Metabolic Pathways

TWiM #169: Breatharian Bacteria

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:01 minutes

The Most Interesting Things (according to students) 

Pathogens contain unique mechanisms which enhance their virulence and bypass their host’s defenses.

This article is not licensed for Creative Commons use; see copyright information. Thus, the abstract and figures cannot be copied here. A version of this paper is available on PubMed at: https://pmc.ncbi.nlm.nih.gov/articles/PMC5562518/

1.2. Main paper; discussion starts at 31:23 minutes

The Most Interesting Things (according to students)

Antarctic bacteria are capable of extracting trace gases from air to use for energy and carbon metabolism.

“Cultivation-independent surveys have shown that the desert soils of Antarctica harbour surprisingly rich microbial communities. Given that phototroph abundance varies across these Antarctic soils, an enduring question is what supports life in those communities with low photosynthetic capacity. Here we provide evidence that atmospheric trace gases are the primary energy sources of two Antarctic surface soil communities. We reconstructed 23 draft genomes from metagenomic reads, including genomes from the candidate bacterial phyla WPS-2 and AD3. The dominant community members encoded and expressed high-affinity hydrogenases, carbon monoxide dehydrogenases, and a RuBisCO lineage known to support chemosynthetic carbon fixation. Soil microcosms aerobically scavenged atmospheric H2 and CO at rates sufficient to sustain their theoretical maintenance energy and mediated substantial levels of chemosynthetic but not photosynthetic CO2 fixation. We propose that atmospheric H2, CO2 and CO provide dependable sources of energy and carbon to support these communities, which suggests that atmospheric energy sources can provide an alternative basis for ecosystem function to solar or geological energy sources. Although more extensive sampling is required to verify whether this process is widespread in terrestrial Antarctica and other oligotrophic habitats, our results provide new understanding of the minimal nutritional requirements for life and open the possibility that atmospheric gases support life on other planets.” (Ji et al. 2017)

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

Snippet Main
Vision and Change Topics
  • Metabolic Pathways (V&C_MP)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • 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 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • 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 12 (ASM_12): Bacteria and Archaea exhibit extensive metabolic diversity, including nitrogen fixation, methane production, and anoxygenic photosynthesis, many of which are unique to these two domains.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall why metals are important for biological processes.
  • Identify the mechanism these bacteria use to control metal toxicity.
  • Recall the process by which these bacteria acquired a pathogenicity island.
 S L
  • Predict how a mutant in a specific YBT function might behave.
 S H
  • List some challenges that make life on Antarctica a struggle for microbes.
  • Identify the data that suggests that bacteria at this site do not utilize photosynthesis.
 M L
  • Predict the outcome of an experiment similar to one discussed in the podcast.
 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

  • Culturing Techniques (6:25–15:20): Microbes were grown in the laboratory using specific culture techniques.  Here, the researchers used both lysogeny broth (LB) and minimal media to grow E. coli.
  • Mutants (13:40 – 15:00): Mutants are strains engineered to have, or naturally have, variant genes.  Here, pathogens engineered to have missing pathogenicity islands were observed to lose their virulence.
  • Mass Spectroscopy (17:20–23:30): Mass spectroscopy is a method to quantify and identify molecules in a mixture.  Here, it was used to measure cellular copper levels
  • Radiolabeling (17:47–23:30): Radioactive molecules can be used to track particular elements or molecules as biological processes occur and produce end products.  Here, carbon 13 (C13) and copper 64 (Cu64) were used as radiolabels.

4.2. Main Paper

  • Metagenomics (35:45–36:15): This is a technique used to isolate environmental DNA, sequence isolated DNA.  Some studies use 16S and 18S rDNA genes to identify the species present and other include whole metagenomes to identify the genes present.  Here, it was used to identify species present and genes present in soil samples from Antarctica.
  • Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) (41:45): This is a method to make DNA copies of RNA.  It uses a RNA-dependent DNA polymerase in the reverse transcription part and a DNA-dependent DNA polymerase in the polymerase chain reaction part.  Here, it was used to construct a cDNA library to find metabolic genes.
  • Gas Chromatography (42:00): This is a chromatography technique to separate, quantify and identify components in a mixture using a vaporized sample.  Here it was used to track metabolites.
  • Radiolabeling (42:20-42:45): Radioactive molecules can be used to track particular elements or molecules as biological processes occur and produce end products.  Here, carbon 14 (C14 ) was used to track the fate of CO2.

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

5.1. Snippet Paper

  • Iron Acquisition (4:40–6:15; 11:35–12:57): Metals, such as iron are enzyme cofactors and iron is rare metal in the environment so its scavenging is important.
  • Urinary Tract Infections (UTI) (6:40–7:55): Urinary tract infections are common.  Half of women will have had a UTI by the time they are 32 years old.
  • Siderophores (8:30–9:25; 11:10–11:35): Siderophores are compounds with high affinity for iron.  Yersiniabactin and Enterobactin were originally categorized as siderophores because they can scavenge iron, but they were found to bind copper.
  • Horizontal Gene Transfer and Pathogenicity Islands (9:35–11:10): Horizontal gene transfer is when DNA, such as pathogenicity islands, is acquired by non-inherited mechanisms, such as transformation and conjugation.
  • Oxidative-Reductive Reactions (12:00–13:30): These are chemical reactions that involve electron transfer.  Redox reactions were discussed in the context of metal toxicity via the Fenton reaction.

5.2. Main Paper

  • Light Independent Reactions (37:55–38:45; 42:50): Rubisco and other “Gas Grabbers” are present, but light dependent reactions were not present.
  • Heterotrophy (38:55–39:30; 40:30–41:00): A heterotroph is an organism that must acquire nutrients from the environment rather than synthesizing them on their own.  H2, CO, and HCO2 are carbon and energy sources for these arctic ecosystems.
  • Phototrophy (46:20–47:05): A phototroph is an organism that converts light energy into chemical energy  to power its cellular processes. Phototrophs were found living under translucent rocks.
  • Biotechnology (47:45–48:35): Biotechnology is the study and use of living organisms to create products for practical purposes.  Here, there was discussion of its application to bioenergy, cleaning pollutants, and fixing excess CO2.

6. Podcast Questions

  1. What is the function of metals, such as copper and iron, for bacterial and eukaryotic cells?
    1. They act as co-factors for essential enzymes.
    2. They serve as primary sources of cellular energy.
    3. They function as structural components of DNA.
    4. They regulate pH directly through chemical buffering.
  2. The study identified the mechanism that the bacteria use to regulate copper toxicity.  Which statement best describes their results?
    1. The bacteria convert copper into a harmless gas that diffuses out of the cell.
    2. The bacteria store excess copper in vacuoles until it is needed for metabolism.
    3. The bacteria eliminate copper by breaking it down into simpler elements.
    4. The bacteria have several proteins that tightly bind and transport the copper.
  3. How do the researchers and the podcasters suggest that the pathogenicity island carrying Ybt came to be present in the urinary tract strain of E. coli?
    1. The pathogenicity island was formed spontaneously through random mutations in the E. coli genome.
    2. It was acquired by horizontal gene transfer, and since it was advantageous to E. coli, it was maintained.
    3. The E. coli strain inherited the pathogenicity island directly from its parent strain during cell division.
    4. It was created by the bacterial CRISPR immune system to help the bacteria evade and survive.
  4. The FyuA and YptPQ work together to bring copper into the bacterial cell. What would be the most likely outcome if the cell had a mutant fyuA gene that encoded a version of FyuA that could not release copper?
    1. FyuA bound to copper would accumulate in the periplasm.
    2. FyuA bound to copper would accumulate at the inner membrane.
    3. FyuA bound to copper would accumulate in the cytoplasm.
    4. FyuA bound to copper would accumulate at the outer membrane.
  5. Which of the following challenges do bacteria overcome to survive in Antarctica? [pick all that apply]
    1. Low nutrients
    2. High UV
    3. High salt
    4. Freeze-thaws
    5. Low humidity
  6. What was the evidence that these microbes do not utilize photosynthesis?
    1. The researchers found no evidence of chloroplast-type structures in their microscopic analysis.
    2. The researchers found no pigments typically used in light absorption for photosynthesis.
    3. The researchers found no light dependent photosynthesis genes in their metagenomic study.
    4. The researchers found no increase in metabolic activity when the microbes were exposed to light.
  7. The podcasters discussed an experiment where bacteria from another site (site B) was mixed with bacteria from this site (site A) and grown at “site A” conditions. They found that site B survivors were those that were able to scavenge trace gases. If the opposite experiment was done, that is mixing site A and site B bacteria and growing at site B conditions, would the outcome be the same or different? Why?
    1. Different; the bacteria in a particular environment are adapted to that environment
    2. Same; the bacteria have identical metabolic pathways regardless of the environment.
    3. Same; the bacteria are highly adaptive and can adjust to any environmental condition.
    4. Different; bacteria from site B are better regulated and will outcompete site A bacteria.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3) 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 the experimental design, including controls and strain differences.
  • Analyze the data to make conclusions about the role of each component in Ybt transport and recycling.
  • Analyze the data to make conclusions about the role of chemical reduction in Ybt transport and recycling.
Experimental Background (Koh et al., Figure 3)

Urinary tract infections (UTI) are common infections with more than 400 million diagnosed each year.  The most common pathogen for UTI is E. coli, called uropathogenic E. coli (UPEC).  There has been a marked rise in antibiotic resistance and virulence in these strains.  Koh et al. (2018) investigate the mechanism used by UTI strains to resist copper toxicity via the metal-binding metallophore called yersiniabactin (Ybt).  In earlier experiments they identified genes/proteins involved in copper transport that are encoded in the acquired Yersinia high pathogenicity island (HIP), including YbtA (a transcription factor), FyuA (an importer protein found in the outer membrane), and YbtPQ (an importer protein found in the inner membrane).  They wanted to determine how these genes/proteins mediated copper uptake in UPEC.  To do this, they constructed several UPEC strains.  All strains had deletions in ybtA, the transcription factor that regulates HIP gene expression (ΔybtA), so that expression of single HIP genes was possible. The first strain had only this mutation (UTI89 ΔybtA). The second strain was engineered to express only fyuA (UTI89 ΔybtA pfyuA). The third strain was engineered to express both fyuA and ybtPQ (UTI89 ΔybtA pfyuA pybtPQ). To determine which components were involved in Ybt uptake and recycling, the researchers provided a Ybt:metal complex to the bacterium in the culture medium. They then quantified the location of the Ybt as: 1) extracellular complexed with a metal (panels a, d, g), 2) cell-associated complexed with a metal (panels b, e, h), or 3) extracellular and metal-free (panels c, f, i). Three metals were tested: copper (Cu(II); panels a-c), iron (Fe(III); panels d-f), and a non-reducible metal, gallium (Ga(III); panels g-i).

7.1.2. Questions

  1. Match the strain with the protein(s) it is making. [0 = expresses none; 1 = expresses YbtA; 2 = expresses FyuA; 3 = expresses YbtPQ; 4 = expresses all (YbtA, FyuA, YbtPQ)]
    1. _____ UTI89
    2. ____ UTI89 ΔybtA
    3. _____ UTI89 ΔybtA pfyuA
    4. _____ UTI89 ΔybtA pfyuA pybtPQ
  2. In this experiment, the control bacterial strain is UTI89 ΔybtA.  This is unusual in that this bacterium has a deletion.  Why is it important that this control strain is ΔybtA?
    1. This strain contains the transcription factor that represses expression of the pathogenicity islands so that the researchers can then express one gene at a time.
    2. This strain lacks the enzyme required to degrade the pathogenicity islands so that the researchers can then express each negative regulator gene at a time.
    3. This strain expresses the transcription factor that activates all pathogenicity islands so that the researchers can then remove one gene at a time.
    4. This strain lacks the transcription factor that induces expression of the pathogenicity islands so that the researchers can then express one gene at a time.
  3. In this experiment, the control bacterial strain is UTI89 ΔybtA.  Where is Ybt (yersiniabactin) located in this strain when copper is the metal (panels a-c)?
    1. Extracellular, complexed to copper
    2. Intracellular, complexed to copper
    3. Extracellular, free from copper
    4. Found equally in all places.
  4. In this experiment, the bacterial strain UTI89 ΔybtA pfyuA expresses an outer membrane protein. Where is Ybt primarily located in this strain when iron is the metal (panels d–f)?
    1. Extracellular, complexed to iron
    2. Intracellular, complexed to iron
    3. Extracellular, free from iron
    4. Found equally in all places.
  5. In this experiment, the bacterial strain UTI89 ΔybtA pfyuA pybtPQ expresses an outer membrane protein and an inner membrane protein. Where is Ybt primarily located in this strain when iron is the metal (panels d–f)?
    1. Extracellular, complexed to iron
    2. Intracellular, complexed to iron
    3. Extracellular, free from iron
    4. Found equally in all places.
  6. The results for copper and iron distribution are similar for each strain.  What do all of these results suggest about the function of FyuA in Ybt:metal transport and recycling?
    1. It is required to prevent the complex from leaving the cell.
    2. It is required to transport the complex into the cell.
    3. It is required to degrade the complex outside of the cell.
    4. It is required to bind the complex without moving it inside.
  7. The results for gallium distribution are shown in panels g-i.  Recall that gallium is a non-reducible metal.  What do these results suggest about how metal-free, extracellular Ybt is formed?
    1. Release of the metal from Ybt requires cleavage.
    2. Release of the metal from Ybt requires transport.
    3. Release of the metal from Ybt requires oxidation.
    4. Release of the metal from Ybt requires reduction.
  8. The results for copper and iron distribution are similar for each strain, but different for gallium. These results suggest two possibilities for the function of YbtPQ. What are they?
    1. Import Ybt:metal from the extracellular space into the cytoplasm.
    2. Import Ybt:metal from the periplasm through the inner membrane.
    3. Export metal-free Ybt through the inner membrane to the periplasm.
    4. Export Ybt:metal from the periplasm into the outer membrane.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features related to this study in stacked bar charts and heat maps.
  • Analyze the data to make conclusions about microbial group abundance and metabolic abilities of species groups.
  • Analyze the data and hypothesize relationships about the abundance of a group and its energy conservation and carbon fixation functions.
  • Propose an experiment to test a hypothesis generated by these data.
Experimental Background (Ji et al., Figure 1)

Antarctica is an extreme desert environment where temperatures range from -80oC (winter) to -30oC (summer) on the high plateau and -15oC (winter) to 0oC (summer) in the coastal area.  It has a high altitude at 9800 feet above sea level and high UV exposures with the snow reflecting solar radiation as well.  You can check out the current weather here: https://www.timeanddate.com/weather/antarctica . Despite this extreme environment microbes are present in the soils in Antarctica.  Ji et al. (2017) set out to learn more about the microbes and microbial communities that populate this environment.  They first collected soils at Robinson Ridge, isolated environmental DNA, and made metagenomic libraries which they sequenced.  They used both 16S and 18S rDNA fragments to identify the microbes present and their relative abundance (panel a), but also used their entire library to reconstruct 23 draft microbial genomes. Knowing that microbes in this extreme environment would have challenges in energy conservation and carbon fixation, they were interested in identifying which genes with those related functions were present and abundant in the reconstructed genomes. Abundance of specific genes per taxonomic group is displayed as a heat map (panel b).

stacked bar and heat map
Figure 1. “Phylogenetic and functional composition of Robinson Ridge soils. a, Microbial community structure of three biologically independent soil samples, as determined by mapping sequence reads onto a database of bacterial, archaeal and eukaryotic 16S/18S rRNA genes. b, Heat map showing the relative abundance of genes encoding key enzymes involved in carbon fixation and energy conservation. The relative abundance was calculated on the basis of the presence or absence of functional genes within the sequenced genomes from three biologically independent soil samples, and is presented relative to the total number of 16S rRNA gene sequences in the total microbial community.” (Ji et al. 2017)

7.2.2. Questions

  1. The relative abundance of each group of microbes is shown as a stacked bar chart (panel a).  Which group’s relative abundance is noted by the green section?
    1. Acidobacteria
    2. Chloroflexi
    3. Proteobacteria
    4. Verrucomicrobia
  2. The relative abundance of each group of microbes is shown as a stacked bar chart (panel a).  Which group is most abundant in all three soils sampled?
    1. Actinobacteria
    2. Cyanobacteria
    3. Eremiobacterioaeta
    4. Thaumarchaeota
  3. The frequency of genes that encode key functions in energy conservation and carbon fixation is shown as a heat map (panel b).  What color would indicate the gene was found at a high frequency?
    1. White
    2. Light orange
    3. Medium orange
    4. Dark orange/red
  4. The frequency of genes that encode key functions in energy conservation and carbon fixation was calculated as genes present relative to total 16S rDNA genes. This was to normalize the data and provide a fair comparison amongst the 23 draft genomes. Based on this, what are the rarest genes/functions across all the groups?
    1. Succinate deyhdrogenase and F1F0-ATPase (ATP synthase)
    2. Group 1h [NiFe]-hydrogenase and Type I NADH dehydrogenase
    3. Ammonia monooxygenase and soluble methane monooxygenase
    4. Cytochrome bd oxidase and Type I [MoCu]-CO dehydrogenase
  5. The frequency of genes that encode key functions in energy conservation and carbon fixation was calculated as genes present relative to total 16S rDNA genes.   Based on this, which group shows the highest frequencies for genes involved in energy conservation and carbon fixation?
    1. Actinobacteria
    2. Cyanobacteria
    3. Eremiobacterioaeta
    4. Thaumarchaeota
  6. What can you hypothesize about the relationship of abundance of microbial groups (panel a) to energy conservation and carbon fixation functions found in that group (panel b)?
    1. The Eremiobacteraeota might be less abundant because they have fewer energy conservation and carbon fixation functions.
    2. The Dormibacteraeota could be abundant because they have a larger number of energy conservation and carbon fixation functions.
    3. The Actinobacteria might be more abundant because they have higher frequencies of energy conservation and carbon fixation functions.
    4. The Chloroflexi group might be more abundant because they have protein synthesis and carbon/nitrogen reduction functions.
  7. Genomic studies such as this are a good place to start, but having a sequence that is similar to a functional gene is limited evidence.  If we wanted to test whether these energy conservation and carbon fixation functions were actually present, what could we do?
    1. Sequence a pure culture of each genome and compare them.
    2. Grow the bacterial sample and test for carbon fixation.
    3. Grow the bacterial sample and observe colony formation.
    4. Grow the bacterial sample and analyze the proteome.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

License

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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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