Structure and Function

TWiM #162: Intracellular Bacteria with Flagella

Podcast and Annotation Information

  • Annotation by Leonardo Baumgartner, Triston Walsh, Nancy Boury, Rebecca Seipelt-Thiemann 
  • Podcast audio by TWiM: Listen to TWiM #162 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #162 Transcript
  • Papers Discussed:
    • Stegen G, Pasmans F, Schmidt B, Rouggaer LO, Praet SV, Schaub M, Canessa S, Laudelout A, Kinet T, Adriaensen C, Haesebrouck F, Bert W, Bossuyt F, Martel A. 2017. Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans. Nature, 544:353–356. doi: 10.1038/nature22059
    • Collingro A, Köstlbacher S, Mussmann M, Stepanauskas R, Hallam SJ, Horn M. 2017. Unexpected genomic features in widespread intracellular bacteria: evidence for motility of marine chlamydiae. The ISME Journal 11:2334–2344. doi: 10.1038/ismej.2017.95 

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 4:00 minutes

The Most Interesting Things (according to students)

A resilient, contagious, and invasive fungus is killing European salamanders at extremely high rates, so don’t import salamanders from Europe or Asia! 

Stegen et al (2017) is not licensed for Creative Commons use, so, the abstract cannot be copied here. Please see the article at the journal’s web page. 

1.2. Main paper; discussion starts at 19:18 minutes

The Most Interesting Things (according to students)

Chlamydial parasites of marine protists have genes encoding flagella, which may confer motility. 

“Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.” (Collingro et al 2017, no changes). 

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

Snippet Main
Vision and Change Topics
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • 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 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 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.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Differentiate between the two types of spores produced by Batrachochytrium salamandrivorans.
  • Recall what makes eradication of B. salamandrivorans a difficult task.
 S L
  • Predict how an organism that is sensitive to a pathogen could evolve to become tolerant to a pathogen.
 S H
  • Compare and contrast motility of Rickettsia and Chlamydia.
  • Explain the concept of parsimony as it relates to genome size.
 M L
  • Speculate as to why some Chlamydia species have genes encoding flagella, if they are obligate intracellular parasites/symbionts.
 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

  • In vivo Infection Experiments (6:00): These are experiments where living organisms are infected experimentally.  Here lab salamanders were infected and are killed by fungus at almost 100% rates.

4.2. Main Paper

  • Single Cell Genomics (26:15–26:35; 30:58–31:29): You can extract and sequence DNA from single isolated cell.  Here the cell was a protist.

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

5.1. Snippet Paper

  • Immune Memory (7:20): European salamanders that survive infection do not generate immune memory to the fungus, so reinfection is possible.
  • Mortality (4:45–7:00): Chytrid fungal infection in European salamanders is almost 100% lethal, even with very low dose of infection due to being naïve. Asian salamanders are resistant due to coevolution.
  • Fungal Spores (8:40–9:15): Chytrid fungus produce 2 types of spores: motile and infectious insistent spores, the latter of which is environmentally-resistant.
  • Epidemiology, Carriers (11:15–12:00): Newts experience lower lethality than salamanders, and toads are asymptomatic carriers
  • Epidemiology, Transmission (9:15–11:15; 12:00–12:40): Spores can attach to bird legs, allowing them to be transported over large distances. Spores may be in soil or on surface of water and can be spread via touch.

5.2. Main Paper

  • Parasite Host Range (19:20–20:31): Chlamydia are ubiquitous, but are only pathogenic to a couple of species.
  • Life Cycles (22:04–22:25): Chlamydia have complex life cycles, with an elementary body (external to the host), and a reticulate body (internal to the host)
  • Parasitism (22:52–23:53; 30:30–30:53): Chlamydia is an obligate intracellular parasite/symbiont. They have small genomes, lack survival genes for nucleotide and amino acid biosynthesis and acquire ATP from their host.
  • Endosymbiosis Theory (23:54–24:53): Chlamydia is thought to have played a role in the establishment of plastids in plant cells
  • Homology/Secretion Systems (29:40–30:10): Type III secretion system components have similarity to flagellar components and the NFT3 secretion system components. (20:34–20:47; 25:23–26:35) Some Chlamydia species (marine/protist symbiont) have genes that encode for flagella proteins. (26:38–27:08)   Rickettsia are also intracellular parasites, and they do have flagella.
  • Motility (27:08–29:27): Rickettsia do not move outside of a cell. Marine Chlamydia may be able to move when outside a cell with their flagella, if they make it
  • Chemotaxis (30:10–30:30): Chlamydia also have chemosensing genes, suggesting their flagella are functional.
  • Omics (32:10–36:06): Future studies should include gene  expression and microscopy to be certain they function

6. Podcast Questions

  1.  How do the two types of Batrachochytrium salamandrivorans spores differ? 

A = Motile; B = Insistent

1. _____ spores can move while _____ spores do not have motility.

2. ____ spores can persist in the environment, but_____ spores are not stable

3. _____ spores are prey for zooplankton while ____ spores are not ingested by predators.

  1. What is not a hurdle to eradicating Batrachochytrium salamandrivorans?
    1. Spores are able to persist for a long time in the environment.
    2. There are many hosts so they have many reservoirs to exist in.
    3. Spores adhere to bird skin and can be transported a long distance.
    4. All of the above are significant hurdles to eradicating this pathogen.
  2. The podcasters note that Asian salamanders are resistant to the chytrid fungus.  While this has not been studied yet, what are some broad ways that the Asian salamanders might have become resistant?
    1. They can sense and avoid contaminated soils.
    2. They digest motile and insistent spores.
    3. They evolved a better immune system.
    4. They are a different species so aren’t affected.
  3. The podcast speakers discuss the different kinds of motility witnessed for Rickettsia and marine Chlamydia. What is the main difference?
    1. Rickettsia are motile only inside host cells, but marine Chlamydia are likely motile outside of a host cell.
    2. Rickettsia are not motile at all in any environment and marine Chlamydia are likely motile in all environments.
    3. Rickettsia are motile due to their plentiful cilia, but marine Chlamydia are likely motile due to a flagellum.
    4. Rickettsia has a more swirling motility and marine Chlamydia has a more wave-like motility.
  4. Elio describes the Chlamydia genome size as exhibiting parsimony.  Which statement is the point he is making?
    1. Genes that are not necessary to maintain a critical function are lost.
    2. Genes with functions in a single process evolve to be near each other.
    3. Genomes with high redundancy have a high fitness compared to those without.
    4. Genomes with less DNA content acquire fewer detrimental mutations.
  5. Why might a species have genes encoding proteins that make up a flagella if they are not motile? Pick all that apply.
    1. The species might have mutations in the genes such that they have a new function.
    2. The species might have evolved from an ancestral species that did have flagella.
    3. The species might produce flagella under certain conditions, but not others.
    4. The species might have evolved new flagella genes hoping to become motile.

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify the different parts of a box and whisker plot.
  • Calculate proportion survival given values for before and after treatment.
  • Analyze data expressed as genomic equivalents to determine the sample with the greater pathogen load.
  • Analyze survival data to evaluate effects of parameters on survival.
  • Predict how changes to spore characteristics might change adhesion and survival.

Experimental Background (Stegen et al., Figure 3BCD)

Batrachochytrium salamandrivorans is a fungal pathogen of Asia salamanders that has been transported to Europe.  Stegen et al (2017) report that this pathogen has nearly 100% mortality in Belgian fire salamanders and that the natural population suffered a steep decline in a short six months.  The authors were interested the fungal characteristics that make this pathogen so deadly for fire salamanders, so they investigate the ability of the fungus to adhere to salamander scales, salamander toes (the control), and goose feet by dipping the different skins into a solution containing spores and quantifying how much adhered using the presence of fungal DNA, as measured by quantitative polymerase chain reaction and reporting this as genomic equivalents (panel b).  They next considered how long encytsted spores could survive and successfully infect salamanders by infecting salamanders with freshly collected spores or spores that were incubated in pond water for 14 or 31 days.  They measured infection of each spore type over 5 weeks by again quantifying infection using the presence of fungal DNA, as measured by quantitative polymerase chain reaction and reporting this as genomic equivalents (panel c).   The final question was whether the different spore types (encysted, passive spores vs. motile, active spores) were consumed differently by micropredators.  They incubated spores with a constant number of zooplankton organisms in a constant volume, then counted the remaining spores at 2 and 4 hours to calculate survival (panel d). 

  • Stegen et al (2017) is not licensed for Creative Commons use, so, the figure cannot be copied here. Please see the article at the journal’s web page. 

7.1.2. Questions

  1. Box and whisker plots (panel b) are used to show visual differences in sample statistics.  What statistical value does the solid horizontal line within the box represent?
    1. The mode
    2. The median
    3. The mean
    4. The quartile
  2. Box and whisker plots (panel b) are used to show visual differences in sample statistics.  What statistical value do the solid vertical lines that extend above and below each box represent?
    1. The range for all values for that sample.
    2. The outliers found for that sample.
    3. The standard error for that sample.
    4. The standard deviation for that sample.
  3. Which sample has the largest variability in sample values (panel b)?
    1. Control
    2. Scale
    3. Toe
    4. None
  4. Compared to the control, which sample(s) show(s) adherence to the skin sample (panel b)? How can you tell?
    1. Scale, as this skin type shows the highest fungal load compared to control.
    2. Toe, as this skin type shows a box area that is nearer the area size of the control.
    3. Neither, the control has a higher adherence value, as shown by the larger box.
    4. Both scale and toe, as they both have high fungal load compared to control.
  5. If you were to identify a chemical compound that could effectively interfere with spore adhesion in a dose-dependent manner and plot it next to the adhesion results in panel b, which box plots in panel b would most resemble your most successful results?
    1. Control
    2. Scale
    3. Toe
  6. Based on the pathogen loads for fresh and incubated spores (panel c), how long can spores survive in pond water? How can you tell?
    1. At least a few hours because you can find fungal genomic DNA present at 2 weeks for t0.
    2. At least 14 days because you can find fungal genomic DNA present at 4 weeks for t1.
    3. At least 31 days because you can find fungal genomic DNA present at 6 weeks for t2.
  7. The authors tested whether the motile (active) or encysted (passive) forms were more affected by predation by calculating a survival score or proportion survival (panel d).  What would be a good representation of proportional survival?
    1. Count of predators at start of experiment/count of predators at end of experiment
    2. Count at fungal spores end of experiment/count of fungal spores at start of experiment
    3. Count of predators at end of experiment/count of predators at start of experiment
    4. Count at fungal spores end of experiment/count of fungal spores at end of experiment
  8. Which two lines should you compare to identify the effect of spore type on predation (panel d) and what does this tell you?
    1. Compare the yellow solid line to the yellow dashed line.  This shows predators do not eat the encysted spores.
    2. Compare the blue solid line to the blue dashed line.  This shows predators do eat the motile spores.
    3. Compare the blue solid line to the yellow solid line. This shows that in the absence of predators, the spores are not eaten.
    4. Compare the blue dashed line to the yellow dashed line. This shows predators do not eat the encysted spores and do eat the motile spores.
  9. If you were able to treat the encysted spores (panel d, yellow) to become motile at 4 hours, then carry out the experiment for another 2 hours, what would you expect to see?
    1. You would see the dashed yellow line continue at 1.0 up to 4 hours, then drop to about 0.5 at 6 hours.
    2. You would see the solid yellow line continue at 1.0 up to 4 hours, then drop to about 0.5 at 6 hours.
    3. You would see both yellow lines continue at 1.0 up to 4 hours, then drop to about 0.5 at 6 hours.
    4. You would see both yellow lines continue at 1.0 throughout the entire 6 hour period.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify components of a model figure
  • Explain the concept of parsimony as it relates to genome size
  • Predict the most closely and distantly related species based on a model figure that identifies components of a large molecular machine and species where they are found.
  • Identify distinct genes and their directions of transcription using a gene diagram illustration.
  • Explain the concept of synteny as it relates to gene/genome conservation.
  • Speculate as to what has occurred that resulted in different Chlamydia species having different numbers of genes that encode flagella components

Experimental Background (Collingro et al., Figure  3)

A group of very successful terrestrial pathogens is the chlamydiae.  These bacteria are obligate intracellular parasites and have reduced genome sizes that reflect their intracellular lifestyle, depending on their host for many metabolic needs.  Studying relatives of pathogenic bacteria can help scientists learn more about pathogens. These investigators found Chlamydia in a marine environment. sequenced their genomes, compared their 16S ribosomal RNA genes to other Chlamydia, and then annotated their protein coding genes to compare these marine species to their terrestrial relatives.  Through these analyses, the authors found a large number of flagella genes present in the marine species that were not present in other Chlamydia species.  The authors illustrate the components present in the flagella and color-code each component with the species where it is present (panel a).  Next, the authors highlight the conservation of flagella genes and their gene order in two of the marine Chlamydia species (panel b), lending further support that these gene clusters are present and function to produce flagella in marine Chlamydia (Collingro et al 2017).  Genes are noted as arrows with the direction of transcription/coding identified by the arrow’s direction.

Graphic representation of flagellar system.
Figure 3. “Flagellar genes detected in chlamydial SAGs. (a) Schematic overview of components of the flagellar apparatus encoded in three different chlamydial SAGs of marine origin. Notably, they contain orthologs for both the flagellar system and the NF-T3SS (which originally evolved from the flagellar system). All other known chlamydiae lack the majority of flagellar genes. (b) Illustration of the syntenic region of a flagellar gene cluster in two SAGs. Contig ends are indicated.” (Collingro et al 2017, no changes).

7.2.2. Questions

  1. If a component in the model figure is shaded in peach, what can you conclude about where the gene encoding this protein is found?
    1. It was found in the marine Chlamydia species AG-110-M15.
    2. It was found in all marine Chlamydia species that were in this study.
    3. It was found in all Chlamydiaceae regardless of species.
    4. It was found in a membrane region: outer membrane or peptidoglycan.
  2. Generally, in a model figure, you have structures and color coding that denote something the authors wish to highlight or summarize.  What would you consider the purpose of this model figure (part a)?
    1. To show which  genes  encode many of the different flagellar types present in nature.
    2. To identify which flagella components are encoded in different Chlamydia species.
    3. To show which Chlamydia species have the ability to use chemosensing with high confidence.
    4. To identify the different genes involved in assembling a flagellum in any bacterial species.
  3. Based on the presence and absence of different flagellar components encoded in the genomes (part a), which two Chlamydia species are most closely related? How can you tell?
    1. AB-751-023 and AG-110-M15 are most closely related because they have more similar genes present than the others (purple, dark green, light green).
    2. AG-110-P3 and AG-110-M15 are most closely related because they have more similar names than the others (AG–110-XXX).
    3. AB-751-023 and AG-110-P3 because their shared genes are mostly encoding outer membrane and cytoplasmic membrane components (greens).
    4. AB-751-023, AG-110-P3, and AG-110-M15 are equally related because they are marine Chlamydia.
  4. How is an individual gene and its direction of transcription denoted in the gene diagram illustration (part b)?
    1. A gene is noted as an arrow that is present for both AB-751-023 and AG-110-M15.  All genes are transcribed left to right.
    2. A gene is noted as an arrow whose arrow head is facing to the right.  The arrowhead denotes the 5’ to 3’ direction of transcription.
    3. A gene is noted as an arrow whose arrow head is either facing to the right or to the left to denote the direction of transcription.
    4. A gene is noted as a line of arrows that is used to transcribe one RNA. The arrow head denotes how the ribosome translates them differently.
  5. What is synteny and how is it highlighted in the genome structure figure (part b)?
    1. Synteny relates to conversation of genes among species including sequence, direction of transcription, and gene order relative to nearby genes.  Syntenic regions are highlighted by purple shadow region.
    2. Synteny relates to conservation of protein sequences across members of a gene family, such as flgN, flgJ, flgI, flgH, flgA, and flgF.  Syntenic regions are highlighted by their peach coloring in the coding region arrows.
    3. Synteny relates to how genes related to a distinct function in the cell can be located near each other in the genome, such as the many flagellar-related genes in the diagram.  Syntenic regions are highlighted by the closeness of the arrows.
    4. Synteny relates to how sections of the genome have been relocated to plasmids in bacteria so they are more easily transferred to other species by horizontal gene transfer.  Syntenic regions are the two DNA fragments.
  6. We see from both part a and b that the marine Chlamydia have many genes encoding flagellar components that are not present in other Chlamydia species.  What concept is represented by this fact?
    1. Synteny
    2. Parsimony
    3. Fitness
    4. Auxotrophy
  7. All Chlamydia species have flagella component genes flhA, fliI, fliH, fliF, and fliA (part a, asterisks), but marine Chlamydia have many more genes encoding components of the flagella, such as flgB, motB, and flgK. Based on these data and your knowledge of evolutionary principles, what is the most likely explanation?
    1. Marine Chlamydia acquired all the flagella genes by horizontal gene transfer, but non-marine Chlamydia acquired only some.
    2. Marine Chlamydia had much more gene duplication to create all the flagella-encoding genes, but only some genes were duplicated in non-marine Chlamydia.
    3. Non-marine and marine Chlamydia both have all the different flagella-encoding genes, but they are non-functional in non-marine Chlamydia.
    4. A common ancestor had all genes encoding flagella components. Marine Chlamydia retained all, but non-marine Chlamydia lost most of them.

8. Paper Information and Licensing

8.1. Snippet paper

  • Stegen G, Pasmans F, Schmidt B, Rouggaer LO, Praet SV, Schaub M, Canessa S, Laudelout A, Kinet T, Adriaensen C, Haesebrouck F, Bert W, Bossuyt F, Martel A. 2017. Drivers of salamander extirpation mediated by Batrachochytrium salamandrivorans. Nature, 544, 353–356. doi: 10.1038/nature22059
  • 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 article at the journal’s web page.   

8.2. Main paper

  • Collingro A, Köstlbacher S, Mussmann M, Stepanauskas R, Hallam SJ, Horn M. 2017. Unexpected genomic features in widespread intracellular bacteria: evidence for motility of marine chlamydiae. The ISME Journal 11:2334–2344. doi: 10.1038/ismej.2017.95 
  • This article is licensed for Creative Commons use using CC BY NC SA 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article’s copyright information.

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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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