Structure and Function

TWiM #210: The Waze of Microbes

Podcast and Annotation Information

  • Annotation by Brian Tran, Jeremy Bullock, Santiago Jacob, Roger Greenwell, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #210 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Bru JL, Rawson B, Trinh C, Whiteson K, Høyland-Kroghsbo NM, Siryaporn A. 2019. PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics. J Bacteriol. 201(23):e00383-19. doi: 10.1128/JB.00383-19.

1. Paper Abstracts

1.1. Snippet paper; there is no snippet paper

The Most Interesting Things (according to students)

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1.2. Main paper; discussion starts at 20:23 minutes

The Most Interesting Things (according to students)

  • Eshel Ben-Jacob from Tel Aviv invented the “social IQ score” of bacteria, and Pseudomonas ranks high on the scale. Social IQ score depends on the number of genes involved in communication.
  • The difference between newtonian and non-newtonian liquids. Viscosity doesn’t change for Newtonian liquids regardless of physical changes such as force application. Viscosity does change for non-Newtonian liquids.
  • The roll and tumble method is not the only way for microbes to move.

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 paper at the journal’s website.

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

Snippet Main
Vision and Change Topics
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  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
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  • 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 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
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 S L
  • n/a
 S H
  • Define quorum sensing,
  • Identify the quorum sensor molecule and its biological role in this study.
  • Identify reasons for changes to standard growth procedures for these experiments.
  • Recall the conclusions from the study regarding Pseudomonas swarming behaviors in response to stressor conditions.
 M L
  • Design an experiment to investigate stress-related swarming behaviors for other motile bacteria.
 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

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4.2. Main Paper

  • Swarming Assay (23:28–24:30): This is an assay to detect motility that is performed on agar that is softer than normal plate agar.   It was used to detect directed bacterial motility towards and away from other bacteria.
  • Mutation/Mutant Strains (43:48–45:17): Mutations, which are DNA changes, were made in different genes to show how the gene contributes to or is required for the swarming/repulsion response.
  • Repulsion Quantification (45:24–48:00): This is a scientific term for quantifying avoidance or “moving away” behavior (repulsing).  The radius of each satellite colony is determined by measuring the center to the two nearest swarming tendrils.
  • Mass Spectrometry (56:57–58:25): This is an analytical technique to quantify and identify molecules present in a mixture using mass and charge.  It was used to see if the PQS (quorum sensor molecule) is diffusing.

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

5.1. Snippet Paper

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5.2. Main Paper

  • Quorum Sensing/Population Communication (24:32–29:10): Microbes communicate information to other individuals using chemical messengers.  Pseudomonas aeruginosa have for of these called quorum sensing systems.  Here, bacterial communication about stress conditions (phage infection or presence of antibiotics) was quantified using a swarming assay.
  • Culture Conditions (55:31–56:15): Culture conditions are the environment, which is an important consideration in an experiment.  Here, solid medium allows for direct visualization of Pseudomonas growth and trajectory changes.  The diffusion kinetics of chemicals in the solid medium also allows the effect of concentration to become apparent.  The choice of solid medium rather than liquid medium allowed these experiments to succeed.

6. Podcast Questions

  1. Quorum sensing is _________________ and the quorum sensor molecule in this study is _________.
    1. A method of bacterial trajectory consensus: CAS
    2. The way bacteria regulate their motility; HAA
    3. A method of extracellular communication; PQS
    4. The way bacteria resist phage infection: RhlAB
  2. Which of the following best defines the role of the Pseudomonas aeruginosa quorum sensor molecule that is discussed in this podcast?
    1. A molecule that degrades antibiotics in stressed environments
    2. A signal that regulates stress responses and bacterial behavior
    3. A protein responsible for flagellar movement during swarming
    4. A nutrient uptake signal activated by rare metal scarcity
  3. What was the reason the researchers changed the plate agar concentration from the normal 1.5% agar to  0.5% agar for their experiments?
    1. To save money on laboratory reagent costs.
    2. To  enable the motility of the Pseudomonas.
    3. To increase the effectiveness of antibiotics.
    4. It allows for better visibility of colonies on plates.
  4. What were the conclusions from this study? [Pick all that apply]
    1. Stressors such as phage infection or antibiotic exposure induce population signaling to warn other bacteria and influence their motility.
    2. Phage infection and antibiotic resistance are regulated by common genes that are present in the genomes of many different species.
    3. Pseudomonas aeruginosa is a rare species that uses quorum sensing to regulate the expression of flagella and pilli genes in distinct ways.
    4. Exposure to stressors causes bacteria to permanently lose their ability to form biofilms, reducing their survival in hostile environments.
  5. How could you investigate whether another motile bacteria, Proteus vulgaris, also uses this quorum sensing system?
    1. Measure the growth rate of P. vulgaris in nutrient-rich media to determine if quorum sensing affects its metabolism.
    2. Sequence the genome of P. vulgaris to identify any genes related to motility and compare them to those in P. aeruginosa.
    3. Co-culture P. vulgaris and P. aeruginosa, then observed them using microscopy to see if they move faster when grown together.
    4. Perform a swarming assay with phage-infected P. aeruginosa at the satellite positions and P. vulgaris in the center.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1B–D and 5C).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in swarming assay agar images and schematics.
  • Identify controls for these experiments and what they indicate.
  • Analyze the data and make conclusions about effects on swarming behaviors towards phage-infected and quorum-deficient strains.
  • Propose an experiment to identify the molecule(s) responsible for any effects.

Experimental Background (Bru et al., Figure 1B-D)

Pseudomonas aeruginosa is a highly adaptive pathogen with many different strategies for survival.  It is a common pathogen of the skin, eye, and lungs, particularly in cystic fibrosis patients where its ability to form and live in biofilms is key to its antibiotic resistance in that location.  Here, Bru et al. (2019) investigate the interconnectedness of another behavior that contributes to its success in many environments, motility, and a virulence regulating quorum sensor system called PQS (Pseudomonas quinolone signal).  The researchers began their study knowing that genes involved in production of quorum sensing molecule PQS  were upregulated in phage-infected P. aeruginosa.  They hypothesized that phage infection would affect PQS and initiate motility changes.  To investigate this behavior, they performed swarming assays where wild-type uninfected (panel B, i), wild-type infected (panel B, ii), or motility-defective uninfected mutant in the quorum sensing system (panel B, iii) P. aeruginosa is spotted at the center of an agar plate and allowed to grow and move (swarm).  Because quorum sensing in an external communication system in bacteria, they were next interested in whether the presence and condition of the bacteria (and thus its quorum communication) might be able to affect swarming behavior.  To do this, they inoculated bacteria onto plates in pairs (panel C) or at multiple positions around the agar plate (see panel D for plates and schematic).

The swarming behaviors were assessed from these images, but the researchers also provide time-lapse videos of these experiments in their supplementary materials, which are available at: on the article’s web page.

  • 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 paper at the journal’s website.

7.1.2. Questions

  1. Different versions and different treatments of P. aeruginosa are used in these experiments.  Which plates in which panels show you the swarming behavior/pattern of only the wild-type P. aeruginosa?
    1. Panel B, image i and panel B, image ii, and panel B, image iii
    2. Panel C, image i and panel C, image ii, and panel C, image iii
    3. Panel D, image i and panel D, image ii, and panel D, image iii
    4. Panel B, image i and panel C, image i and panel D, image i
  2. Controls are an important part of any experiment.  The swarming behaviors of the different bacteria are examined in paired cultures on agar plates, with data shown in panel C.  What controls are shown in these data and what does each tell us? [Pick all that apply]
    1. Plate C i is a control.  It shows us how swarming behaviors look on the same plate.
    2. Plate C ii is a control.  It shows us how different swarming behaviors look on the plate.
    3. Plate C iii is a control.  It shows us how swarming and non-swarming look together.
    4. Plate C iii is a control.  It shows us how quorum-capable and -defective look together.
    5. All of these plates are the necessary controls for the plates that are shown in panel D.
  3. Based on all of the data in these panels, what can you conclude about how phage infection affects swarming? [Pick all that apply]
    1. Wild-type P. aeruginosa avoid/are repulsed by phage-infected P. aeruginosa.
    2. Phage-infected P. aeruginosa avoid/are repulsed by wild-type P. aeruginosa.
    3. Wild-type P. aeruginosa swarm more towards phage-infected P. aeruginosa.
    4. Phage-infected P. aeruginosa swarm more towards wild-type P. aeruginosa.
    5. Swarming avoidance/repulsion requires the quorum system in the partner.
    6. Motility/swarming increases require the quorum system  in the partner.
  4. How could you determine which molecules are diffusing into the agar from the phage-infected P. aeruginosa that are responsible for this effect?
    1. Make cell extracts of phage-infected P. aeruginosa, fractionate them, and test fractions for swarming differences.
    2. Make cell extracts of uninfected and phage-infected P. aeruginosa and analyze them using mass spectrometry.
    3. Measure the optical density of the agar to detect changes in bacterial concentration caused by diffusing molecules.
    4. Use a microscope to observe the agar surface for visible changes in texture or color near the phage-infected colonies.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in swarming assay agar images.
  • Analyze the data and make conclusions about production of the quorum sensor molecule for each strain.
  • Propose additional controls for these experiments that would help with interpretation.

Experimental Background (Bru et al., Figure 5C)

Pseudomonas aeruginosa is a highly adaptive pathogen with many different strategies for survival.  It is a common pathogen of the skin, eye, and lungs, particularly in cystic fibrosis patients where its ability to form and live in biofilms is key to its antibiotic resistance in that location.  Here, the researchers investigate the interconnectedness of another behavior that contributes to its success in many environments, motility, and a virulence regulating quorum sensor system called PQS (Pseudomonas quinolone signal).  The researchers had identified that phage-infection and antibiotic-treatment induced production of the PQS quorum sensor molecule and that release of this molecule into the medium repulsed laboratory strains of P. aeruginosa.  To determine the clinical relevance of this behavior, the researchers next tested a hypervirulent strain of P. aeruginosa isolated from a Liverpool epidemic (LESB58; panel C, left image) as well as a mucoid isolate from a cystic fibrosis patient (P2m; panel C, center image), and another common laboratory strain (MPA01; panel C, right image).  Additionally, P2m was pre-treated with the antibiotic kanamycin to induce stress and the MPA01 strain was pre-treated with the antibiotic gentamicin to induce stress.  Swarming assays were performed by inoculating wild-type (PA14) into the center of an agar plate and then inoculating the other strains at six spots near the plate periphery (satellites).

The swarming behaviors were assessed from these images, but the researchers also provide time-lapse videos of these experiments in their supplementary materials, which are available at: https://journals.asm.org/doi/10.1128/jb.00383-19

  • 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 paper at the journal’s website.

7.2.2. Questions

  1. The behavior of wild-type P. aeruginosa towards other strains is shown in the figure images.  What is the strain designation for the wild-type strain and where on the plates is it inoculated?
    1. LESB58; the left image
    2. MPA01: the right image
    3. P2m; the plate periphery
    4. PA14; center of each plate
  2. Which of the following strains show evidence of swarming behaviors?
    1. Untreated MPOA1
    2. antibiotic -treated MPOA1
    3. Untreated P2m
    4. antibiotic -treated P2m
    5. Untreated LESB58
    6. antibiotic -treated LESB58
    7. Untreated PA14
    8. antibiotic -treated PA14
  3. The results from this experiment show that the hypervirulent Liverpool strain (LESB58) repulses the wild-type strain’s swarming in the absence of any other treatment.  What does this indicate about strain?
    1. The strain likely already produces the quorum molecule PQS.
    2. The strain lacks the ability to respond to quorum sensing signals.
    3. The strain likely avoids swarming due to reduced motility genes.
    4. The strain responds to lack of stress by suppressing movement.
  4. Controls are an important part of any experiment. Two of the three new strains are treated with antibiotics to induce stress, while one was not. This makes drawing conclusions somewhat difficult. How could you improve the thoroughness of this experiment? What would these additions tell you?
    1. Add gene expression data for environmental stress, flagella, and pili-related genes. It would tell you if transcription levels affect bacterial motility.
    2. Add swarming assays for LESB85 + antibiotic, P2m alone, and MPAO1 alone.  It would tell you if P2m and MPAO1 already produce the PQS molecule.
    3. Add growth curves for all strains with and without antibiotics. It would tell you if antibiotics affect replication rates of the indicated strains.
    4. Use microscopy techniques to image all strains on agar plates. It would tell you if antibiotics change cell shape, such as forming protrusions or blebs.
  5. The results from this experiment show that the clinical mucoid strain repulses the wild-type strain’s swarming when pre-treated with kanamycin.  How can you tell that antibiotic-induced stress was necessary for this effect for this strain?
    1. We can assume the strain is stressed because antibiotics like kanamycin or gentamycin always cause metabolic stress.
    2. We can conclude the P2m strain is resistant to antibiotic stress because it still repels swarming when tested with wild-type.
    3. We can tell the strain is not stressed because it shows normal behavior of repulsing swarming in the wild-type PA14 bacteria.
    4. We can’t tell whether antibiotic induced stress or whether the strain is already stressed because there is no untreated comparison.

8. Paper Information and Licensing

8.1. Snippet paper

  • n/a

8.2. Main paper

  • Bru JL, Rawson B, Trinh C, Whiteson K, Høyland-Kroghsbo NM, Siryaporn A. 2019. PQS Produced by the Pseudomonas aeruginosa Stress Response Repels Swarms Away from Bacteriophage and Antibiotics. J Bacteriol. 201(23):e00383-19. doi: 10.1128/JB.00383-19.
  • 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 paper at the journal’s website.

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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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