Microbial Ecology

TWiM #286: Integrons and Invasion

Podcast and Annotation Information
  • Annotation by Andrea Ayala-Lopez, Brittney Heist, Thomas Salazar, Rebecca Seipelt-Thiemann, Mel Melendrez-Vallard and Angela Wilson.
  • Podcast audio by TWiM: Listen to TWiM #286 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Sandoval-Quintana E, Lauga B, Cagnon C. 2023. Environmental integrons: the dark side of the integron world. Trends Microbiol. 31(5):432-434. doi: 10.1016/j.tim.2022.01.009.
    • Chowdhury R, Pavinski Bitar PD, Chapman HM, Altier C. 2023. Salmonella Invasion Is Controlled by Competition among Intestinal Chemical Signals. mBio. 14(2). doi: 10.1128/mbio.00012-23.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:13 minutes

The Most Interesting Things (according to students)

Environmental integrons are able to tell which microbes can suit them best and are able to withstand environmental changes. It fascinates me that they are aware of this and have evolved to not choose a microbe that will lead to death.

In general, how an integron’s ability to support microbes by giving them antibiotic resistant genes is a reason antibiotic resistance has become so widespread.

The abstract cannot be copied from the Cell manuscript due to licensing restrictions, but an open access version of the manuscript is available in the HAL open access archive: Access the article online [PDF].

1.2. Main paper; discussion starts at 22:40 minutes

The Most Interesting Things (according to students)

Salmonella can gauge where it is along the track from our mouth to our anus by sensing the ratio of fatty acids to formic acid. It’s in the Peyer’s patch, cells with collections of tissues in the ileum, that Salmonella can invade. Salmonella is also uniquely adapted to survive in the formic acid-rich environment of the distal large intestine, while many other bacteria and viruses are in fact, inactivated by it.

E. coli is one of the gut microbes that produce formic acid as a metabolic byproduct, as well as hydrogen gas, which both serve as primary nutrient growth factors for other microorganisms and help moderate the gut microbiome.

“The intestine is a complex, ever-changing environment replete with an array of signaling molecules. To colonize such a complex organ, pathogens have adapted to utilize specific cues from the local environment to intricately regulate the expression of their virulence determinants. Salmonella preferentially colonizes the distal ileum, a niche enriched in the metabolite formic acid. Here, we show that the relatively higher concentration of this metabolite in the distal ileum prevents other signals from repressing Salmonella invasion in that region. We show that imported and un-metabolized formic acid functions as a cytoplasmic signal that competitively binds to HilD, the master transcriptional regulator of Salmonella invasion, thus preventing repressive fatty acids from binding to the protein. This results in an increased lifetime of HilD and subsequent de-repression of invasion genes. This study demonstrates an important mechanism by which Salmonella utilizes competition among signals in the gut to its advantage as a pathogen.” (Chowdhury et al 2023, no changes)

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)
  • Microbial Ecology (V&C_ME)
  • Metabolic Pathways (V&C_MP)
ASM Fundamental Statements
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect 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.
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify key components defining an integron
  • Describe how integrons contribute to the adaptability of environmental microbes
  • Identify which source is likely to have more diverse integrons and why.
 S L
  • Design an experiment to identify integrons in a microbiome community.
 S H
  • Identify the Salmonella proteins involved in intestinal invasion
  • Explain how HilD’s binding partner regulates its function.
 M L
  • Predict whether Salmonella invasion might or might not occur in another organism.
 M H

1Papers: Snippet (S) or Main (M)
2Learning 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

  • Fluorescent Transcription/Translation Fusion (9:14–9:43): This is a method/technique to quantify gene expression. You can quantify transcription or translation by using a fusion of your gene of interest with the Green Fluorescent Protein (GFP), which can be more sensitive than directly quantifying specific RNA or protein.  Before this technique was established, a Β-galactosidase fusion cassette with an enzyme assay would have been used.

4.2. Main Paper

  • Electrophoretic Mobility Shift Assay (EMSA) (33:08–39:53; 47:35–47:43): This is an assay to determine whether a protein binds DNA.  If a protein binds the DNA, it slows progression of the DNA fragment through agarose gel. Here, it is was used to examine HilD binding and whether the presence or absence of formic acid affected HilD binding to a target promoter.  It was also used to test the hypothesis that pre-incubation of bacteria with formic acid prevents other fatty acids from causing dissociation of nucleic acid targets of the HilD protein and repressing the invasion pathway.
  • Oral Inoculation (40:43–41:18): This is an inoculation route for infecting an organism.  Here, mice were fed a meal inoculated with a strain of Salmonella genetically modified to contain a fluorescent GFP reporter molecule bound to the sicA gene involved in the invasion cascade.
  • Dissection (41:18–41:34): This is a technique to isolate the tissue of interest.  Here, the ileum (large intestine) was dissected to measure the amount of the GFP reporter present, which estimates the fraction of the Salmonella population that had begun expressing of the invasion gene in each of the mice.
  • Gene Expression  (47:35–47:43): Fluorometric gene expression assays were used to measure the response of bacteria to formic acid by quantifying the expression of the invasion gene reporter system.

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

5.1. Snippet Paper

  • Regulation of Gene Expression (3:20–3:45): Integrons have exogenous genes so in order to ensure correct genetic elements they have gene cassettes which are expressed at different levels depending on how close they are to the promoter. The genes start as independent recombination and convert into circular DNA.
  • Mechanisms of Pathogenesis (3:46–4:01; 4:28–4:31): Integrons were originally known for their antibiotic resistance genes about 20 years ago. This is due to their ability to change parts of DNA into proteins (capture open reading frames) and their site-specific recombination in a host.
  • Gene Structure (4:54–5:52): There are three key parts of the integron: the integrase gene, primary recombination site (attI) and the attC site. The integrase gene is part of the tyrosine recombinase family and allows for DNA recombination. Furthermore, the primary recombination site (attI) is the attachment site where new genes can be inserted. The attC site(s) is/are in the chromosome and carries the gene, most importantly the antibiotic resistance gene in the integron. Then, the enzyme integrase recognizes these sites and joins them together and the gene is expressed.
  • Promoter Sequences (6:00–6:22): Another important aspect of the integron is the two types of promoters it has, the Pc and PintI. The Pc is an outward oriented promoter that originates from the chromosome. It is responsible for directing transcription of captured genes. The PintI is embedded into the integron.

5.2. Main Paper

  • Virulence Factors (42:28–42:37): Bacteria employ many different mechanisms of invading a host or causing disease. In this case, Salmonella has multiple toxin secretion systems, some products of which must be injected into host cells to deliver proteins that prompt infection and allow proliferation of the pathogen in the host.
  • Regulation of Gene Expression (42:54–43:26): Instead of constantly (constitutively) expressing the genes involved in virulence and invasion, Salmonella differentially expresses these genes in response to environmental cues, namely higher concentrations of formic acid in the ileum, that allow more efficient use of cellular resources. After invasion is accomplished and infection has been established, the pathogen represses expression of these genes to reserve metabolic resources and prioritize reproductive functions instead.
  • Transcription (45:06–45:27): The master gene in charge of regulating expression of the invasion pathway genes in Salmonella can have its transcription repressed by fatty acids (larger than formic acid). Blockage of transcription of this gene causes transcription of all invasion genes downstream of it to be downregulated.
  • Mechanisms of Pathogenesis (47:04–47:33): Salmonella relies on the presence of certain compounds in its host environment to mount an effective and efficient pathological attack on the host. In the presence of high concentrations of formic acid, gene expression is retained, which is why infection at the distal ileum appears to be the most common location of the start of the pathology caused by Salmonella.

6. Podcast Questions

  1. Where are the three components of an integron?
    1. the Pi promoter
    2. attl site
    3. Integrase gene
    4. the Pc promoter
    5. attC site
  2. The podcasters discuss how integrons contribute to the adaptability of environmental microbes.  What analogy did they use to describe this ability and how does it fit?
    1. They compared them to building with Lego blocks because they can be used to assemble groups of genes that aid in the microbes survival.
    2. They compared them to using a colander because they can be used to filter genes that are beneficial for virulence from those that are not.
    3. They compared them to using a shovel and pail because they can be used to collect many sequences that may or may not be beneficial.
    4. They compared them to working a jigsaw puzzle because they can be used to form a single, functional structure from small component parts.
  3. The podcasters discuss that integrons of a certain type are likely to be more diverse.  Which ones were described and what was the reasoning?
    1. Integrons from desert microbes because they encounter a greater variety of water selection pressures.
    2. Integrons from aquatic microbes because they encounter a greater variety of temperature selection pressures.
    3. Integrons from environmental microbes because they encounter a greater variety of selection pressures.
    4. Integrons from clinical microbes because they encounter a greater variety of antibiotic selection pressures.
  4. The podcasters mention the microbiome near the end of the snippet discussion and postulate that integrons might be present in the microbiome.  How could you determine if integrons were present in microbes of a microbiome community that has been sequenced?
    1. Search for microbial genes that are expressed at much higher levels than other genes.
    2. Search for antibiotic resistance genes because we know they can be transmitted.
    3. Search for F plasmids that can be transferred using conjugation methods.
    4. Search DNA sequences that encode the integrase, a key component of the integron.
  5. Which Salmonella proteins are involved in invasion? [pick all that apply]
    1. HilA
    2. HilD
    3. FocA
    4. SicA
  6. How does the binding partner of HilD influence its function? [pick all that apply]
    1. When bound to fatty acids, HilD cannot bind DNA and cannot support expression of invasion genes.
    2. When bound to formic acid, HilD binds DNA and promotes expression of invasion genes.
    3. When bound to fatty acids, HilD promotes expression of surface virulence proteins FocA and PlbF.
    4. When bound to formic acid, HilD phosphorylates FocA, which activates import of formic acid.
  7. The soil nematode C. elegans has a single species populating its gut microbiome, E. coli.  Based on these data, would you expect Salmonella invasion to occur if nematodes were infected with Salmonella? What is your reasoning?
    1. No, based on the discussion, E. coli produces fatty acids, which inhibit invasion.
    2. Yes, based on the discussion, E. coli would transfer virulence plasmids allowing invasion.
    3. No, based on the discussion, E. coli populates the niche and Salmonella could not infect.
    4. Yes, based on the discussion, E.coli produces formic acid, which promotes invasion.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet (Figure 1) and one from the main paper (Figure 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in a schematic/model diagram.
  • Match the basic structures and functions of an integron.
  • Describe the relationship between the components of the integron’s gene cassettes.
  • Predict the impact of removing and adding components to an integron.
Experimental Background (Sandoval-Quintana et al. Figure 1)

Integrons are mechanisms used by bacteria to capture genetic material via the function of an integrase enzyme.  Integrons enable bacteria to acquire new genes and phenotypes. They are of particular interest to clinical microbiologists because they are a significant way antibiotic resistance and other virulence-related genes can be transmitted via horizontal gene transfer. In this review, Sandoval-Quintana et al. (2023) created a schematic showing the structure and functional components of an integron (Sandoval-Quintana et al., 2023).

  • The figure cannot be copied from the Cell manuscript due to licensing restrictions, but noncommercial Creative Commons licensing (CC BY-NC) is available for a version of the manuscript and is presented in the HAL open access archive [PDF].

7.1.2. Questions

  1. Match each integron component to its general function. Functions may be used more than once, or not at all. (1 = promoter; 2= recombination site; 3= gene cassette; 4 = integrase enzyme; 5= gene encoding integrase)
    1. ________ intI 
    2. ________ IntI
    3. ________ attl
    4. ________ Pc  
    5. ________ PintI 
    6. ________ attC
  2. What does the arrow thickness in the figure represent?
    1. The level of expression for the gene cassette.
    2. The direction of transcription for the gene cassette.
    3. The level of recombination for the gene cassette.
    4. The likelihood of transmission by horizontal gene transfer.
  3. What component drives expression of the integrated gene cassettes?
    1. attl
    2. Pc  
    3. PintI 
    4. attC
  4. If the intI gene was deleted from the integron, what would be the impact? Select all that apply.
    1. Integration would not occur
    2. Recombination be random
    3. No transcription would occur
    4. Excision would not occur
  5. If a new cassette (pink) was added and located between the dark blue and green gene cassettes, which gene cassettes would have an expression level greater than green? Select all that apply.
    1. dark blue
    2. pink
    3. medium blue
    4. gold

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in the experimental design, strain naming, and bar graph representation of the data.
  • Evaluate the data and make conclusions about the effect of formic acid on Salmonella invasion gene expression.
  • Analyze the data and make conclusions about the genes/proteins necessary for formic acid’s effects on Salmonella invasion gene expression.
  • Hypothesize the formic acid-related invasion ability of a specific strain based on these data.
Experimental Background (Chowdhury et al., Figure 2)

Salmonella is an enteric pathogen that is usually contracted by ingesting food or water contaminated with fecal material.  It typically colonizes the distal ileum where it causes diarrhea but can spread beyond the intestine and become a life-threatening infection. Enteric pathogens such as Salmonella must tightly regulate expression of virulence-related genes to enable survival in this resource-limited, quickly changing niche.  One feature of the distal ileum, where Salmonella invade, is the high level of the metabolite formic acid.  To investigate how formic acid might affect expression of Salmonella virulence genes related to confining invasion to the ileum, Chowdhury et al. (2023) first engineered a Salmonella strain to have a bioluminescent reporter gene that should be expressed when “invasion gene” expression is activated.  This virulence gene for invasion is hilA and the reporter gene system is called hilA-luxCDABE.  The researchers then engineered several strains with additional gene mutations related to formic acid import and biosynthesis: focA (formic acid import protein), pflB (synthesis of endogenous formic acid), and both focA and pflB.  They exposed these Salmonella strains to a control solvent (DMSO), a fatty acid that represses invasion (c2-HDA), or the fatty acid supplemented with formic acid (Formic acid + c2-HDA).  Bioluminescence, which indicates activation of the invasion reporter gene system (hilA-luxCDABE), was quantified (Area Under the Curve; AUC) and normalized to the DMSO control for each strain and treatment condition (panel A).  Having identified formic acid effects, they next were interested to determine whether formic acid metabolism was necessary for the effect.  To do this, they constructed another Salmonella strain with mutations in each of three formic acid metabolism genes: fdnG, fdoG, fdhF.  They again exposed the strains to control solvent (DMSO), a fatty acid that represses invasion (c2-HDA), or the fatty acid supplemented with formic acid (Formic acid + c2-HDA) and quantified activation of the invasion gene reporter system, hilA-luxCDABE (panel B).

Two bar charts comparing AUC across different treatments in salmonella. DMSO saw the greatest AUC in each category except pflB, where formic acid saw a greater effect.
Figure 2: “Import of formic acid, but not its metabolism, is required to rescue the expression of invasion genes. (A and B) Expression of the invasion gene hilA was determined in Salmonella strains with different treatments (preincubation with 10 mM formic acid before adding 500 nM c2-HDA or an equal volume of DMSO) shown using a hilA-luxCDABE transcriptional reporter fusion. Luminescence was normalized to bacterial culture density. The AUC for each treatment was calculated. The AUC for DMSO treatment was set to 100%, and others were normalized accordingly. Bars show the normalized AUC ± SD (n = 5). Differences between the indicated treatments were calculated with the Mann-Whitney test. **, P < 0.01; ns, not significant. “(Chowdhury et al 2023, no changes)

 

7.2.2. Questions

  1. What notation in these bar graphs tells you the comparison being made is statistically significant?
    1. ns
    2. whiskers
    3. bar height
    4. asterisks
  2. Match the strain identifier with the its description (in panel A and panel B). (1 = Wild-type; 2= focA; 3= plfB; 4 = focA plfB; 5= fdnG fdoG fdhF)
    1. ________ is mutant for the formic acid import protein
    2. ________ is mutant for each gene encoding encoding formic acid metabolism proteins
    3. ________ is mutant for the gene encoding the formic acid biosynthesis protein
    4. ________ contains hilA-luxCDABE
    5. ________ contains a wild-type version of the gene encoding the formic acid biosynthesis protein
    6. ________ contains a wild-type version of the gene encoding the formic acid import protein
    7. ________ contains a wild-type version of the gene encoding formic acid metabolism proteins
  3. Which panel(s) and strain(s) indicate(s) the normal hilA gene expression pattern and bioluminescence values that you should use for comparison purposes?
    1. panel A, wild-type
    2. panel A, focA pflB
    3. panel B, wild-type
    4. panel B, fdn fdoG fdhF
  4. The researchers confirmed that fatty acid treatment (c2-HDA) of wild-type Salmonella represses hilA gene expression (panel A; left most panel; orange bar).  What effect does combining fatty acid and formic acid have on Salmonella invasion gene expression?
    1. Formic acid enhances the hilA repression.
    2. Formic acid reduces the hilA repression.
    3. Formic acid stops the hilA repression entirely.
    4. Formic acid has no effect on hilA repression.
  5. Considering the strain data noted in panel A and B, which genes and functions, if any, are necessary for formic acid’s effect on invasion gene expression (hilA)? [pick all that apply]
    1. focA, formic acid import is necessary
    2. pflB; formic acid biosynthesis is necessary
    3. fdnG fdoG fdhF; metabolism is necessary
    4. None of the listed genes are necessary
  6. Based on these data, would you expect a Salmonella strain that had mutations in fdnG, fdoG, fdhF, and focA to be able to invade? What is your reasoning?
    1. No; the strain would be mutant for a large number of formic acid-related genes
    2. Yes; the combination of mutations cancels out the repression of hilA gene expression
    3. No; the formic acid import protein, not the metabolism genes, is necessary for invasion
    4. Yes; these mutations are in separate genes so there will be complementation

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Chowdhury R, Pavinski Bitar PD, Chapman HM, Altier C. 2023. Salmonella Invasion Is Controlled by Competition among Intestinal Chemical Signals. mBio.14(2):e0001223. doi: 10.1128/mbio.00012-23.
  • 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 the article on the journal’s web page.

License

Icon for the Creative Commons Attribution 4.0 International License

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.

Share This Book