Structure and Function

TWiM #229: Dirt Is Not So Simple

Podcast and Annotation Information

  • Annotation by Jesse Blick, Analyse Keaten, Isabella Torres, Kevin Nguyen, Jeremy Ritzert, Rebecca Seipelt-Thiemann, and Mel Melendrez-Vallard
  • Podcast audio by TWiM: Listen to TWiM #229 Podcast
  • Podcast transcript by Otter.ai and edited by Darian Taylor and Marvin Romo: Access Podcast Transcripts
  • Papers Discussed:
    • Hoskisson PA and Seipke RF. 2020. Cryptic or Silent? The Known Unknowns, Unknown Knowns, and Unknown Unknowns of Secondary Metabolism. mBio. 11:10.1128/mbio.02642-20. DOI: 10.1128/mBio.02642-20
    • Pazicky S, Dhamotharan K, Kaszuba K, Mertens HDT, Gilberger T, Svergun D, Kosinski J, Weininger U, Löw C. 2020. Structural role of essential light chains in the apicomplexan glideosome. Commun Biol. 3(1):568 DOI: 10.1038/s42003-020-01283-8

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:39 minutes

The Most Interesting Things (according to students)

  • It was interesting how erythromycin is interlinked to clavulanic acid and its effects on the body and what it does as an antimicrobial. Also, the mining of aquatic variants of Actinobacteria for antimicrobials was interesting.
  • Despite sequencing a bacterial genome, 1/3 of genes lack functional annotations. We know it is a protein coding gene, but we do not know what it does or produces.

“Microbial natural products, particularly those produced by filamentous Actinobacteria, underpin the majority of clinically used antibiotics. Unfortunately, only a few new antibiotic classes have been discovered since the 1970s, which has exacerbated fears of a postapocalyptic world in which antibiotics have lost their utility. Excitingly, the genome sequencing revolution painted an entirely new picture, one in which an average strain of filamentous Actinobacteria harbors 20 to 50 natural product biosynthetic pathways but expresses very few of these under laboratory conditions. Development of methodology to access this “hidden” biochemical diversity has the potential to usher in a second Golden Era of antibiotic discovery. The proliferation of genomic data has led to inconsistent use of “cryptic” and “silent” when referring to biosynthetic gene clusters identified by bioinformatic analysis. In this Perspective, we discuss this issue and propose to formalize the use of this terminology.” (Hoskisson and Seipke 2020, no changes)

1.2. Main paper; discussion starts at 26:01 minutes

The Most Interesting Things (according to students)

  • Calcium is a significant source of minerals for the essential light chain complexes because it increases the stability of them as a whole. Discussing gliding and comparing it to a grappling hook movement and how it can pull itself along, thrust itself into other cells and is absolutely necessary for infection.
  • A gliding protein that moves in a similar fashion to how muscles contract and extend to allow bacteria to glide across surfaces.

“Gliding, a type of motility based on an actin-myosin motor, is specific to apicomplexan parasites. Myosin A binds two light chains which further interact with glideosome associated proteins and assemble into the glideosome. The role of individual glideosome proteins is unclear due to the lack of structures of larger glideosome assemblies. Here, we investigate the role of essential light chains (ELCs) in Toxoplasma gondii and Plasmodium falciparum and present their crystal structures as part of trimeric sub-complexes. We show that although ELCs bind a conserved MyoA sequence, P. falciparum ELC adopts a distinct structure in the free and MyoA-bound state. We suggest that ELCs enhance MyoA performance by inducing secondary structure in MyoA and thus stiffen its lever arm. Structural and biophysical analysis reveals that calcium binding has no influence on the structure of ELCs. Our work represents a further step towards understanding the mechanism of gliding in Apicomplexa.” (Pazicky et al. 2020, no changes)

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Metabolic Pathways (V&C_MP)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 23 (ASM_23): The health of the environment and all organisms (microbes, plants, humans, other animals) are closely linked and interdependent, as described by the One Health paradigm.
  • Fundamental Statement 25 (ASM_25): Microbes are used as models that provide fundamental knowledge about life processes.
  • 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 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define key terms and relationships among terms related to the topic of secondary metabolism, such as “cryptic gene clusters” and “biosynthetic pathways.”
  • Recall the common biological processes that secondary metabolites affect.
  • Identify why laboratory and natural environments are more and less likely to activate expression of biosynthetic gene clusters.
 S L
  • Predict the condition that would be most likely to activate expression of a biosynthetic gene cluster.
 S H
  • Identify infectious diseases caused by apicomplexans.
  • List the structural components of the glideosome.
 M L
  • Predict the strain with the competitive advantage given different strains.
 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

  • Microbial Growth (9:15): There are many assays to quantify microbial growth. The podcasters discuss using turbidity, mass/protein measurement, and ability to form a colony.

4.2. Main Paper

  • X-Ray Crystallography (33:35): A technique used to determine the 3-dimensional (3D) structure of molecules using the process of diffraction as they X-rays pass through a crystal. The authors used this technique to visualize the glideosome.
  • Nuclear Magnetic Resonance (NMR) (33:36): This is a technique used to study structure, dynamics and interactions of molecules using a strong magnetic field and radio waves.  This technique was used to visualize the conformation of proteins within the glideosome.
  • Circular Dichroism (33:37):  This technique complements structural discernment techniques like X-ray crystallography and NMR focused on secondary structure content of proteins and peptides by measuring differences in absorption of polarized light.
  • Small Angle X-ray Scatter (33:38): This technique measures nanoscale density differences in a sample which can be quantified. The authors use it here to confirm the size of various proteins and structures within the glideosome that they were characterizing.

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

5.1. Snippet Paper

  • Novel Antimicrobials (6:19–7:44): Organisms such as Streptomyces have a lot of natural products just waiting for science to discover and develop as antimicrobials.
  • Microbial Metabolism and Ecology (7:45–8:20): Primary versus secondary metabolism within a biosynthetic pathway where they start to run out of energy and start to express these products that serve as defense mechanisms for the microbes to cement their role in the niche they live in. It differs from primary metabolism because it doesn’t happen ‘normally’ it has to happen as a result of an ‘event’. Secondary metabolism doesn’t happen ‘normally’; it happens when the population experiences a change within their niche.
  • Quorum Sensing/Cell Signaling (9:45–10:14): Bacterial communication is discussed in connection with secondary metabolism.
  • Operons and Gene Expression (10:15–11:27): The podcasters discussed operons within the context of secondary metabolism and how things are added to the nucleic acids to trigger positive gene expression via regulons.
  • Antimicrobials (12:10–16:51): Substances that display unique metabolic properties that can be exploited as medications. Example: Erythromycin structure, Clavulanic acid etc.

5.2. Main Paper

  • Microbial Diversity (27:18–27:57): Apicomplexan organisms (like those that cause malaria–Plasmodium or Toxoplasma gondii) and their diversity was discussed.
  • Bacterial Motility (27:58–28:10): Gliding motility, different from flagellar motility or movement via pili.
  • Cell Structure (28:11–29:36): Pili was discussed with respect to how bacteria move throughout their environments. The glideosome is two sets of membranes (IMC and Plasma membrane) with a space between them (space). Inside the space is a large complex of proteins made up of actin, myosin among the proteins that span the space between the two membranes. Use this machinery to ‘thrust’ themselves into host cells which makes the structure critical for infection and could be a target of therapeutics.

6. Podcast Questions

  1.  What is a cryptic gene cluster?
    1. A gene cluster that is expressed at different levels under different conditions
    2. A gene cluster that is expressed at the same level, is known and widely studied
    3. A gene cluster with an unknown function or not expressed under standard conditions
    4. A gene cluster in the chloroplast or nuclear genome that is related to photosynthesis
  2. What is/are the role(s) that secondary metabolites play in microbial organisms, as discussed in the podcast? [Pick all that apply]
    1. Defense mechanisms
    2. Basic cellular functions
    3. Cell division and replication
    4. Cell communication
    5. Energy storage
  3. Identify the following statements as true or false regarding the relationship of environment and activation of biosynthetic gene clusters.
    1. ______ Laboratory environments often provide the competition necessary to active cryptic gene clusters.
    2. ______ Natural environments have specific and complex conditions that likely activate cryptic gene clusters
    3. ______ Laboratory environments activate cryptic gene clusters only when different carbon sources are used.
    4. ______ Natural environments provide constant conditions that suppress activation of cryptic gene clusters.
    5. ______ Laboratory environments are unlikely to activate cryptic gene clusters because conditions do not vary.
  4. Based on the discussion, which condition is most likely to trigger the normal activation of a cryptic gene cluster that encodes enzymes necessary for production of a secondary metabolite?
    1. Expression of a common sigma factor and RNA polymerase
    2. Exposure to a mutagen to produce mutations to the genome
    3. Incubation in a defined medium and  consistent temperature
    4. Growth in medium supplemented with environmental solutes.
  5. Which infectious diseases are caused by apicomplexans? [Pick all that apply]
    1. Malaria
    2. Ebola
    3. Onchocerciasis
    4. Toxoplasmosis
    5. Candidiasis
  6. Select the correct list of glideosome components.
    1. IMC, plasma protein, actin, myosin, ELCs, and proteins that span the two membranes
    2. Actin and myosin filaments anchored to a membrane via a lipid-anchored adaptor protein
    3. IMC, plasma protein, actin, myosin, ELCs, and proteins that span one of the membranes
    4. IMC, plasma protein, ELCs and proteins that span the two membranes to form channels
  7. If an newly infected organism contained two different strains, one with a functional MyoA (myoA+) and one with a non-functional MyoA (myoA-), which strain would likely be found in greater abundance in late infection?
    1. myoA+ ;  the rigid structure allows them to withstand the immune response.
    2. myoA- ; the reduction in protein synthesis would be an energy saving process.
    3. myoA+ ; gliding is unaffected which is involved in the successful spread of infection
    4. myoA- ; myoA is an inhibitor of gliding, so gliding is faster for strains lacking myoA.

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 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify the importance of BGC and secondary metabolites for humans and bacteria that produce the metabolites.
  • Identify key features of the schematic and experimental background, including abbreviations and specific group identifiers.
  • Analyze the schematic to distinguish what defines the terms cryptic and silent, and identify them when given specific data.
  • Analyze the schematic to classify biosynthetic gene clusters when given their expression and product, and vice versa.

Experimental Background (Hoskisson and Seipke, Figure 1)

Microbial products are widely varied and play roles in microbial communities.  A great number therapeutically valuable microbial products are produced by Actinobacteria, such as antimicrobial compounds.  A typical workflow for finding these microbial compounds is to screen microbes, but many biosynthetic gene clusters are not expressed at all times and not necessarily when grown in laboratory conditions.  This suggests that a wealth of putative valuable metabolites are yet unknown and uncharacterized.  One advance in investigating new compounds was in identifying uncharacterized biosynthetic gene clusters (BGCs) when the first Streptomyces genome was sequenced in 2002, Streptomyces coelicolor. Subsequent analysis of genome sequence identified putative BGCs, which are closely located groups of genes on a genome that might work together to create specific secondary metabolites. Secondary metabolites are metabolic compounds that are not crucial for the function of normal growth but are useful other processes, such as defense mechanism or communication.  More recently, other Strreptomyces genomes have been sequenced and analyzed.  It is estimated that more than 11,000 natural compounds are yet uncharacterized from this genus alone.  In this perspective paper, Hoskisson and Seipke (2020) clarify some nomenclature (particularly silent BGCs and cryptic metabolites), as well as use schematics to relay features regarding the interplay between environment, BGC, and microbial products.

Expresed and unexpressed BGC and their relations to known unknowns, unknown unknowns, known knowns, etc.
Figure 1: “A summary of the description of various permutations of the cryptic or silent nature of secondary metabolism.”(Hoskisson and Seipke 2020, no changes)

 

7.1.2. Questions

  1. Why is the identification of BGCs and secondary metabolites clinically significant?
    1. They are a source of new antimicrobials.
    2. They are valuable for metal chelation therapy.
    3. They are compounds with no carbons.
    4. They identify phage evolution partners.
  2. What role do secondary metabolites have for the bacterial producer?
    1. They restructure the inorganic molecules.
    2. They synthesize novel compounds.
    3. They act as a defense molecules.
    4. They decompose organic materials for fuel.
  3. What does the abbreviation BGC indicate in this figure?
    1. Basal Ganglia Classification
    2. Biogeochemical Cycle
    3. Blood Glucose Concentration
    4. Biosynthetic Gene Cluster
  4. A BGC that is not silent and produces a product that is not cryptic would be categorized as ___________.
    1. Known known
    2. Unknown known
    3. Known unknown
    4. Unknown unknown
  5. Based on the schematic, when should the term “silent”  be used when talking about BGCs?
    1. When BGCs are known
    2. When BGCs are hidden
    3. When BGCs are silent
    4. When BGCs are expressed
  6. Based on the schematic, what is the difference between cryptic and silent?
    1. Cryptic describes metabolites that have unknown aspects; silent describes BGC are not expressed.
    2. Cryptic describes BGC that are not expressed; silent describes BGC that are known or characterized.
    3. Cryptic describes metabolites that are found in media; silent describes BGC that lack promoter elements.
    4. Cryptic describes active metabolites from BGC; silent describes inactive metabolites produced from BGC.
  7. If you were to identify a putative BGC in a newly sequenced genome, but you were unable to observe its expression or its product, what category would you put this in?
    1. Known known
    2. Unknown known
    3. Known unknown
    4. Unknown unknown
  8. The synthesis of many microbial compounds is unknown. What category would you put this in?
    1. Known known
    2. Unknown known
    3. Known unknown
    4. Unknown unknown

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in the schematic and background.
  • Analyze the schematic to make predictions about the effect of removing different proteins.

Experimental Background (Pazicky et al 2020, Figure 1)

Apicomplexan parasites are single-celled eukaryotes that cause many devastating diseases.  For example, apicomplexan Toxoplasma gondii, the causative agent of toxoplasmosis, affects about 30% of the world’s population, while apicomplexan Plasmodium spp. cause more than 400,000 malaria-related deaths per year.   Apicomplexans are motile microbes and use a specific type of motility called gliding that requires a complex of proteins called a glideosome. The glidesosome aids the parasite in invading cells by grasping host components and pulling itself along. The current model of T. gondii glideosome structure is that it is a membrane-embedded multi-protein complex composed of six proteins (MyoA, essential light chain ELC, myosin light chain MLC1, GAP40, GAP45 and GAP50) found between the parasite’s plasma membrane (PM) and the inner membrane complex (IMC).

Simple figure depicting glideosomes in the T. gondii.

Figure 1: “Schematic representation of the current model of the glideosome and its localization in the T. gondii intermembrane space. Actin polymerization occurs between the plasma membrane (PM) and the inner membrane complex (IMC) whereas myosin A is part of the glideosome, which binds the essential light chains ELC and myosin light chain MLC1 (called myosin tail interacting protein, MTIP, in Plasmodium spp.). Myosin A and its light chains further interact with glideosome associated proteins GAP40, GAP45 and GAP50, which anchor the glideosome in the outer membrane of the inner membrane complex. On the other side, glideosome associated connector (GAC) facilitates the association of actin filaments with surface transmembrane proteins such as MIC2.” (Pazicky et al 2020, no changes)

7.2.2. Questions

  1. Where the glideosome’s cellular location?
    1. Inside the inner membrane complex layer, aligned with the lamina
    2. Between the plasma membrane and the inner membrane complex
    3. Inside the plasma membrane layer in the periplasm of the parasite
    4. Outside the plasma membrane, anchored to it by the  MIC2 protein
  2. What color is the structure that represents the MyoA protein?
    1. maroon
    2. blue
    3. yellow
    4. orange
    5. pink
    6. brown
    7. purple
  3. In this schematic, which area would be nearest the target host cell membrane?
    1. nearest GAP50
    2. nearest GAP 45
    3. nearest MIC2
    4. nearest MyoA
  4. Which of the following proteins are found within a glideosome? [Pick all that apply]
    1. ELC
    2. GAC
    3. GAP50
    4. GAP30
    5. MIC2
  5. Based on the schematic, what is the function of the GAP45 protein?
    1. It plays a role in anchoring MIC2 in the plasma membrane
    2. It provides stability to actin stabilization during invasion
    3. It plays a role in ion transport across the inner membrane
    4. It anchors the glidesosome across the membrane and IMC
  6. If you were to delete MIC2 in a strain of this parasite, what would be the likely effect on the glideosome structure?
    1. The GAC would be unanchored from the plasma membrane.
    2. Actin filaments would not be able to associate with GAC.
    3. ELC would be unable to associate and phosphorylate MLC1.
    4. GAP40 would dissociate the connection of glideosome to the IMC.
  7. If you were to delete MyoA in a strain of this parasite, what would be the likely effect on the gliding behavior?
    1. Gliding is unaffected.
    2. Decreased ability to glide.
    3. Unable to glide.
    4. Increased ability to glide.

8. Paper Information and Licensing

8.1. Snippet paper

  • Hoskisson PA and Seipke RF. 2020. Cryptic or Silent? The Known Unknowns, Unknown Knowns, and Unknown Unknowns of Secondary Metabolism. mBio., 11: doi: 10.1128/mBio.02642-20
  • 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’s information on its web page.

8.2. Main paper

  • Pazicky S, Dhamotharan K, Kaszuba K, Mertens HDT, Gilberger T, Svergun D, Kosinski J, Weininger U, Löw C. 2020. Structural role of essential light chains in the apicomplexan glideosome. Commun Biol. 3(1):568. doi: 10.1038/s42003-020-01283-8
  • 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’s information on its web page.

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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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