Information Flow and Genetics

TWiM #283: Quorum Sensing in the Gut

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:09 minutes

The Most Interesting Things (according to students)

  • The protein MvfR’s regulatory role in intestinal permeability. MvfR is identified as a key driver of intestinal permeability, not just RhlR and LasR, indicating that targeting this regulator could be a potential therapeutic strategy to mitigate gut barrier dysfunction caused by P. aeruginosa infections.
  • Plasmids can influence bacterial behavior through translational regulatory crosstalk, allowing them to enhance bacterial resistance and virulence.

“Quorum sensing (QS) is a highly conserved microbial communication mechanism based on the production and sensing of secreted signaling molecules. The recalcitrant pathogen Pseudomonas aeruginosa is a problematic nosocomial pathogen with complex interconnected QS systems controlling multiple virulence functions. The relevance of QS in P. aeruginosa pathogenesis is well established; however, the regulatory interrelationships of the three major QS systems, LasR/LasI, MvfR (PqsR)/PqsABCD, and RhlR/RhlI, have been studied primarily in vitro. It is, therefore, unclear how these relationships translate to the host environment during infection. Here, we use a collection of P. aeruginosa QS mutants of the three major QS systems to assess the interconnections and contributions in intestinal inflammation and barrier function in vivo. This work reveals that MvfR, not LasR or RhlR, promotes intestinal inflammation during infection. In contrast, we find that P. aeruginosa-driven murine intestinal permeability is controlled by an interconnected QS network involving all three regulators, with MvfR situated upstream of LasR and RhlR. This study demonstrates the importance of understanding the interrelationships of the QS systems during infection and provides critical insights for developing successful antivirulence strategies. Moreover, this work provides a framework to interrogate QS systems in physiologically relevant settings.” (Singh et al. 2023)

1.2. Main paper; discussion starts at 31:26 minutes

The Most Interesting Things (according to students)

  • RsmQ uses environmental cues to determine whether or not to stay put and spread to the progeny of the cell, or to transfer to a different cell via conjugation.
  • The swarming motility of Pseudomonas fluorescens is regulated by RsmQ. RsmQ shifts P. fluorescens towards a sessile, biofilm-producing lifestyle while repressing swarming motility.

“Beyond their role in horizontal gene transfer, conjugative plasmids commonly encode homologs of bacterial regulators. Known plasmid regulator homologs have highly targeted effects on the transcription of specific bacterial traits. Here, we characterize a plasmid translational regulator, RsmQ, capable of taking global regulatory control in Pseudomonas fluorescens and causing a behavioral switch from a motile to a sessile lifestyle. RsmQ acts as a global regulator, controlling the host proteome through direct interaction with host mRNAs and interference with the host’s translational regulatory network. This mRNA interference leads to large-scale proteomic changes in metabolic genes, key regulators, and genes involved in chemotaxis, thus controlling bacterial metabolism and motility. Moreover, comparative analyses found RsmQ to be encoded on many divergent plasmids isolated from multiple bacterial host taxa, suggesting the widespread importance of RsmQ for manipulating bacterial behavior across clinical, environmental, and agricultural niches. RsmQ is a widespread plasmid global translational regulator primarily evolved for host chromosomal control to manipulate bacterial behavior and lifestyle.” (Thompson et al 2023, no changes).

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Information Flow and Genetics (V&C_IFG)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define quorum sensing.
  • Identify the quorum sensor regulator and the effect that pathway on Pseudomonas aeruginosa virulence.
  • Describe the quorum sensor pathway-related gene regulation events and mechanisms that impact gut permeability.
 S L
  • Analyze hypothetical double and single mutants data to distinguish the relationship/function of two genes in a regulatory cascade.
 S H
  • Identify the mechanism used by RmsQ to alter host cell translation.
  • Recall how nutrient levels influence RmsQ-related host cell gene expression and plasmid dispersion.
 M L
  • Predict how a hypothetical RsmQ inhibitor would likely affect P. fluorescens characteristics.
 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

  • Immunofluorescent Staining (6:30–9:30): The technique is used to visualize the location of proteins in cells or tissues.  It can use antibodies tagged with fluorescent dyes to identify locations of specific proteins or tagged molecules to identify leakage of one tissue compartment into another, such as the gut and circulatory system.

4.2. Main Paper

  • In Silico Analysis (31:03–31:52: The term in silico  means that this analysis was done using computational methods.  Here, it involved using computer methods to compare sequences and  was done to determine whether or not the plasmid pQBR103 is the only one that has obtained an rsmQ gene.
  • Reusable DNA Capture Technology (REDCAT) (33:16–34:15): This method tests to see how much protein of interest is bound to a particular substrate.
  • Motility Assay (40:32–41:15): This method tests to see if a microorganism can move independently (motility).

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

5.1. Snippet Paper

  • Quorum Sensing (2:00–10:00): A bacterial communication mechanism that regulates the expression of virulence genes. In Pseudomonas aeruginosa, virulence is controlled through three key regulatory systems that work synergistically: the LasR and RhlR synthase/transcription factor receptor pairs, and the MvfR (PqsR) system.
  • Biofilm and Planktonic Growth (10:00–20:00): Biofilms are structured microbial communities where bacteria, such as P. aeruginosa, attach to surfaces and communicate through quorum sensing. They exist in a stationary state, embedded in a self-produced matrix that offers protection and plays a critical role in antibiotic resistance.

5.2. Main Paper

  • Regulation of Gene Expression Mechanisms (28:58–29:11) Plasmid chromosome crosstalk is where a regulatory protein encoded on a plasmid can affect the expression of host chromosomal genes.
  • Proteomics (41:32–41:44): Using proteomic data, it was determined that one of the major impacts of RsmQ expression on P. fluorescens was on the utilization of carbohydrates.
  • Conjugation (41:45–42:28): RsmQ protein can transfer to other cells via conjugation. Its decision to do so depends on the nutrient availability around the cell
  • Chemotaxis (43:21–43:45): Cells of P. fluorescens that contain RsmQ on the plasmid utilize local nutrient cues to dictate the activation of chemotaxis machinery. Either activate the flagellar apparatus and become motile or remain put to acquire more amino acids and carbohydrates.

6. Podcast Questions

  1. What is quorum sensing?
    1. A method of regulating temperature using proximity and number of cells in bacterial colonies.
    2. A method of evading the immune system in the host intestine gut using extracellular receptors.
    3. A method of communication using small secreted molecules to regulate bacterial phenotypes.
    4. A method of bacterial reproduction that speeds up DNA replication under specific conditions.
  2. Which quorum sensing regulator is primarily responsible for Pseudomonas aeruginosa’s effect on intestinal permeability and how is the permeability altered?
    1. LasR; It strengthens the intestinal barrier, blocking solute diffusion.
    2. MvfR; It disrupts intestinal tight junctions, increasing permeability.
    3. RhlR; It enhances mucosal inflammation, decreasing permeability.
    4. PqsR; It destroys the ability of the gut to acquire acidic nutrients.
  3. Which statement describes the gene regulation events related to the ability of Pseudomonas aeruginosa to affect inflammation and gut permeability through quorum sensing?
    1. MvfR triggers intestinal inflammation and gut permeability and activates expression of LasR and RhlR which affect tight junctions and gut permeability.
    2. LasR activates expression of RhlR, and together trigger intestinal inflammation. RhlR also activates expression of MvfR, causing systemic infection.
    3. RhlR activates expression of MvfR and LasR which initiates biofilm formation. MvfR then initiates degradation of RhlR reducing intestinal permeability.
    4. MvfR and RhlR are activated by environmental factors, causing intestinal inflammation, which then activates LasR which breaks down tight junctions.
  4. The podcasters note that the researchers used combinations of mutant genes, which they called triple mutants, double mutants, and single mutants of all three regulator genes (mvfR, lasR, and rhlR).  If mutation in two different genes individually shows no effect, but mutation of both together (double mutant) shows an effect, what can you say about the function of these two genes?
    1. The two genes work independently of each other in different gene pathways.
    2. The two genes are involved in different parts of the same regulatory cascade.
    3. The two genes work together as heterodimers in the same regulatory cascade.
    4. The genes have the same function so you have to mutate both to see any effect.
  5. How does RsmQ inhibit translation of host proteins?
    1. It binds and blocks the peptidyl (P) site in the ribosome.
    2. It blocks assembly of the 50S and 30S ribosomal subunits
    3. It binds and blocks the ribosome binding site of mRNAs.
    4. It activates a RNase that degrades all bacterial RNAs.
  6. When the environment is poor in nutrients, RsmQ inhibits translation of  _____–related genes and induces ________ to disperse the plasmid.
    1. chemotaxis; pili formation
    2. metabolism; conjugation
    3. DNA replication; cell division
    4. motility; transformation
  7. If you were to treat a strain of  P. fluorescens with a small molecule inhibitor of RmsQ in a low nutrient environment, which of the following phenotypes would you expect? [pick all that apply]
    1. Swarming
    2. Non-motile/biofilm
    3. High transformation frequency
    4. Low transformation frequency
    5. High conjugation frequency
    6. Low conjugation frequency
    7. High metabolism/low chemotaxis protein expression
    8. Low metabolism/high chemotaxis protein expression

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 6).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify experimental design features, including variable types and  and controls in an experiment.
  • Identify important features of the bar charts used to display experimental results.
  • Match the study measure with its experimental question.
  • Analyze the bacterial survival/dissemination, inflammation, and intestinal permeability data to make conclusions about how each gene contributes to each phenotype.
Experimental Background (Singh et al., Figure 1)

Pseudomonas aeruginosa is a highly versatile and adaptive pathogen, infecting a large number of sites in humans including the eyes, skin, lungs, and intestines. The versatility of this pathogen is in part due to its ability to change its characteristics using quorum sensing pathways.  Quorum sensing uses bacteria-generated small molecule communication systems to initiate bacterial changes in response to the environment. Additionally, P. aeruginosa is resistant to many antibiotics, so identifying the mechanisms of its infection and pathogenesis at each site is important to identify more ways to combat it. In this paper, Singh et al. (2023) wanted to characterize the roles of, and interactions among, the three quorum sensing pathways in P. aeruginosa gut infection.  To do this, the researchers first constructed bacteria with deletions in each of the three quorum pathway regulator genes singly (ΔmvfR, ΔlasR, ΔrhlR), in two of the genes at a time (ΔmvfR::ΔlasR, ΔlasR::ΔrhlR, ΔmvfR::ΔrhlR), and a triple mutant for all three (ΔmvfR::ΔlasR::ΔrhlR).

The next step in this characterization used a mouse infection model.  In this protocol, they first burned the dorsal skin of the mouse, then infected the skin with each variant strain, the wild-type strain (PA14), or no bacterium (burn alone).  They quantified living bacteria dissemination to the ileum (panel A) and colon (panel B) using a plate count method and report their data as colony forming units (CFU). Knowing that P. aeruginosa induces inflammation, they also quantified levels of the inflammation marker TNF-ɑ (TNF-a; panel C) in mice infected with the different strains.  Lastly, knowing that P. aeruginosa can escape the intestine and cause widespread sepsis, they also quantified gut integrity using a fluorescent marker (FITC-dextran) that remains localized to the gut so long as the intestinal barrier is intact, but enters the blood stream when gut integrity is lost (panel D).  Although not indicated in the figure legend, statistical significance is achieved when P <0.05 for most biology experiments.

Figure 1. “Dissemination of P. aeruginosa from the site of infection to the ileum (A) and colon (B). The inflammatory response in the ileum TNF-α levels (C) increased when mice were infected with ΔlasR, ΔrhlR, and WT strains, whereas the TNF-α level was similar to burn alone in ΔmvfR, double, and triple mutants. (D) The flow of the FITC-dextran from the intestinal lumen to the systemic circulation was not increased significantly in the ΔmvfR single mutant in comparison to burn alone, whereas the WT, ΔlasR, ΔrhlR, and double and triple mutants increased intestinal permeability. Each dot represents data from one mouse. The error bars denote ± SEM. Significance was assessed by Kruskal-Wallis nonparametric test with Dunnett’s posttest applied. Exact P values are provided. A portion of the underlying data for the WT, burn alone, and ΔmvfR were previously published in reference 23. These data were reused and reanalyzed, as these experiments were performed in parallel to experiments with the other single, double, and triple mutants.” (Singh et al. 2023, no changes)

7.1.2. Questions

  1. Why were the PA14 and burn alone included in this study?
    1. These P. aeruginosa strains are the experimental treatment groups
    2. These P. aeruginosa strains are both the negative control groups
    3. PA14 is the negative control, while burn alone is a positive control
    4. Burn alone is the negative control, while PA14 is the positive control
  2. Match the data panel (A, B, C, D) with its experimental question.
    1. ______How do the mutations affect the ability of P. aeruginosa to escape the gut?
    2. ______How do the mutations affect the ability of P. aeruginosa to localize and grow in the ileum?
    3. ______How do the mutations affect the ability of P. aeruginosa to induce inflammation?
    4. ______How do the mutations affect the ability of P. aeruginosa to localize and grow in the colon?
  3. What do the bar height and the whiskers on the bars indicate?
    1. Mean; standard deviation
    2. Median; standard error
    3. Mean; standard error
    4. Median; standard deviation
  4. Statistical significance is notated here as the P value on the lines above bars in each bar chart. Compare wild-type (PA14) and the bacteria lacking mvrR (ΔmvfR).  Which infection phenotypes are statistically different between wild-type and ΔmvfR strain?  What is your evidence?
    1. CFU in ileum and CFU in colon; P > 0.05
    2. TNF-α and intestine permeability; P < 0.05
    3. TNF-α; CFU in ileum and colon; P < 1
    4. All of them are statistically significant; P > 0.1
  5. Which single gene has the greatest impact on P. aeruginosa dissemination and growth in the ileum?
    1. mvfR
    2. lasR
    3. rhlR
    4. None impact this alone
  6. Which single gene has the greatest impact on P. aeruginosa inflammation in the ileum, as reported by TNF-α level?
    1. mvfR
    2. lasR
    3. rhlR
    4. None impact this alone

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of experimental design including variable types and controls in an experiment.
  • Identify relevant bacterial phenotypes.
  • Analyze the data to conclude the impact of mutating and overexpressing rsmQ on genes on bacteria phenotypes.
Experimental Background (Thompson et al., Figure 6AB)

Plasmids are circular extrachromosomal pieces of DNA that play significant roles in horizontal gene transfer and are widely known to contribute to bacterial phenotypes, such as antibiotic resistance.  Thompson et al. (2023) discovered a plasmid that encodes a regulatory protein that is a homolog of a genome-encoded protein RmsQ. When the plasmid-encoded rmsQ was expressed in Pseudomonas fluorescens, it affected the expression of over 700 genes encoded on the bacterial genome.  This paradigm-shifting phenomenon is called plasmid-chromosome crosstalk (PCC).  These researchers found that RsmQ binds mRNAs and uses translation inhibition to impact gene expression at the protein level, so they were next interested in investigating how the plasmid-encoded RsmQ affected two bacterial phenotypes involved in the Pseudomonas lifestyle: biofilm formation and swarming.  First, they engineered bacteria to have a plasmid that either : 1) expressed RsmQ at high levels when an inducer called IPTG was present (pME6032-rsmQ) or 2) did not have rsmQ (pME6032). They also engineered bacteria to have a plasmid that expressed RsmQ under its native promoter (pQBR103Km) or the same plasmid without rsmQ (pQBR103Km-ΔrsmQ). To quantify effects on swarming behavior they employed a motility assay where each of the bacteria noted above, as well as P. fluorescens with no plasmid, were inoculated at the center of the agar plate and allowed to grow/move for 48 (panel a) or 72 hours (panel b). Note that panel a has media with and without IPTG and panel b has LB (lysogeny broth; a standard nutrient-rich medium; KB is a typo of LB) and M9GluCAS medium (minimal medium with glucose, carbenicillin, ampicillin, and streptomycin; a nutrient poor medium).

motility assay
Figure 6. “Motility … [is] impacted by RsmQ. (a) The 48-h swarming motility assays for SBW25 containing pME6032 +/- rsmQ. (b) The 72-h swarming motility assays for SBW25 cells either plasmid free (-) or carrying pQBR103Km (+) or pQBR103Km-ΔrsmQ grown on 0.5% agar plates with media as indicated. Quantification of swarming of 4 biological replicates in triplicate can be found in S5 Fig….” (Thompson et al 2023, image cropped to panels a and b only; legend edited to include panels a and b only).

7.2.2. Questions

  1. Which of the agar plates in panel b show the highest motility?
    1. column 1
    2. column 2
    3. column 3
    4. top row
    5. bottom row
  2. For the experiment shown in panel a, _____ is/are the control(s). [pick all that apply]
    1. No IPTG and pME6032
    2. No IPTG and pME6032-rsmQ
    3. 1 mm IPTG and pME6032
    4. 1 mm IPTG and pME6032-rsmQ
    5. There are no controls.
  3. Which agar plate would have the bacteria that express high levels of RsmQ and what induces that expression?
    1. Panel a top left; pME6032
    2. Panel a bottom left; IPTG
    3. Panel a top right; pME6032
    4. Panel a bottom right; IPTG
    5. Panel a top row; pME6032
    6. Panel a bottom row; IPTG
  4. Which agar plate would have the bacteria that express native levels of RsmQ?
    1. Panel b column 1
    2. Panel b column 2
    3. Panel b column 3
    4. Panel b top row
    5. Panel b bottom row
  5. RsmQ changes the behavior of P. fluorescens when overexpressed on a high copy plasmid (panel a). Based on these data, which lifestyle would P. fluorescens with high RsmQ expression likely have? What is your evidence?
    1. A parasitic, host-dependent lifestyle; the growth is reduced when rsmQ is present and expressed
    2. A fast-growing, nutrient rich lifestyle; the motility is increased when rsmQ is present regardless of expression
    3. A dormant, spore-forming lifestyle; the growth is reduced when rsmQ is present and expressed
    4. A sessile, biofilm-forming lifestyle; the motility is decreased when rsmQ is present and expressed
  6. Which of the following statements best fits the results of the motility assays performed on rich (LB) and nutrient poor (M9GluCAS) media (panel b)? Pick all that apply.
    1. The addition of any of the plasmids increases swarming and motility, but only on poor medium.
    2. Bacteria that contain neither of the plasmids have high swarming on both rich and poor media.
    3. Deletion of rsmQ from the plasmid increases swarming compared to with the plasmid on rich medium.
    4. The deletion of rsmQ from the plasmid is effective in rescuing swarming motility on both rich and poor media.
    5. Deletion of rsmQ from the plasmid does not affect swarming on poor medium compared to with the plasmid

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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