  5. If the researchers were to repeat their experiment using another meat, such as pork or chicken, how would you expect the results to change?  What is your reasoning?
    1. The levels of pathogenic bacteria would initially be the same, but a different meat would interact with PAA differently so it is not possible to predict the outcome.
    2. The pH levels and spoilage rates would likely change, but the bacterial species present would likely not change since spoilage environmental microbes are common.
    3. The specific bacteria would likely change, but not the conclusions that pH level drops with storage, because different species of animals carry different microbes.
    4. The spoilage rate would increase due to smaller cuts of meat being treated.  This would be due to a larger surface to mass ratio for the bacteria to populate.
  6. Where does Bacillus subtilis divide during vegetative growth versus sporulation?
    1. At the mid-cell; Near one pole
    2. Near one pole; At both poles
    3. At both poles; At the forespore
    4. At the forespore; At the mid-cell
  7. Match the protein involved in Bacillus subtilis cell division with its function/description? (1 = FtsA; 2= FtsZ; 3= SpoIIE)
    1. ______ Protein involved in Z ring formation
    2. ______ Protein that makes another protein adhere to the membrane
    3. ______ Protein that regulates the spore/vegetative switch
  8. What improvement did the cryo-focused ion beam milling bring to the experiment?
    1. To kill the bacteria so imaging was more detailed
    2. To freeze the cells so confocal imaging could be done
    3. To thin the cell membrane so imaging was feasible
    4. To stain the membranes so they were distinguishable
  9. The podcasters describe the model put forth by the researchers. You can see this model in Figure 9d of the paper if you’d like. In sporulation cell division, the FtsA/FtsZ filaments localize to the mother cell’s side of the cell. In vegetative cell division, the FtsA filaments are found near the cell membrane and the FtsZ filaments are found away from the cell membrane.  Which type of cell division would you expect to find if you engineered a version of FtsA to remain bound to the main cell membrane? Why?
    1. Vegetative; FtsA is found at the membrane in vegetative cell division
    2. Sporulation; FtsA is found at the mother’s cell side in sporulation division
    3. No cell division; the functional association of FtsA with FtsZ would be disrupted
    4. There is not enough data to answer this question.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 2) and one from the main paper (Figure 7EF).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of growth curves, including their display and phase names.
  • Identify key aspects of experimental design, including the variable types in an experiment and control groups.
  • Evaluate the data to make conclusions about the impact of temperature on growth of total and specific groups of bacteria.
  • Evaluate the data to make conclusions about the impact of PAA on growth of total and specific groups of bacteria.
Experimental Background (Yang et al. 2021, Figure 2)

Food spoilage is a significant and costly waste of resources, estimated to cost the average American nearly $3000 a year.  Food spoilage also has environmental repercussions by adding to land fills and increasing greenhouse gases.  Spoilage occurs due to microbial growth that can include pathogens such as E. coli O157:H7.  In fact, cattle are a significant reservoir of this pathogen, making safe beef processing methods particularly important for food security.  In this study, Yang et al. (2021) investigated the ability of a low exposure to peroxyacetic acid (PAA) to affect spoilage and microbiome composition on a specific cut of beef, subprimal ribeye, when stored vacuum-packed at temperatures used by consumers (4oC in panel A; 2oC in panel B; -1oC in panel C) for up to 180 days.  The steaks were treated with 200 parts per million PAA or no treatment (control).  Because pH was significant in prior experiments, they focus here on quantifying living bacteria using colony forming units (CFU) and provide data for total aerobic bacteria counts (TAC) and two specific groups: lactic acid bacteria (LAB) and Enterobaceriaceae (ENT).

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

7.1.2. Questions

  1. The growth curve for PAA-treated meat stored at 4oC is displayed in panel ___ and the total aerobic counts are shown by the ____ curve.
    1. A; red
    2. B; red
    3. A; blue
    4. B; blue
  2. The dependent variable for the experiment results displayed on just the left side of panel B is _____ while the independent variable for this experiment is ______.
    1. Time; ENT counts
    2. PAA treatment; time
    3. Time; PAA treatment
    4. Bacterial counts; time
  3. The negative control for this experiment is ______ and the positive control for this experiment is ______.
    1. No PAA treatment; no positive control
    2. 0 days vacuum ; 104 days vacuum
    3. No negative control; PAA-treatment
    4. Total aerobic counts; no positive control
  4. How does the exponential bacterial growth compare for control and PAA-treatment meats stored at -1oC?
    1. Exponential growth is delayed by 40 days in PAA-treated meats compared to untreated.
    2. Exponential growth is shortened by about 20 days in PAA-treated compared to control.
    3. Exponential growth is lengthened by 20 days in PAA-treated meats compared to untreated.
    4. Exponential growth does not differ between them, but rapidly changes to stationary phase.
  5. Which storage temperature affects bacterial growth the most (in the absence of PAA)?
    1. 4°C
    2. 2°C
    3. -1°C
    4. None; all affect it equally
  6. Which bacterial group appears to be most impacted by PAA-treatment?  What is your evidence that supports your conclusion?
    1. Total aerobic; the growth curve is misshapen in PAA-treatment compared to the control at 4°C.
    2. Enterobacteriaceae; CFU counts are lower in PAA-treated meats compared to the control at 2°C.
    3. Lactobacillus; CFU counts are lower in PAA-treated meats compared to the control -1°C.
    4. Total aerobic; CFU counts are lower in PAA-treated meats compared to the control at -1°C.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of bar plots as related to this experiment.
  • Identify key features of experimental design, including dependent and independent variables in the experiment.
  • Analyze the data to make conclusions about the difference in specific sporulation features for vegetative and sporulation.
  • Predict the growth condition, septal location, and septal wall type when given specific hypothetical characteristics.
  • Hypothesize advantages for a particular septum type based on the data.
Experimental Background (Khanna et al. 2021, Figure 7 EF)

Spores are highly resistant products of bacteria and yeast, including pathogen species.  They are generated when species encounter specific stress environments.  Because they are more resistant to decontamination processes, the process of sporulation is a target of pathogen containment procedures.  Bacillus subtilis is a good model system to study sporulation as it has two types of cell division: vegetative and sporulation. During vegetative growth, which is typical in nutrient-rich conditions, the cell division septum is formed at the midcell, producing two daughter cells of equal sizes. However, during sporulation, which occurs under stress, the cell division septum is formed closer to one pole to produce a smaller forespore and a larger mother cell.  The forespore matures after being engulfed by the mother cell.  Scientists knew that septum formation involved the FtsZ, FtsA, and SpoIIE proteins, but the outer structures of this organism had, up until this point, been too thick to use high resolution microscopy to visualize and study formation of these septa.  Khanna et al. (2021) developed a milling technique to thin the structures so sporulation could be studied microscopically.  To learn more about the structures and features of vegetative and sporulation septa, they quantified several cell division features in vegetative (veg) and sporulating (spo) growth, including septal length and the distance spanned by FtsZ, which is the protein that forms the contracting ring to separate the cells.  Their data are presented as bar graphs.

Two bar charts, both with veg much higher than spo.
Figure 7. “FtsAZ filaments during sporulation and septal thickness measurements.  … (E, F) Bar graphs depicting (E) septal thickness and (F) distance spanned by FtsZ bundle in wild-type vegetative and sporulating cells. For both, error bars indicate standard deviation. Each dot indicates a sample point. (****p≤0.0001, unpaired t-test). Scale bars: (A) 200 nm, (B, C) 25 nm. See also Figure 7—figure supplements 1–5.”(Khanna et al. 2021, cropped image and removed figure legend text to include only panels E, F).

7.2.2. Questions

  1. The dependent variable for the experiment in panel E is ______ and the independent variable for the experiment in panel E is ______.
    1. Vegetative growth; sporulation
    2. Septal thickness; growth conditions
    3. Aporulation; vegetative growth
    4. Growth conditions; septal thickness
  2. The results of septal thickness and FtsZ bundle are shown in panels D and E, respectively.  There are some results that are statistically significantly different.  What feature in the bar plot allows you to tell which are?
    1. The standard error whiskers
    2. The mean bar height
    3. The asterisks at the top
    4. The value of the y-axis
  3. What is the difference in average septal thickness for vegetative and sporulating growth?
    1. 26 nanometers
    2. 35 nanometers
    3. 45 nanometers
    4. 60 nanometers
  4. Based on these data, what would you predict to be the cellular location of the septum in a cell that has a thinner septum?
    1. At the mid-cell
    2. Near one pole
    3. At both poles
    4. At the forespore
  5. If you found that the distance spanned by the FtsZ bundle was 45 nm, which type of septal wall would be most likely forming? What is your evidence?
    1. Sporulation;  the distance for sporulation is dependent of FtsZ bundle which is found in this range
    2. Vegetative; the distance for vegetative has less error than sporulation, as noted by the whiskers
    3. Sporulation;  the distance for sporulation is calculated by multiplying the bundle by filament numbers
    4. Vegetative; the distance for vegetative averages much higher (about 50) than sporulation (about 22)
  6. Why might the particular septum size (panel E) for sporulation and spore maturation be an advantage over the septum size for vegetative growth?
    1. It allows faster for DNA replication
    2. It eases engulfment of the forespore
    3. It strengthens the spore membrane
    4. It promotes symmetrical cell division

8. Paper Information and Licensing

8.1. Snippet paper

  • Yang X, Wang H, Hrycauk S, Klassen MD. 2021. Effects of Peroxyacetic Acid Spray and Storage Temperature on the Microbiota and Sensory Properties of Vacuum-Packed Subprimal Cuts of Meat. Appl Environ Microbiol. 87(11):e03143-20. doi: 10.1128/AEM.03143-20
  • 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

  • Khanna K, Lopez-Garrido J, Sugie J, Pogliano K, Villa E. 2021. Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation. Elife. 10:e62204. doi: 10.7554/eLife.62204.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See https://elifesciences.org/articles/62204#copyright

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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