Information Flow and Genetics

TWiM #189: Salmonella BonJovi

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:50 minutes

The Most Interesting Things (according to students)

Fungi are an incredibly diverse group with a myriad of industrial uses and ecological effect.

Snippet-related paper: “Fungi comprise approximately 20% of all eukaryotic species and are connected to virtually all life forms on Earth. Yet, their diversity remains contentious, their distribution elusive, and their conservation neglected. We aim to flip this situation by synthesizing current knowledge. We present a revised estimate of 2–3 million fungal species with a “best estimate” at 2.5 million. To name the unknown >90% of these by the end of this century, we propose recognition of species known only from DNA data and call for large-scale sampling campaigns. We present an updated global map of fungal richness, highlighting tropical and temperate ecoregions of high diversity. We call for further Red List assessments and enhanced management guidelines to aid fungal conservation. Given that fungi play an inseparable role in our lives and in all ecosystems, and considering the fascinating questions remaining to be answered, we argue that fungi constitute the next frontier of biodiversity research.” (Niskanen et al. 2023)

1.2. Main paper; discussion starts at 31:43 minutes

The Most Interesting Things (according to students)

Salmonella can stop expressing flagellar proteins when infecting host cells so as to evade an inflammatory response.

“Bacterial two-component regulatory systems (TCS) couple the detection of niche-specific cues with adaptive gene expression to optimize fitness. In Salmonella Typhimurium (STM), the SsrA-SsrB TCS regulates virulence genes needed for survival within host cells, yet the impact of this TCS on regulatory evolution in this pathogen remains incompletely understood. Here, we show that SsrB alters a transcriptional network controlling bacterial motility to limit inflammasome activation during host cell infection. Using comparative RNA sequencing between STM and S. bongori (SBG) engineered to express SsrB, we show that SsrB represses flagellar gene expression in STM but activates this pathway in SBG, which has evolved in the absence of SsrB. Motility repression in STM is driven by an SsrB-binding region upstream of flhDC that appears to have evolved in STM following divergence from SBG. These data reveal a divergent regulatory circuit in non-coding DNA that reduces flagellar gene expression to evade host defenses.” (Ilyas et al. 2018).

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_ME)
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
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 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.
  • Fundamental Statement 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental Statement 4 (ASM_4): Phylogenetic trees best reflect the evolutionary relatedness of all organisms although microbial lineages may be difficult to define due to horizontal gene transfer or lack of conserved genes

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the mutual benefits in mycorrhizal relationships.
  • Explain the significance of the phrase “Yeast are the E. coli of the eukaryotic world.”
 S L
  • Propose an experiment to explore how monoculture and polyculture affect the spread of plant fungal pathogens.
 S H
  • List the major differences between S. enterica serovarTyphimurium and S. bongori.
  • Explain the benefits of being able to regulate gene expression.
 M L
  • Speculate why the specific base content of pathogenicity island DNA was a “flag” for horizontal gene transfer.
 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

  • n/a

4.2. Main Paper

  • Transcriptomics (45-32–47:50) :This is a technique used to examine all the transcribed genes in a sample, usually to compare between samples to identify differentially expressed genes (DEG).  It is also called RNA sequencing (RNA-seq).  Here, the researchers used it to Isolate and sequence total RNA in cell to find genes regulated by Ssrb.
  • Swimming Assays (48:45–50:08): This assay measures bacterial motility.  The researchers used this assay to determine Salmonella with/without SSR system.
  • Mouse Studies (57:26–58:47): Mouse models of infection are commonly used for pathogen studies.  Here the bacteria lacking SSRB (SSRB-) were found to continue to produce flagella after internalization.
  • Electrophoretic Mobility Shift Assay (EMSA) (54:25–54:53): Commonly known as a “gel shift assay”, this assay is used to determine if a protein binds nucleic acids, such as a DNA binding protein.  If the protein binds, the DNA migrates more slowly in electrophoresis due to the increase in mass compared to “naked” DNA.  So, protein binding is seen as a “shift up” on the gel.

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

5.1. Snippet Paper

  • Yeast (6:30–7:00): “E. coli of the eukaryotic world” studied a lot, 7% of yeast genes can be substituted by human genes.
  • Uses of Fungi (7:00): As of 2018, there are about 200 species of fungi are thought to be hallucinogenic (9:00–11:30), Fungi play huge roles in our diet- edible mushrooms, yeast used in fermentation, many other uses in the food industry, (11:30) Antibiotics, statins, cyclosporin and many other substances obtained from fungi, (12:25) Mold is used to make Vitamin B12 and a lot of enzymes; precursor for plastic production also obtained from fungi, (12:30–16:30) Fungal-derived enzymes are important, e.g. turning crop waste into ethanol.
  • Detritovores (16:45–18:00; 19:45–20:25): Fungi are major decomposers; both of dead organic matter and inorganic pollutants.
  • Fungal Symbiosis (18:00–19:45):  Mycorrhizal relationships with trees obtain sugar in exchange for defense and increasing the reach of trees.
  • Molecular Grenades (20:25): Phanerochaetes excrete reactive oxygen species (ROS) that destroy lignin.
  • Pathogens (21:40 -2 4:50): Fungi cause ecologically important diseases all across the world, especially for plants.

5.2. Main Paper

  • Salmonella (34:00–38:00) :Salmonella are facultative intracellular parasites; Salmonella disable motility in order to prevent inflammasome expression.
  • Horizontal Gene Transfer (38:00–41:12): Horizontal gene transfer is a mechanism where organisms acquire DNA from other individuals that is not a parent-offspring relationship.  It is responsible for S.  enterica serovar Typhimurium obtaining pathogenicity islands and is the only major difference between S. enterica serovar Typhimurium and S. bongori. – S. enterica serovar Typhimurium can survive inside immune cells, when S. bongori can’t.
  • Two Component System (40:30–41:12): Two component systems common in bacteria and use two components to sense and respond to environmental signals.  One component is the external sensor and the other component is a regulator.  SsrB is the regulator part of a two component system.
  • Gene Expression Regulation (41:40–42:11): Gene expression regulation is how the transcript levels are controlled in a cell.  For transcription, this usually involves binding of proteins at promoters.  Transcription factor (SsrB) is activated in acidic conditions, contributing to the collective fitness of S. enterica serovar Typhimurium once inside the host.
  • Promoter:(52:37–53:05): Promoters are DNA locations where proteins that influence transcription can bind.  The Salmonella flagellar promoter is a hotspot for motility regulation.

6. Podcast Questions

  1. Elio states that “yeast is the E. coli of the eukaryotic world.”  What is the point he is making with this statement?
    1. Both yeast and E. coli are well-studied organisms.
    2. Yeast and E. coli are both single-celled organisms.
    3. Yeast and E. coli share many of the same genes.
    4. Both yeast and E. coli are pathogens of humans.
  2. In the tree-fungus mycorrhizal relationship, _________ are gained by the tree and _______ is gained by the fungus.
    1. sugars and hormones; water
    2. calcium and lithium; sodium
    3. water and salts; endorphins
    4. water and nutrients; sugars
  3. The podcasters note that industrial farming uses plant monocultures (e.g., one kind of pea) and that makes crops more susceptible to fungal disease than a diverse variety of crops (polyculture; here, many varieties of peas).  The best experiment to test the spread of a plant pathogen in monoculture compared to polyculture in nature might be:
    1. Grow 100 plants of two different varieties of peas in separate greenhouses, expose both greenhouses to a plant pathogen, and quantify disease in all 200 plants.
    2. Grow a 5 acre field of multiple varieties of peas, expose all the plants to a plant pathogen and quantify disease in each of the plants by taking root sections and performing microscopy.
    3. Grow 10 fields each of single or  multiple varieties of peas, expose the beginning field to a pathogen and quantify how long it takes for the last field to become infected.
    4. Grow different fungal pathogens with a slurry made from the leaves of different pea varieties and quantify the growth of the pathogen using spectroscopy and microscopy.
  4. Two species of Salmonella are studied in the main paper, S. enterica serovar Typhimurium and S. bongori.  The podcasters note a number of ways these two species are alike and differ.  Match the statement below with the species. (S. enterica serovar Typhimurium  = SeT;  S. bongori = Sb; Neither = N; Both = B)
    1. Is motile
    2. Can produce flagella
    3. Is a facultative intracellular parasite
    4. Can’t replicate in immune cells
    5. Has a two component system
    6. Has pathogenicity islands
    7. Has two chromosomes
    8. Is the ancestral species
  5. Another type of motility structure is the pilus, hair-like structures on the cell surface.  Bacterium A has the ability to control expression of its pili genes in response to an environmental cue while bacterium B expresses its pili genes all the time.  Which statements are advantages that these hypothetical bacteria might have based on the podcast discussion of gene regulation? Pick all that apply.
    1. Bacterium A saves energy by not producing RNAs and proteins when they are not needed.
    2. Bacterium B saves energy by not producing any of the transcription machinery proteins.
    3. Bacterium A could evade the immune system better by stopping production of surface pili.
    4. Bacterium B could use pili to move around in the cytoplasm of an infected immune cell.
  6. The podcasters noted that the base composition (G-C content) of the pathogenicity islands identified this DNA as resulting from horizontal gene transfer.  What about base composition suggests horizontal gene transfer?
    1. Bacterial DNA bases differ from archaea and from eukaryotes, so researchers can identify the larger genetic group of sample DNA.
    2. Different species have the same bases, but different frequencies of bases, so researchers can identify DNAs that evolved separately.
    3. Pathogenic species have specific chemical organizations to the bases, so researchers can identify base and gene order for a species.
    4. Different species have different bases in their DNA and genome topology, so researchers can tell the species origin of genetic material.

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Describe how a tree image can be a visual for evolutionary classification.
  • Identify key features of clades.
  • Analyze a phylogenetic tree to make inferences about evolutionary relationships.
Experimental Background (Snippet-related paper, Niskanen et al. 2023)

Fungi have many roles in Earth’s ecosystems including improving nutrient uptake in plants and decomposing organic materials.  They also have been co-opted to produce bioactive compounds and are used to make many foods and materials; they were used to make Legos!  They are also responsible for some diseases such as Chytridiomycosis (frogs), Cryptococcosis (humans), and white pine blister rust (trees).  The authors (Niskanen et al. 2023) estimate that 90% of fungal species are not known, but use phylogenetic analyses of the currently known genomes and multiple genetic markers to construct the relationships among fungal phyla in the kingdom Fungi.

Phylogenetic tree.

Figure 1. “Synopsis of the current classification of phyla in the kingdom Fungi, compiled from [several references] …  Some conflicts in the placement of individual phyla across these studies are indicated (dashed line): Glomeromycota has been either placed as sister to Asco-, Basidio-, and Entorrhizomycota in multimarker studies or nested with Mucoromycota in phylogenomic approaches; Blastocladiomycota appears as an early emerging lineage in some phylogenomic approaches or as a supported sister to Sanchytriomycota next to Zoopagomycota, Entomophthoromycota, and Kickxellomycota or included in the clade formed by these latter three phyla in other phylogenomic and in multimarker studies. The phylum Caulochytriomycota, proposed for the genus Caulochytrium is considered a synonym of Chytridiomycota. Background colors set apart major clades.” (Niskanen et al. 2023)

7.1.2. Questions

  1. Why is a tree image good for representing taxonomic and genetic relationships? Pick all that apply.
    1. Genetically similar groups are located near each other, such as Monoblepharomycota and Sanchytriomycota.
    2. Genetically similar groups are on the same branches, such as Basidiobolomycota and Olipodomycota.
    3. Genetically dissimilar groups are found randomly in the tree, such as Glomeromycota and Mucoromycota.
    4. The group that is most genetically distant splits off the tree at the earliest time, such as Rozellomycota.
  2. Groups that are most closely related have a _______ proportion of similar DNA or amino acid sequences and will be found coming off ___________ node.
    1. higher; the same
    2. lower; the same
    3. higher; a different
    4. lower; a different
  3. Based on this tree, what is the most evolutionarily distant phylum of all Fungi?
    1. Apheliodomycota
    2. Basidiomycota
    3. Glomeromycota
    4. Rozellomycota
  4. Mark the following statements as true (T) or False (F).
    1. ________ Membership in a clade is based on membrane characteristics, such as lipid content.
    2. ________ Members of a clade are likely to have similar characteristics because they have similar genomes.
    3. ________ Members of a clade likely have a common ancestor from which they all evolved.
    4. ________ Members of a clade can be distributed across many evolutionary heritages.
  5. The clade represented in sky blue contains Ascomycota, Entorrhizomycota, Basidiomycota. Which clade is most evolutionarily related to the sky blue clade?
    1. Aphelidomycota (dark purple)
    2. Chtridomycota/Neocallimastigomycota/Nomoblepharomycota (tan-green)
    3. Glomerulomycota (peach)
    4. Sanchytriomycota/Bastocladiomycota (medium blue)

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Match components of two component systems.
  • Calculate the log2 fold change in gene expression given formulae and data.
  • Analyze quantitative reverse transcription-polymerase chain reaction (qRT-PCR) data to make conclusions about which species shows repression and activation of genes.
  • Predict electrophoretic mobility shift assay results given a specific scenario.
  • Analyze electrophoretic mobility shift assay data to identify whether a protein binds DNA or not.
  • Predict the effect of mutations on the two component system.
  • Identify the calculations and/or molecular aspects represented in each graph.
Experimental Background

Salmonella enterica serovar Typhimurium (STM) is a pathogen that causes gastrointestinal disease.  Many genes involved in Salmonella virulence were acquired by horizontal gene transfer of DNA sections called pathogenicity islands, including a two component regulatory system. In acidic conditions, which occur after invasion for this intracellular pathogen, component one (SsrA) phosphorylates component two (SsrB) which regulates expression of genes.  In this study, Ilyas et al (2018) were interested in the difference between STM and an ancestor species that lacks a native SsrB (Salmonella bongori; SBG). They engineered both bacteria to constitutively produce SsrB (SsrB-positive cells; SsrB+) or to not produce any SsrB (SsrB-negative cells; SsrB–).  In this experiment, they wanted to examine which flagella genes are expressed differently depending on the expression of SsrB in the two species, so performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR; panel A).  They compared RNA from SsrB+ and SsrB– STM and also compared RNA from SsrB+ and  SsrB– SBG.  Here, a log2 ratio indicates decreased expression as a negative value and increased expression as a positive value.  Following this result, they next wanted to identify how SsrB was controlling RNA levels. Since SsrB is a regulator protein, they reasoned that it could be acting as a regulator of transcription by binding flagella gene promoters.  To investigate this possibility, they performed electrophoretic mobility shift assays (EMSA) using promoter (P) DNA from the putative target flagella gene operon flhDC of STM or of SBG and purified SsrB protein (panel C). Here, labeled, unbound DNA moves through a gel based on its mass and charge.  If protein binds the DNA, the mass increases and the migration is slowed resulting in a “shifted” band.

Bar graph and gel shift images.
Figure 3. “SsrB Antagonizes Transcription of Flagellar Genes in STM by Binding PflhDC (A) Transcription of flagellar genes is repressed by SsrB in STM and activated in SBG, measured by RT-qPCR and expressed as log2 fold change in transcript levels relative to rsmC. Data are mean ± SEM from n = 3. (B) Expression of PflhDCSBG does not show SsrB-dependent repression of STM flhD, measured by RT-qPCR. Data are mean ± SEM from n = 6; ∗p = 0.03 by Wilcoxon test. (C) Electrophoretic mobility shift assay (EMSA) of SsrBc incubated with PflhDC from either STM or SBG. Protein-DNA complexes are indicated with an asterisk.” (Ilyas et al. 2018, cropped to panels A-C).

7.2.2. Questions

  1. The two component system in this study involves an acidic environment, SsrA, SSrB, and transcription of genes encoding proteins for the  flagella as noted in the figure background.  Staphylococcus pneumoniae has a two component system where CiaH senses penicillin levels and interacts with CiaR which induces production of a penicillin binding protein (Barrett and Hoch 1998).  Match the components of the Staphylococcus system to its equivalent in the Salmonella system and its general description.
General Descriptors: Staphylococcus system components:
Environmental cue = E; Outcome = O; Regulator molecule:= R; Sensor molecule = S penicillin binding protein production = b; CiaH = h; penicillin level = p; CiaR = r,
    1. _______ SsrA
    2. _______ Control transcription of genes for flagella
    3. _______ SSrB
    4. _______ acidic environment

Barrett JF, Hoch JA. 1998. Two-component signal transduction as a target for microbial anti-infective therapy. Antimicrob Agents Chemother. 42(7):1529-36. doi: 10.1128/AAC.42.7.1529.

Use this text for the next two questions (2 and 3)

The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay used to generate the data in panel A is a measure of RNAs for each gene represented on the x-axis.  The measure is a log2 conversion of RNA present in SsrB+ cells (STM) divided by RNA present in SsrB-negative cells (SBG), e.g. log2(STM fliA RNA/SBG fliA RNA).

  1. If there was 8 times more fliA RNA in STM cells compared to SBG cells, what would be the value of the bar?
    1. 2
    2. 3
    3. 4
    4. 8
  2. If there was 7 times less flgE in STM cells compared to SBG cells, what would be the value of the bar?
    1. -1.7
    2. -2.4
    3. -2.8
    4. -7
  3. Based on the results of the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in panel A, which bacterium reduces the level of flagella gene transcription when SsrB is present compared to when SsrB is absent?  What is your evidence?
    1. STM because transcript levels for genes involved in flagella formation are positive in SsrB+ bacteria.
    2. SBG because the RNA levels for flagella genes decrease as you move along the different operons in the genome.
    3. STM because the log2 of RNA levels for flagella genes in SsrB+ bacteria compared to SsrB– is negative.
    4. SBG because RNA levels as visualized by log2 of SsrB+ bacteria RNA compared to SsrB– RNA is negative.
  4. An electrophoretic mobility shift assay, such as the one in panel C, depends on the interaction of two molecules.  Let’s suppose that we are looking at the interaction of a DNA and a protein.  How would the mass of the complex change for “naked” DNA, one protein bound to the DNA, and two proteins bound to the DNA?
    1. The more mass, the faster the complex will migrate in electrophoresis, so order from fastest to slowest would be two proteins bound, one protein bound, “naked” DNA.
    2. The more mass, the slower the complex will migrate in electrophoresis, so order from fastest to slowest would be two proteins bound, one protein bound, “naked” DNA.
    3. The more mass, the faster the complex will migrate in electrophoresis, so order from fastest to slowest would be “naked” DNA, one protein bound, two proteins bound.
    4. The more mass, the slower the complex will migrate in electrophoresis, so order from fastest to slowest would be “naked” DNA, one protein bound, two proteins bound.
  5. Based on the results of the electrophoretic mobility shift assay in panel C, which species’ promoter DNA (PflhDC) does SsrB bind and what is your evidence?
    1. Both STM’s and SBG’s PflhDC because the DNA fragment bands are found in both assays so both DNAs are bound by SsrB protein.
    2. STM PflhDC because as when SsrB protein in mixed with the DNA, it binds and creates a higher mass complex that migrates more slowly
    3. SBG’s PflhDC because SsrB protein doesn’t inhibit the migration of the promoter DNA as shown by the consistent DNA migration in the gel.
    4. Neither promoter DNA is bound by SsrB protein because you can see free, fast migrating-PflhDC in both gels regardless of the protein presence.
  6. This two component system works because SsrA is a kinase that adds a phosphate group to SsrB when the bacterium is in an acidic environment, which activates it and causes repression of genes needed for flagella production. If the STM bacterium were to acquire a mutation that destroys the kinase activity of SsrA, what would you expect if this mutant bacterium infected an immune cell?
    1. It would be unable to make flagella regardless of entering the cell.
    2. It would make flagella all the time regardless of entering the cell.
    3. It would make flagella only when it was present inside of the cell.
    4. It would make flagella only when it was outside of the immune cell.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Ilyas B, Mulder DT, Little DJ, Elhenawy W, Banda MM, Pérez-Morales D, Tsai CN, Chau NYE, Bustamante VH, Coombes BK. 2018. Regulatory Evolution Drives Evasion of Host Inflammasomes by Salmonella Typhimurium. Cell Rep. 25(4):825-832.e5. doi: 10.1016/j.celrep.2018.09.078.
  • 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://www.sciencedirect.com/science/article/pii/S2211124718315389?via%3Dihub

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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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