Information Flow and Genetics

TWiM #222: Biosensors in Bacteria

Podcast and Annotation Information
  • Annotation by Madison Hayes, Alexis Austin, Dillon Nguyen, Angela Wilson, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #222 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • McLean JS, Bor B, Kerns KA, Liu Q, To TT, Solden L, Hendrickson EL, Wrighton K, Shi W, He X. 2020. Acquisition and Adaptation of Ultra-small Parasitic Reduced Genome Bacteria to Mammalian Hosts. Cell Rep. 32(3):107939. doi: 10.1016/j.celrep.2020.107939
    • Dieudonné A, Prévéral S, Pignol D. 2020. A Sensitive Magnetic Arsenite-Specific Biosensor Hosted in Magnetotactic Bacteria. Appl Environ Microbiol. 2020 Jul 2;86(14):e00803-20. doi: 10.1128/aem.00803-20

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 6:07 minutes

The Most Interesting Things (according to students)

  • TM7x which is a strain of Ca. Nanosynbacter lyticus is classified as an obligate-epibiont, meaning it requires a host to survive. When attempting to grow this organism, researchers found that while the microbe was inherently fastidious and required enriched media, it also required the presence of its prey organism because true to its parasitic epibiont nature, it can only replicate with nutrients from its host.
  • TM7x also proved it can remain viable and re-infect host bacterium after temporary separation from its basibiont (the organism it lives on top of). This discovery of quorum sensing between TM7x and its host bacterium Actinomyces odontolyticus that allow for the parasitic obligate-epibiont relationship have proven how these ultra-small organisms have survived for so long despite missing capacities for de novo biosynthesis of many essential compounds.

“The first cultivated representative of the enigmatic phylum Saccharibacteria (formerly TM7) was isolated from humans and revealed an ultra-small cell size (200–300 nm), a reduced genome with limited biosynthetic capabilities, and a unique parasitic lifestyle. TM7x was the only cultivated member of the candidate phyla radiation (CPR), estimated to encompass 26% of the domain Bacteria. Here we report on divergent genomes from major lineages across the Saccharibacteria phylum in humans and mammals, as well as from ancient dental calculus. These lineages are present at high prevalence within hosts. Direct imaging reveals that all groups are ultra-small in size, likely feeding off commensal bacteria. Analyses suggest that multiple acquisition events in the past led to the current wide diversity, with convergent evolution of key functions allowing Saccharibacteria from the environment to adapt to mammals. Ultra-small, parasitic CPR bacteria represent a relatively unexplored paradigm of prokaryotic interactions within mammalian microbiomes.” (McLean et al. 2020).

1.2. Main paper; discussion starts at 30:57 minutes

The Most Interesting Things (according to students)

  • Bacteria that have natural magnets that help steer their motility were engineered to produce light in response to arsenic so that low cost, low resource, field-level arsenic detection could be done.
  • Freeze drying was able to preserve the sensors for longer periods of time without damaging its sensitivity to arsenic detection.

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract and figures cannot be copied here.

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Information Flow and Genetics (V&C_IFG)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 27 (ASM_27): The extent of microbial diversity is largely unknown, and exploration of this diversity is critical to understanding microbes and their role in the biosphere.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Define the relationship between an epibiont and a basibiont
  • Identify the reasons E. coli became a model organism.
 S L
  • Predict how abundance in metagenomic studies is affected by gene copy number.
  • Predict a bacterium’s lifestyle based on its genome features.
 S H
  • Recall the symptoms of arsenic poisoning.
  • Define the relationship of sensitivity and specificity to accuracy of a test.
 M L
  • Analyze hypothetical operon response data to identify those with promise for a biosensor.
  • Determine how scientists determined which bacteria would be the best candidate for their testing.
 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

  • Fluorescence in situ Hybridization (FISH) (18:19–18:51; 20:19–20:50): Fluorescence in situ hybridization (FISH) is a technique that detects and locates specific DNA sequences by using fluorescently labeled probes mixed with target DNA that bind to their complementary DNA sequence. This allows for genetic mapping to identify gene expression and abnormalities. FISH has been used in this paper to correlate cell size with reduced genomes of bacterial parasites by probing the G3, G5, G6, and SR1 groups of the CPR lineage.

4.2. Main Paper

  • Magnetic Concentration of Bacteria (40:45–41:30; 58:00–59:00): A technique where bacteria are concentrated using a magnet, and boosts detection sensitivity without requiring a filter or a centrifuge. Scientists engineered magnetobacteria to produce light when it’s exposed to arsenic, but since the arsenic concentrations in contaminated water can be so low, the light may be faint. This technique also works well in not only field settings, but low resource areas.
  • LuxCDABRE Operon and Reporter Cassette (47:45–48:30; 1:08:00): This is the operon for a bioluminescent reporter system.  In this study, bacteria are engineered to produce light in response to arsenic exposure using this reporter.  There are two main advancements.  First, they use the entire operon, the luxCDABE operon, which is a cluster of genes found in naturally light-producing bacteria like Photorhabdus luminescens or Vibrio fischeri.  The genes luxA and luxB encode the actual light-producing enzyme (luciferase). The genes luxC, luxD, and luxE encode proteins needed to produce the substrate for the luciferase.  Using all five genes means that researchers do not have to provide an external substrate, which is common for bioluminescent reporters to work. They investigate arsenic-regulated promoters to use and test two.  This allows them to use an instrument to measure light to determine how much arsenic is present based on how intense the glow is. This works well in low resource environments and can be paired with magnetism to amplify the signal.
  • Freeze Drying with Cryoprotectants (59:30 – 1:01:30): A useful method used to preserve living bacteria. Since freeze drying alone can be harsh on cells and damage their membranes and DNA, scientists add cryoprotectants that act as a cushion to protect the cells during the process. This helps by preventing ice crystals from forming inside of the cells and stabilizing proteins and membranes during drying and rehydration. This technique makes it possible to ship biosensors without refrigeration and enables a simple “just add water” like kit for quick arsenic testing.

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

5.1. Snippet Paper

  • Human Oral Microbiome (6:10–8:17): This microorganism originated in the environment and eventually evolved to survive in the human oral cavity as part of normal flora.
  • Quorum Sensing (10:50–11:36): This is cell-to-cell communication that allows bacteria to mass control their behaviors. This is seen in cultivating TM7 because it can only grow when sensing its prey organism is in its vicinity.
  • Structure and Function (13:25 -15:00; 25:00–27:00): TM7 has a specialized secretory system common in all Saccharibacteria that aid in defenses, proliferation, and overall survival.
  • Evolution (17:45): TM7 steals nutrients out of its prey bacteria Actinomyces, and may grow and thrive in various environments even without a basibiont.

5.2. Main Paper

  • Effects of Metals on Microbes (33:08–36:16): Scientists made sure the bacteria were suitable for an aqueous solution with metalloid ions (arsenic).
  • Effects of Metals on Humans (34:20–36-20): Exposure to arsenic can lead to dark or thick skin, heart disease, cancer; it affects our mammalian cell longevity and disrupts with the pyruvate-dehydrogenase complex, energy is disrupted, and cells can be in a state of apoptosis.
  • Genetic Engineering (36:46–59:00):  Scientists were able to engineer the bacteria to produce bioluminescence in response to arsenic.
  • Gene Regulation Mechanisms (36:46–59:00): Scientists were able to regulate expression of the microbe’s bioluminescence; tested to see if the reporter was expressed over 24 hours; arsenic needs to be reduced to +3 to be able to be detected, arsenate’s valence state is +5 and the operon won’t turn on unless reduced.

6. Podcast Questions

  1. A _______ is a species that lives on the surface of another species, which is called the ______.
    1. Basibiont; epibiont
    2. Basibiont; habitont
    3. Epibiont; basibiont
    4. Habibiont; epibiont
  2. The podcasters discuss why E. coli became a bacterial model organism. What are the reasons they noted? [pick all that apply]
    1. It is easy to grow in the laboratory.
    2. It has many non-pathogenic strains.
    3. It is most prevalent in the environment.
    4. Knowledge using it accumulated.
    5. It is very large and easy to see.
  3. The researchers noted that Tm7x and other similar bacteria have a single copy of the 16S rDNA gene, while most other bacteria have many copies of this gene.  This is the gene often used to identify species in metagenomic sequencing studies. What would be the impact of this?\
    1. The abundance of these bacteria is underestimated.
    2. The abundance of these bacteria is correctly estimated.
    3. The abundance of these bacteria is overestimated.
    4. The bacterial abundance can’t be quantified like this.
  4. You find three bacteria and characterize their genomes.  Your findings for a number characteristics are noted in the table for each.  Which is most likely to be a parasitic bacterium?  What is your evidence?
Bacterial Size (nanometers) Genome Size (million base pairs)  Total Gene Number Number of Amino Acids Synthesized Genes involved in Nucleotide Synthesis
Species 1 275 0.9 706 4 2
Species 2 2000 5.0 4200 10 30
Species 3 900 1.4 2700 19 25
    1. Species 2 is most likely to be a parasitic bacterium due to lack of ability to synthesize its own amino acids for translation.
    2. Species 1 and 3 are most likely to be parasitic bacteria because they are both small in physical size and have small genomes.
    3. Species 1 is most likely to be a parasitic bacterium due to lack of ability to synthesize its own amino acids and nucleotides.
    4. Species 3  is the most likely to be a parasitic bacterium due to its small physical size coupled with a small genome size.

5. What are the symptoms of arsenic poisoning? [pick all that apply]

    1. Thicker skin
    2. Cancer
    3. Lightened skin
    4. Abdominal pain
    5. Heart disease
    6. Numbness

6. When measuring testing accuracy, we often quantify several measures related to true positives and true negatives.  Which statement is true about these measures?

    1. Specificity quantifies how well a test finds true positives and negative predictive value quantifies how well a test finds true negatives.
    2. Positive predictive value quantifies how well a test finds true positives and sensitivity quantifies how well a test finds true negatives.
    3. Positive predictive value quantifies how well a test finds true positives and negative predictive value quantifies finding true negatives.
    4. Sensitivity quantifies how well a test finds the true positives and specificity quantifies how well a test finds the true negatives.

7. Let’s say you wanted to develop a similar biosensor to detect mercury.  You tested three different operons for regulation by mercury at 0 to 100 parts per million mercury (Hg) and the quantified data for your RNA detection are shown below.  Which operon has a promoter that you’d want to consider using?

0 ppm Hg 10 ppm Hg 25 ppm Hg 50 ppm Hg 100 ppm Hg
merABC 275 290 400 300 250
hgwDEF 25 28 30 100 130
appAGR 10 107 705 1090 1502
    1. merABC; it has a high level of signal across the experimental range of Hg concentrations
    2. hgwDEF; it starts fairly low and increases along with increasing mercury concentrations
    3. appAGR; it starts low and increases steadily with the increasing mercury concentrations.
    4. None of the above

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 2B).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features in fluorescent in situ hybridization (FISH) microscopy images and gel images of polymerase chain reaction (PCR), including sample identification.
  • Analyze the data to make conclusions regarding the presence/absence, as well as the localization of each Saccharibacter group in the oral samples.
  • Analyze the data to make conclusions regarding its likely symbiotic lifestyle.
  • Analyze the data to make conclusions about the bacterial size and its ability to be removed by filter sterilization.
  • Propose additional controls that would improve the experiment.
Experimental Background (McLean et al., Figure 3)

Saccharibacteria are a group of bacteria that are members of the Candidate Phyla Radiation (CPR) and are very prevalent in nature, but are difficult to cultivate in the lab.  They have unusual characteristics including a very reduced genome, an epibiont lifestyle, a single copy of the typical species biomarker gene 16S rDNA gene, and are a mere 200-300 nanometers in size.  By comparison, E. coli is 500 nanometers wide and 2000 nanometers long.  In this study, McLean et al. (2020) identified and characterized Saccharibacteria (also known as T7M) sequences in metagenomic samples including in a fossilized human tooth plaque deposit called a dental calculus.  They identified six strain groups which they named G1 through G6.  Since they found sequences for these small bacteria in ancient tooth plaque, they were interested in determining whether modern humans harbor these bacteria as well.  To do this, they used fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) to identify whether Saccharibacteria were present in modern human tongue scrapings and saliva.  They used probes (FISH) and primers (PCR) that are specific for the G1, G3, G4, and G5 Saccharibacteria, and the SR1 phylum. Microscopy images of the fluorescence attributable to Saccharibacteria or SR1 (panel A, column 1, red), fluorescence of total DNA including other oral bacteria and/or human cells in the sample (panel A, column 2, green), and the merged fluorescence (panel A, column 3) are displayed.  Additionally, polymerase chain reaction using primers specific for the Saccharibacteria group or SR1 were used to determine whether they were present in saliva (panel B, lane a), saliva supernatant (panel B, lane b), and saliva after filtration with a 0.45 micron filter (panel B, lane c) for two individuals (1 and 2).

Microscopy and gel photos.
Figure 3. “New Groups from the Human Oral Cavity Are Ultra-small Epibionts. (A) Human saliva and tongue samples were stained using fluorescence in situ hybridization (FISH) probes that specifically targeted the G1, G3, G5, and G6 lineages of TM7 phylum, as well as the SR1 phylum. Column 1 is TM7 staining with lineage-specific probes (red), column 2 is total DNA stain with Syto9 dye (green), and column 3 is merged images (magnified images indicate ultra-small bacteria). All scale bars are 5 μm. (B) PCR amplification of G3, G5, G6, and SR1 16S rRNA with lineage-specific primers, using template DNA isolated from original saliva samples, saliva supernatants, and filtrate passing through 0.45 μm filters (see STAR Methods) confirming ultra-small cell sizes.” (McLean et al. 2020, no changes).

 

7.1.2. Questions

  1. What color fluorescence indicates Saccharibacteria are present and in which columns of panel A do you see it? Pick all that apply.
    1. Red; column 1
    2. Green; column 2
    3. Red; column 3
    4. Green; column 3
  2. Which groups of Saccharibacteria that were tested for are present in the oral samples?  What is your microscopy evidence?
    1. G1, G3, G5.  We can see red fluorescence in these samples.
    2. G1, G3, G5, SR1.  We can see red fluorescence in these samples.
    3. All of them.  We can see green fluorescence in these samples.
    4. All of them.  We can see red fluorescence in these samples.
  3. Which groups of Saccharibacteria are associated with other bacteria in the oral samples? What is your microscopy evidence?
    1. All of them.  We can see overlapping green and red fluorescence in all samples.
    2. All of them.  We can see red fluorescence at the surface of bacteria (green).
    3. G1, G3, G5.  We can see red fluorescence overlapping the green fluorescence.
    4. G1, G3, G5, SR1.  We can see red fluorescence at the surface of bacteria (green).
  4. Which Saccharibacteria lifestyle is supported by the microscopy localization evidence?
    1. Basibiont because they are the habitat for other bacteria.
    2. Endosymbiont because they live and grow inside other bacteria.
    3. Epibiont because they live on the surface of other bacteria.
    4. Endosymbiont because they live and grow inside eukaryotes.
  5. What is different about the samples used for lanes a and c polymerase chain reaction methodology (panel B)?
    1. a is saliva from a male and c is saliva from a female
    2. a is total saliva and c is 0.45 micron filtered saliva
    3. a is total saliva and c is saliva sterilized by autoclave
    4. a is saliva supernatant and c is enzyme-digested saliva
  6. Which individual’s saliva is positive for which groups of Saccharibacteria, according to the PCR evidence (panel B)?
    1. Individual 1: all and individual 2: all but SR1.
    2. Individual 2: all and individual 1: G3 and G5.
    3. Both are positive for all Saccharibacteria.
    4. Both are negative for all Saccharibacteria.
  7. Based on these data, would you be able to filter-sterilize a solution using a 0.45 micron filter to remove these bacteria?
    1. Depends; one of the groups is filtered
    2. Depends; two of the groups are filtered
    3. Yes; all groups are removed by the filter
    4. No; all groups pass through the filter.
  8. There are no controls in either of these experiments.  What would have been good controls to use and what would they tell us? [pick all that apply]
    1. Negative control, no probe in FISH to show background red and green fluorescence levels.
    2. Positive control,  use a FISH probe for a known oral bacterium to show example staining.
    3. Negative control, no template in PCR to show there is no DNA contaminating the reagents.
    4. Positive control,  use purified DNA from the species in PCR to show where fragments should be.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in a bar chart.
  • Identify the purpose of key elements in the engineered biosensor.
  • Analyze the data to make conclusions about arsenic sensitivities for the promoters used in the biosensor and about which biosensor would be best for further development.
  • Analyze the data and identify problems if specific biosensors were deployed in the field as they currently are.
Experimental Background (Dieudonné et al., Figure 3AB)

Arsenic contamination of drinking water is a significant problem worldwide, but particularly in areas that rely on well water.  Symptoms of arsenic poisoning for humans include diarrhea, heart problems, and cancer.  Arsenic inhibits a key enzyme in ATP generation, so limiting exposure is important, however, systems for monitoring arsenic levels can be expensive, difficult to use, not very sensitive, and are typically not used in the field.  Here, the researchers are aiming to develop a sensitive, low cost, easy to use alternative by engineering a biosensor to detect arsenic.  Through a series of experiments, they identified two magnetobacteria that could grow in the presence of arsenic (Magnetospirillum magneticum [AMB-1] and M. gryphiswaldense [MSR-1] and four promoters regulated by arsenic (Pred, Pmet, ParsR1, ParsRM).  They engineered each promoter to a reporter cassette operon of five genes that encode proteins that will report activation as bioluminescence.  They tested the ability of the bacteria containing one each of their test constructs to produce light in response to different arsenic concentrations (panels A and B).  In this experiment light level was normalized to culture density.  The background signal (light when no arsenic was present) was removed.  The biosensors are named for the bacterium_promoter.

  • This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract and figures cannot be copied here.
7.2.2. Questions
  1. Which panel and which color bars indicate the results for the Magnetospirillum magneticum  bacterium that has the Pred construct?
    1. Panel A, blue
    2. Panel B, blue
    3. Panel A, orange
    4. Panel B, orange
  2. What feature shows the mean of each sample and timepoint?
    1. Asterisks
    2. Whiskers
    3. The y-axis
    4. Bar heights
  3. What is the difference between the biosensor strains in panel B?
    1. They are variants of the same Magnetobacterium.
    2. They have different arsenic-responsive promoters.
    3. They contain different bioluminescent operons.
    4. They are different species of Magnetobacteria.
    5. They are tested against different arsenic compounds.
  4. What patterns of arsenic-induced light do you see in panel A for biosensors named AMB_Pred and AMB_Pmet? What does this indicate for these arsenic-responsive promoters? [pick all that apply]
    1. Light increases as arsenic increases for both; both work equally well.
    2. Light increases early, peaks, and decreases for AMB_Pred; dose impacts Pred.
    3. Light increases as arsenic increases for AMB_Pmet; dose influences Pmet.
    4. AMB_Pmet detects arsenic at the lowest levels; Pmet is most sensitive.
    5. AMB_Pred detects arsenic at the lowest levels; Pred is most sensitive.
    6. Light decreases as arsenic increases for both; neither work well.
  5. Based on these data, which biosensor bacterium would be the best choice to continue in testing as an arsenic detector? What is your evidence?
    1. AMB_Pred; it has the smallest error bars overall.
    2. AMB_Pmet; it coordinates well with arsenic level.
    3. MSR_ParsR1; it detects the lowest concentrations.
    4. MSR_ParsRM; it produces the most bioluminescence.
  6. What kind of error would you see if you used the AMB_Pred at higher concentrations? Why would this be a problem?
    1. False negative; at higher concentrations the biosensor would not show arsenic contamination accurately.
    2. False positive; at lower concentrations the biosensor would not show arsenic contamination accurately.
    3. False negative; at higher concentrations the biosensor would not show arsenic contamination accurately.
    4. False positive; at lower concentrations the biosensor would not show arsenic contamination accurately.

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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