Metabolic Pathways

TWiM #272: Metabolism’s Got Rhythm

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:22 minutes

The Most Interesting Things (according to students)

  • Soil bacteria express natural products encoded by Biosynthetic Gene Clusters (BGCs), providing potential sources for new antibiotics.
  • CRISPR elements may be strategically positioned at various distances from the transcriptional start point to control gene expression.

“Natural products derived from the genus Streptomyces are a well-established and rich source of bioactive compounds, including many antibiotics in current clinical use. The rapid rise of antibiotic-resistant bacteria presents a critical and escalating threat to public health worldwide, intensifying the need for novel antibiotic discovery. These compounds are synthesized by biosynthetic gene clusters (BGCs), which encode complex metabolic pathways. However, a major challenge in utilizing *Streptomyces* for drug discovery is that many BGCs remain silent or are only weakly expressed under standard laboratory conditions, limiting the ability to identify and harness new chemical entities. In this study, we propose an innovative strategy to overcome this limitation by activating silent BGCs through the reprogramming of their endogenous regulatory mechanisms using synthetic gene regulators based on the CRISPR-Cas system. We optimized CRISPR interference (CRISPRi) and developed CRISPR activation (CRISPRa) systems specifically for use in Streptomyces, enabling precise and programmable gene repression and activation. By utilizing these systems, we were able to successfully activate a silent BGC by modulating its native regulatory network, leading to the production of novel bioactive compounds. This work represents a significant advancement in the synthetic biology toolbox for Streptomyces, providing a robust and programmable method for the activation of cryptic biosynthetic pathways. Our approach opens new avenues for exploring the vast, untapped reservoir of natural products encoded by silent BGCs, with implications for the discovery of new antibiotics and other therapeutic agents. The methods and findings described here offer a promising solution to the global challenge of antibiotic resistance by unlocking new chemical diversity for pharmaceutical development.” (Amuoroso et al, 2022 no changes)

1.2. Main paper; discussion starts at 22:45 minutes

The Most Interesting Things (according to students)

  • Light impacts metabolism activity and gene expression  in non-photosynthetic bacteria.
  • Fixed physiological states are maintained after growth under cycling conditions.

“Environmental conditions  such as sunlight, fluctuate on a daily basis due to the Earth’s rotation, and non phototrophic organisms can benefit from the ability to detect, adapt to, or even anticipate these periodic changes. Sunlight plays a crucial role in driving phototrophic metabolism, influencing redox conditions and generating substrates that support nonphototrophic organisms. Additionally, circadian rhythms in host organisms, such as daily variations in body temperature, can also impact the local environment in which colonizing bacteria reside. In this study, we explored how cycles of light/darkness and temperature variations influence biofilms formed by Pseudomonas aeruginosa PA14, an opportunistic pathogen. Using a respiratory indicator dye, we observed that zones of enhanced dye reduction emerged in biofilm regions formed during dark intervals and at lower temperatures. This pattern formation occurred regardless of the type of light used, including blue, red, and far-red light. To identify the underlying mechanisms, we conducted a mutant screen targeting potential sensory proteins and identified two mutants with defects in pattern formation specifically under red light cycling conditions. We further discovered that the distinct physiological states of biofilm subzones, shaped by specific light and temperature conditions, were maintained even when these conditions were cycled. Moreover, light/dark and temperature fluctuations were found to influence the expression of genes involved in primary metabolic pathways and redox balance, including genes encoding components of the electron transport chain. Consistent with this, we demonstrated that cbb3-type oxidases contributed to the dye reduction observed under cycling light/dark conditions. In summary, our findings reveal that cyclic variations in light exposure and temperature have long-lasting effects on redox metabolism in biofilms formed by a nonphototrophic, pathogenic bacterium. This highlights the importance of environmental dynamics in shaping microbial physiology and offers new insights into how pathogens like Pseudomonas aeruginosa can adapt to fluctuating host and environmental conditions.” (Kahl et al, 2022, 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)
  • Information flow and genetics (V&C_IFG)
  

  • Metabolic pathways (V&C_MP)
  • Information flow and genetics (V&C_IFG)
  • Microbial ecology (V&C_ME)
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  16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals
  • Fundamental Statement 13(ASM_13):Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes
  • 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 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the functions and components of CRISPR-Cas.
  • Recall how discovery of new biosynthetic gene clusters can aid modern medicine.
  • Define regulatory elements of gene expression.
 S L
  • Propose an experiment based on a hypothesis generated in the podcast discussion.
  • Predict gene expression outcomes when using CRISPR to modify existing regulatory mechanisms.
 S H
  • Identify the role of triphenyl tetrazolium chloride (TTC).
  • Identify the primary role of the electron transport chain.
 M L
  • Propose an experiment to confirm a specific gene’s contribute to light effects.
 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

  • CRISPR Technology (7:33–14:01):  CRISPR is a bacterial defense system against viruses that has been engineered to enable genome editing.  Here, the researchers modify this system  to design precise insertions that activate the expression of silent targeted Biosynthetic Gene Clusters  (BGCs) in Streptomyces venezuelae.
  • Reporter Gene  (9:30–11:15): This is a molecular technique/method to quantify or visualize gene activation.  A promoter and a reporter gene are fused in frame to the coding region of a fluorescent protein so that when the promoter is activated, fluorescence is observed.  Here, a reporter strain with a constitutive promoter fused to the reporter gene mCherry (fluorescent) was constructed to measure transcription repression.

4.2. Main Paper

  • Triphenyl Tetrazolium Chloride Reduction (30:00–39:00): Triphenyl tetrazolium chloride (TTC) is a redox indicator to indicate cellular respiration is occuring. TTC is colorless but will turn red when it is reduced by components of the electron transport chain (ETC).
  • Microdissection (40:00–41:00): Laser capture microdissection microscopy is a way to capture very small samples with high precision.  Here, it was used to take samples from different zones of the biofilm to test for gene expression.
  • RNA Sequencing Analysis (40:00–41:00): RNA sequencing, also known as transcriptome analysis, is a method of identifying and quantifying all RNAs being expressed in a population of cells at particular time.  Here, it was used on the samples collected from microdissection to analyze and compare gene expression levels to determine differentially expressed genes (DEG).

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

5.1. Snippet Paper

  • Regulation of Gene Expression (3:56–22:45): Genes can be activated or repressed using CRISPR-Cas.
  • CRISPR Cas (7:33–14:01): The concept involves testing CRISPR elements at various positions from the transcriptional start site to determine optimal inhibition of gene expression.

5.2. Main Paper

  • Regulation of Gene Expression (19:50–end time): Gene expression can be regulated by outside stimuli such as light and temperature.
  • Electron Transport Chain (26:05–end time): A series of protein complexes involved in redox reactions during cellular respiration.
  • Biofilm vs Planktonic growth (25:46-33:48): Pseudomonas aeruginosa can form biofilms in the lungs of cystic fibrosis patients, and on plates in the lab.

6. Podcast Questions 

  1. Which of the following describes CRISPR-Cas? [pick all that apply]
    1. Includes an enzyme that cuts DNA and a guide RNA
    2. uses viral RNA to induce the SOS DNA repair system
    3. Is a defense mechanism in bacteria against viruses
    4. Can be used as a tool for gene and genome editing
  2. Identifying novel biosynthetic gene clusters may lead to the discovery of new_____
    1. Antibodies
    2. Vaccines
    3. Antibiotics
    4. Toxins
  3. In the podcast, the speakers describe how some of these biosynthetic gene clusters are regulated by a repressor. What would be the most accurate definition of a repressor?
    1. A protein that prevents gene expression
    2. A protein that prevents the repression of gene expression
    3. A guide RNA that prevents gene expression
    4. A guide RNA that prevents the repression of gene expression
  4. The researcher developed the CRISPR system to enable them to find biosynthetic gene clusters that might produce bacterial metabolites with antibiotic properties.  If the researchers wanted to test their metabolites for antibiotic function, what would be a good experiment for them to do?
    1. A bacterial competition assay; grow bacteria in a co-culture, one expressing and one not expressing the metabolite, and monitor the ratio of survivors.
    2. A reporter activation assay; use a reporter gene fused to the promoter for common antibiotic biosynthesis genes, look for activation with metabolite exposure.
    3. A disk diffusion assay; grow bacteria in a lawn on an agar plate with paper disks impregnated with different metabolites and monitor the zone of clearing.
    4. An enzyme-linked immunosorbent assay; make dilutions of the metabolite and incubate with antibodies to common antibiotics, look for a loss of signal.
  5. The scientists of this study used guide RNA to interfere with transcription. What would you predict would happen to gene expression of a gene if a guide RNA was designed to complement the operator region of the gene.
    1. Gene expression would be off because the CRISPR-Cas system would interfere with RNA polymerase binding.
    2. Gene expression would be off because the CRISPR-Cas system would interfere with repressor binding.
    3. Gene expression would be on because the CRISPR-Cas system would facilitate RNA polymerase binding.
    4. Gene expression would be on because the CRISPR-Cas system would interfere with repressor binding.
  6. What is the role of triphenyl tetrazolium chloride (TTC) in the experiments described in the podcast?
    1. it is the electron donor in the electron transport chain
    2. it is the electron acceptor in the electron transport chain
    3. it is a sensor for temperature changes
    4. it is a sensor for light intensity changes
  7. What is the primary role of the electron transport chain?
    1. Synthesis of proteins
    2. Convert pyruvate into glucose
    3. Convert ATP to ADP
    4. Generate a proton gradient
  8. The researchers used transcriptome (RNA profiling analysis) to identify genes overexpressed and repressed in the light/dark conditions. If you were to investigate a single gene for its role in a light-enhanced response, what could you do to show whether this gene/protein is needed for the light-enhancement of cellular metabolism?
    1. Delete the gene from the genome and assay for cellular respiration in light and dark conditions.  It should not be able to respond to light.
    2. A reporter activation assay; use a reporter gene fused to the promoter for a light responsive gene and look for activation in light conditions.
    3. A light diffusion; grow bacteria at differing distances from bright ight conditions and monitor the level of cellular respiration relative to distance.
    4. Construct a pool of bacteria engineered to have a transposon integrated into each gene, then assay each strain for its ability to respond to light.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 2AB) 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 key features in schematics and bar graphs.
  • Identify key features in experimental design, including controls and variables for this experiment.
  • Evaluate the data to make conclusions about the guide RNA-activation domain pairings that enable gene activation.
Experimental Background (Ameruoso et al., Figure 2AB)

Microbial products make diverse chemical products that play roles in microbial communities.  A great number of these products also have therapeutic value, such as antimicrobial compounds.  A typical workflow for finding these microbial compounds is to screen environmental microbes, but many biosynthetic gene clusters are not expressed at all times.  This suggests that a wealth of putative valuable metabolites are missed and thus yet unknown and uncharacterized.  In this study,  Ameruoso et al. (2022) develop and validate CRISPR-based systems to enable expression of silent gene biosynthetic clusters in Streptomyces venezuelae. They developed two systems: one for repression to enable Biosynthetic Gene Cluster (BGC)  expression by removing repressing transcription of an inhibitor protein (CRISPRi), and one for activation to enable BGC cluster expression by activating transcription (CRISPRa).  In this experiment they test the efficacy of their designed CRISPRa system, which is a fusion of a DNase-deficient Cas9 and one of three possible transcription activation domains (AD): the N-terminal domain of the α subunit of RNA polymerase (αNTD), the ω subunit of RNA polymerase (ω), or the RbpA transcription factor (RbpA) (panel A).  To determine activation, they fused a fluorescent reporter gene mCherry to a synthetic promoter called SP10 and integrated this DNA segment into the bacterial genome (panel A and upper area of panel B).  Guide RNAs were synthesized to recognize the template strand, the non-template strand, or a non-coding region of the genome (off-target) (panel B). To determine which, if any, activation domain and guide RNA pair could activate gene expression, fluorescence of the bacteria with the genomic reporter system and specific dCas9-guide RNA pairings was quantified and normalized the the culture density (panel B).

schematics and bar graphs
Figure 2. “Creating a CRISPR activation (CRISPRa) for Streptomyces venezuelae. (A) Schematic of CRISPRa mechanism. An activator domain (AD, colored purple) is translationally fused to a dCas9 via a flexible linker. The CRISPRa complex binds upstream of a target promoter to recruit RNA Polymerase (RNAP) and activate transcription of the target gene. (B) The N-terminal domain of the α subunit of RNAP (αNTD) can serve as an AD for CRISPRa in Streptomyces. Schematic of sgRNA binding sites used that target the non-template (NT) and template (T) strand upstream of a promoter driving mCherry expression. The indicated distances reflect the number of nucleotides intervening between the 5’ end of the PAM (not included) and the TSS (also not included). Fluorescence characterization of S. venezuelae cells conjugated with CRISPRa plasmid variants using different AD. Statistical significance was calculated using two-tailed unpaired Welch’s t-test. Statistically significant differences compared to the no-CRISPRi are shown as asterisks (***P-value < 0.005). ” (Ameruoso et al., 2022, text and image cropped to include only panels A and B)

7.1.2. Questions

  1. What does the purple circle in the schematic diagram indicate (panel A)?
    1. This is the general activation domain, which is one of three being tested in this experiment.
    2. This is the actinomycin binding domain, which is linked to the Cas9 DNase molecule.
    3. This is the alanine-aspartic acid (AD) rich domain of the RNA polymerase alpha subunit.
    4. This is a fusion of the N-terminal and ω domains of RNA polymerase and the RbpA protein.
  2. Based on the schematic (panel A), how will the researchers know that activation by a particular guide RNA-AD pair was successful?
    1. Fluorescence will be repressed.
    2. Fluorescence will be increased.
    3. The gene will be edited.
    4. RNA polymerase will bind to the DNA.
  3. Activation of gene expression by each guide RNA-AD pair is shown in panel B.  In the experiment with the α subunit of RNA polymerase (αNTD), _______ is/are the negative control(s).
    1. Off target
    2. Template & non-template
    3. Control
    4. Control & off-target
  4. What feature in the graph (panel B) shows the average activation of each guide RNA-AD pair?
    1. Asterisks
    2. Whiskers
    3. Bar height
    4. The y-axis
  5. Based on the data shown, which guide RNA-AD pair is successful in activating gene expression? [pick all that apply]
    1. template guide RNA and the N-terminal domain RNA polymerase α subunit (αNTD)
    2. both template and non-template guide RNAs and the ω subunit of RNA polymerase (ω)
    3. both template and non-template guide RNAs and the RbpA transcription factor (RbpA)
    4. non-template guide RNA and the N-terminal domain RNA polymerase α subunit (αNTD)

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in schematics, colony growth, line graphs, and detrended line graphs.
  • Identify key features in experimental design for this experiment.
  • Evaluate the data to make conclusions about the impact of environmental cycling on biofilm cellular respiration
Experimental Background (Kahl et al., Figure 1)

An organism’s ability to respond to different environment conditions is important for its growth and survival.  Light affects many biological processes, most notably photosynthesis and circadian rhythms, but there is increasing evidence of broader effects by light. Here, Kahl et al. (2022) follow up their previous study that found biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa was affected by light exposure and that there is a protein necessary for this response coordinated light and redox information.  To test the idea that light affect redox metabolism, the researchers grew P. aeruginosa on medium containing the redox indicator triphenyl tetrazolium chloride (TTC).  TTC is colorless, but forms a red precipitate when reduced by electron transport chain (ETC) components (panel A).  Therefore, the red precipitate is a marker for active cellular respiration.  To examine patterns of cellular respiration in different environment conditions over time, colonies were inoculated and grown under different light/dark and temperature conditions (schematic; panel B).  Colony color from the center inoculation spot to the leading edge was visualized (colony pictures) and quantified (line graphs) for constant dark (panel C), constant light (panel D), light/dark cycling (panel E), temperature cycling (panel F), and light/dark/temperature cycling (panel G).  Additionally, to visualize the cyclic nature of the patterns visible in the colony images, the TTC data were detrended* (far right side of panels E-G), which means that the larger downward trend was removed.

schematics, colony images, and line graphs

Figure 1:  “Pseudomonas aeruginosa Δphz colony biofilms grown under light/dark or temperature cycling conditions form patterns of TTC reduction. (A) Schematic showing PA14 electron transport chain (ETC)-dependent reduction of TTC, which produces a red precipitate. (B) Schematic of the experimental setup examining the effect of biofilm growth under constant or cycling conditions (with light or dark conditions indicated by the sun and moon). (C and D) (Left) Δphz biofilm grown under the indicated condition. Right: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. (E to G) Left: Δphz biofilm grown under the indicated conditions. Middle: Quantification of red color intensity (i.e., TTC reduction) at the indicated distance from the biofilm center for a radius of the biofilm. (Right) Results of detrending analysis applied to the linear portion of the data (generated using BioDare2) (132). For all experiments, the concentration of TTC in the growth medium was 0.004%. Experiments were performed with biological triplicates, and representative data are shown. Scale bars = 2.5 mm. “(Kahl et al 2022, no changes).

7.2.2. Questions

  1. Why was triphenyl tetrazolium chloride (TTC) added to the medium for all of the experiments?
    1. To measure biofilm thickness
    2. To indicate active metabolism
    3. To stain cell nuclei of living cells
    4. To inhibit the formation of biofilm
  2. What coloring would indicate active metabolism is occurring in that region of the colony?
    1. alternating color
    2. brown color
    3. cream color
    4. red color
  3. In each colony/bioflim, where should the “oldest” cells be found?
    1. outside edge
    2. center
    3. left side
    4. right side
  4. Which of the constant conditions shows higher cellular respiration?
    1. Constant dark (panel C)
    2. Constant light (panel D)
    3. Both show high levels
    4. Neither shows a high level
  5. Which of the cycling conditions shows an effect on cellular respiration? [pick all that apply]
    1. light/dark (panel E)
    2. temperature (panel F)
    3. light/dark and temperature (panel G)
    4. None show cycling effects
  6. Based on the detrended data, which cycling condition shows a greater effect on cellular respiration?
    1. light/dark (panel E)
    2.  temperature (panel F)
    3. light/dark and temperature (panel G)
    4. None, all show about equal effects

8. Paper Information and Licensing

8.1. Snippet paper

  • Ameruoso A, Villegas Kcam MC, Cohen KP, Chappell J. 2022. Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces. Nucleic Acids Res.50(13):7751-7760. https://doi.org/10.1093/nar/gkac556
  • This article is licensed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

8.2. Main paper

  • Kahl LJ, Eckartt KN, Morales DK, Price-Whelan A, Dietrich LEP. 2022. Light/Dark and Temperature Cycling Modulate Metabolic Electron Flow in Pseudomonas aeruginosa Biofilms. mBio13:e01407-22. https://doi.org/10.1128/mbio.01407-22
  • This article is licensed under the terms of the Creative Commons CC BY 4.0 license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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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