Information Flow and Genetics

TWiM #176: Elio Has Lots of Colanic Acid

Podcast and Annotation Information
  • Annotation by Lauren Ballard, Kaitlyn Wesselink, Benjamin Walsh, Nancy Boury, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #176 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #176 Transcript
  • Papers Discussed:
    • Caforio A, Siliakus MF, Exterkate M, Jain S, Jumde VR, Andringa RLH, Kengen SWM, Minnaard AJ, Driessen AJM, van der Oost J. 2018. Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane. Proc Natl Acad Sci U S A. 115(14):3704-3709. doi: 10.1073/pnas.1721604115
    • Han B, Sivaramakrishnan P, Lin CJ, Neve IAA, He J, Tay LWR, Sowa JN, Sizovs A, Du G, Wang J, Herman C, Wang MC. 2017. Microbial Genetic Composition Tunes Host Longevity. 2017. Cell. 169(7):1249-1262.e13. doi: 10.1016/j.cell.2017.05.036

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 11:49 minutes

The Most Interesting Things (according to students)

  • Through the use of bioengineering, scientists have genetically modified Escherichia coli to have the membrane lipid structure of archaea, creating an archaebacterium that is more able to withstand environmental challenges than an ordinary E. coli organism.
  • Speculation is best done over a beer.

“One of the main differences between bacteria and archaea concerns their membrane composition. Whereas bacterial membranes are made up of glycerol-3-phosphate ester lipids, archaeal membranes are composed of glycerol-1-phosphate ether lipids. Here, we report the construction of a stable hybrid heterochiral membrane through lipid engineering of the bacterium Escherichia coli. By boosting isoprenoid biosynthesis and heterologous expression of archaeal ether lipid biosynthesis genes, we obtained a viable E. coli strain of which the membranes contain archaeal lipids with the expected stereochemistry. It has been found that the archaeal lipid biosynthesis enzymes are relatively promiscuous with respect to their glycerol phosphate backbone and that E. coli has the unexpected potential to generate glycerol-1-phosphate. The unprecedented level of 20–30% archaeal lipids in a bacterial cell has allowed for analyzing the effect on the mixed-membrane cell’s phenotype. Interestingly, growth rates are unchanged, whereas the robustness of cells with a hybrid heterochiral membrane appeared slightly increased. The implications of these findings for evolutionary scenarios are discussed.” (Caforio et al. 2018)

1.2. Main paper; discussion starts at 25:20 minutes

The Most Interesting Things (according to students)

Multiple studies have shown that colanic acid, and the effects it has on the mitochondria, plays a major part in the elongation of life for numerous model organisms. Colanic acid may be the secret to immortality! 😉 … and Elio has lots :p

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)
  • Structure and Function (V&C_SF)
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • 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 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 16 (ASM_16): Genetic variation can influence microbial structures and their functions.
  • Fundamental Statement 24 (ASM_24): Microbes and their communities are essential for supporting all life as we know it.
  • Fundamental Statement 25 (ASM_25): Microbes are used as models that provide fundamental knowledge about life processes.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify lipid features associated with archaea and bacteria.
  • Recall how the archaebacterium hybrid appears be more equipped to resist environmental challenges than a wild-type Escherichia coli.
 S L
  • Predict the challenges of treating a hypothetical pathogenic archaebacterium hybrid if it were to become a pathogen.
 S H
  • Define microbiome.
  • List the characteristics of a model organism.
  • Recall how manipulated colanic acid levels affected lifespan, disease models, and/or  mitochondria.
 M L
  • Predict the impact of altering gut colanic acid levels on the lifespan of humans.
 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

  • Genetic Alteration/Bioengineering (14:23–15:19) The method by which the Escherichia coli was implanted with the archaeal lipid/membrane genes, and how the hybrid was produced.  The distinction of lipids between bacteria and archaea has to do with survival. The conversion of Escherichia coli into an archaebacterium is performed by cloning the archaeal lipid genes into the E. coli using a high-level expression system.
  • Optical Density (17:48–18:03): This is a spectrophotometry method commonly used for estimating the concentration of bacteria or other cells in a liquid.

4.2. Main Paper

  • Gene Knockout Library (28:57–29:07, 30:58–31:17): A collection of 3983 different E. coli deletion mutants. The deleted genes are of all different classes including transcription, translation, metabolism, respiration, membrane transport, proteases, chaperones, etc.
  • Extending the Lifespan of C. elegans (29:21–29:38): E. coli deletion mutants are fed to C. elegans until adulthood and the lifespan of the worms is measured. The normal lifespan is 25-28 days and 21 different mutants increased the lifespan by 5-6 days.

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

5.1. Snippet Paper

  • Membrane Lipids (Phospholipids) (12:50–13:27; 13:38–42):  Bacteria have a normal lipid structure, where the lipids attach at the 3’ carbon, whereas archaea lipids attach at the 1’ carbon. Archaeal lipids are attached by an ether link while bacterial lipids are attached by an ester link. Some archaea have a lipid monolayer instead of a lipid bilayer.
  • Extremophiles (13:52–14:03): Organisms (often archaea): which are able to survive in the most extreme temperatures, salinity, pH levels, etc.
  • Genetic Overexpression (18:09–18:22): When the archaebacterial genes are overexpressed in the Escherichia coli, the cell morphology drastically changes as they become elongated and ‘funny looking.’
  • Archaea vs. Bacteria (18:47–19:31): Archaea are much more resilient/resistant to environmental challenges than bacteria, and the archaebacterial hybrid produced in this experiment were much more resistant to environmental challenges than E. coli typically is.
  • Ester vs. Ether Linked Lipids (19:47–20:15): The ester linked lipids are much stronger than the ether linked lipids, which allows for more efficient transformations, fermentations in mass production processes.
  • Last Universal Common Ancestor (LUCA) (20:21–20:35): The last universal common ancestor is the most recent population of organisms from which all organisms now living on Earth have a common descent, the most recent common ancestor of all current life on Earth.

5.2. Main Paper

  • Model Organism (28:00–28:28): C. elegans (a nematode) is a model organism for experiments to predict how treatments will affect humans
  • Microbiome (28:31- 28:41): The gut microbiome is defined as the complete number/variety of microorganisms, bacteria, viruses, protozoa, and fungi, and their collective genetic material present in the gastrointestinal tract. C. elegans has a gut microbiome of n=1.
  • Polysaccharide Regulation (35:14–36:12): Colanic acid is a polysaccharide secreted by many enterobacterial species that is biosynthesized and regulated by a gene cluster, a transcriptional factor called RscA, and a protease called LON.
  • Biofilm Formation (37:18–37:43): Colanic acid is needed to promote biofilm formation. Theoretically, could this promotion of biofilm formation be a factor of the elongation of life?
  • Mitochondrial Fragmentation/Fission (38:54–39:21; 42:25–42:50): The attenuated fragmentation of the mitochondria as a result of increased production of colanic acid has shown to increase the lifespan of several model organisms. Worms that are defective at mitochondrial fission are unaffected by the colanic acid.
  • Colanic Acid (40:39–40:49): Colanic acid is a repeating unit of glucose, galactose, fucose, and glucuronic acid decorated by pyruvate and acetate. When fed in excess to C. elegans, colanic acid increases their lifespan by attenuating to mitochondrial fragmentation.
  • Endocytosis (41:45–42:07): A way of taking up material from the cell’s exterior. Mutant worms who are unable to endocytose do not have their life span prolonged
  • Heat Shock Response (42:57–43:42): A response that is induced by stress on the organism (i.e. viral proteins) to which the cell responds by shutting down translation in an effort to prevent the production of misfolded proteins. In the case of colanic acid, the acid is inducing the misfolding protein response and the effectors that respond in order to rectify the situation are those that prolong life
  • Metabolism (45:27–45:58): Chorismate metabolism is the precursor for aromatic metabolites. Deficiencies in chorismate derivatives ubiquinone and folate which have shown to allow E. coli mutants to prolong the lifespan of C. elegans.

6. Podcast Questions

  1. Match the following lipid characteristics to archaeal lipids (A) or bacterial lipids (B).
    1. Attachment is at the 3’ carbon of the glycerol backbone –
    2. Attachment is at the 1’ carbon of the glycerol backbone –
    3. Lipids are phospholipids –
    4. Lipids are fatty acids or isoprenoids –
    5. Ether link to glycerol backbone –
    6. Ester link to glycerol backbone-
    7. Sometimes have a lipid monolayer –
    8. Always have a lipid bilayer –
  2. The podcasters discuss that archaea are often found in extreme environments and that their lipids might enable better survival.  What did the researchers find when they tested the hybrid’s growing conditions?  [pick all that apply]
    1. The hybrid survived more heat than wild-type E. coli.
    2. The hybrid replicated much better than wild-type E. coli.
    3. The hybrid survived freezing better than wild-type E. coli.
    4. The hybrid survived fermentation better than wild-type E. coli.
    5. The hybrid survived butanol better than wild-type E. coli.
  3. The podcasters noted several applications for the hybrid type bacteria including transformation efficiencies and fermentation processes. However, this also brings up treatment challenges if a hybrid became a pathogen.  What are some safety and/or pathogen treatment concerns based on the discussion? [pick all that apply]
    1. There may be more horizontal gene transfer of antibiotic resistance genes.
    2. The immune response element of generating heat may be ineffective.
    3. They may be able to survive common food safety/sterilization processes.
    4. They may have a lower infectious dose due to a higher replication rate.
  4. What is a microbiome?
    1. All of the microorganisms that live in the human gut.
    2. A small habitat’s population of anaerobic bacterial species.
    3. A population of gram positive, aerobic microbes.
    4. All microorganisms that live in a particular environment.
  5. Two model organisms were used in the main paper study, E. coli and C. elegans, which led to a discussion of model organisms.  Which features made C. elegans a good study organism for this work? [pick all that apply]
    1. They have a circular and sequenced genome.
    2. They have a single gut microbiome species.
    3. They are a very long-lived worm species.
    4. They are easy to grow in the laboratory.
    5. Mutant strains already exist for this species.
  6. The study found that high/higher levels of colanic acid increased ________________ . [pick all that apply]
    1. Lifespan
    2. Reproduction
    3. Mitochondrial fragmentation
    4. Metabolism
    5. Calorie restriction
    6. Insulin-like growth factors
    7. Disease model symptoms
  7. The study found that high/higher levels of colanic acid decreased ________________ . [pick all that apply]
    1. Lifespan
    2. Reproduction
    3. Mitochondrial fragmentation
    4. Metabolism
    5. Calorie restriction
    6. Insulin-like growth factors
    7. Disease model symptoms
  8. Based on the results discussed for the main paper that apply to C. elegans, another distant nematode species, and fruit flies, what would be a reasonable treatment to try to increase lifespan in humans?
    1. Feed people a supplement that inhibits bacteria that produce colanic acid.
    2. Feed people a supplement that increases anaerobic gut microbiome species.
    3. Feed people a supplement that increases bacteria that produce colanic acid.
    4. Feed people a supplement that inhibits all gut bacteria and increases fungi.

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 key features of the experimental background, chromatography images, and stacked bar charts that are relevant for this experiment.
  • Identify key experimental design features for the bioengineered hybrid bacterium and growth media that are relevant for this experiment.
  • Analyze the data to make conclusions about lipid molecule abundance.
  • Analyze the data to make conclusions about the success of engineering a hybrid archaea-like bacterium
Experimental Background (Calforio et al., Figure 1)

Archaea, Bacteria, and Eukarya are the three domains of life and each has distinct characteristics.  Evolutionary microbiologists such as Caforio et al (2018) have an interest in exploring features of the last common ancestor (LUCA) from which these three domains might have evolved.  In this study, they focus on the membrane differences between archaea and bacteria, specifically the lipids.  Their goal was to engineer a bacterium with the type of membrane that LUCA might have had, that is, a hybrid archaeal-bacterial membrane with archaeal unsaturated archaetidylglycerol (AG) and archaetidylethanolamine (AE), which are the counterparts of bacterial phosphatidylglycerol (PG) and phosphatidylethanolamine (PE).  To attempt this they added several genes encoding isoprenoid biosynthesis genes of the non-mevalonate isoprenoid biosynthesis pathway (MEP-DOXP) (idi alone or idi, ispD, ispF, dxs), as well as ether lipid synthases (EL genes), and bacterial polar head group synthesis genes (psd, pgsA, pgpA, pssA). They also engineered regulated expression of the MEP-DOXP pathway so that increased expression would occur in the presence of the chemical isopropyl-β-d-1-thiogalactopyranoside (IPTG). Finally, to enhance use of the correct carbon for archaeal lipids (C1) when additions to the glycerophosphate backbone occurred, they also made a mutant that is defective in that addition by deletion of the araM gene.  To test the lipids produced by these strains, including the effect of IPTG induction of the MEP-DOXP pathway and presence/absence of araM, they quantified the lipids in wild-type E. coli, the engineered strain with araM (MEP/DOXP+EL+), and the engineered strain lacking araM (MEP/DOXP+EL+AraM-) with and without the inducer IPTG.  Quantitation used thin layer chromatography plates (TLC; panel A); relative abundance was calculated for each lipid and is displayed as a stacked bar chart (panel B).

thin layer chromatography plate and stacked bar chart
Figure 1. “TLC-based quantitation of in vivo archaeal lipid synthesis. (A) TLC of lipid extracts from wild-type E. coli [JM109 (DE3)] heterochiral mixed membrane E. coli (MEP/DOXP+EL+) induced early during growth (OD600 = 0.0) with different IPTG concentrations and incubated until stationary phase, and the E. coli strain harboring the entire ether lipid pathway but lacking the araM gene (MEP/DOXP+EL+AraM−) treated similarly. (B) Relative quantitation of the spots detected in the TLC. AG, archaetidylglycerol; CL, cardiolipin; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.” (Calforio et al. 2018)

7.1.2. Questions

  1. Identify which lipids are archaeal (A) and which are bacterial (B).
    1. AE =
    2. AG =
    3. PE =
    4. PG =
  2. The relative abundances of each lipid are noted in panel B.  Which lipid’s abundance is represented by the orange color?
    1. unsaturated archaetidylglycerol
    2. cardiolipin
    3. phosphatidylglycerol
    4. phosphatidylethanolamine
  3. Which gene is theorized to influence the attachment to the phosphoglycerate backbone?
    1. idi
    2. araM
    3. pgsA
    4. dxs
  4. IPTG is used in the media for some of the cultures.  What role does it play in this experiment?
    1. It inhibits the expression of the ether linkage pathway genes.
    2. It induces high expression of ether linkage pathway genes.
    3. It induces high expression of the MEP/DOXP pathway genes.
    4. It inhibits the expression of the MEP/DOXP pathway genes.
  5. Which lane/bar shows the abundance of each lipid in wild-type E. coli?
    1. The first lane/bar; JM109; DE3 with no IPTG
    2. The second lane/bar; MEP/DOXP+EL+; no IPTG
    3. The third lane/bar; MEP/DOXP+EL+; 5 IPTG
    4. The last lane/bar; MEP/DOXP+EL+AraM-; 10 IPTG
  6. Using either panel, which engineered E. coli with which IPTG concentration shows the highest relative abundance of the archaeal lipid?
    1. MEP/DOXP+EL+; no IPTG
    2. MEP/DOXP+EL+; 5 IPTG
    3. MEP/DOXP+EL+; 10 IPTG
    4. MEP/DOXP+EL+; 50 IPTG
    5. MEP/DOXP+EL+; 100 IPTG
    6. MEP/DOXP+EL+AraM-; 10 IPTG
  7. The researchers had hypothesized they could influence the attachment to the phosphoglycerate backbone by making a specific bioengineering change?  Did this change allow them to increase the level of archaeal lipids as they hypothesized?  What is your evidence?
    1. Yes; there was as much archaeal lipid in the araM- strain as the araM+ strain at the same IPTG
    2. No; there was much less archaeal lipid in the araM– strain than the araM+ strain at the same IPTG
    3. Yes; there was much more archaeal lipid in the araM– strain than the araM+ strain at the same IPTG
    4. No; there was not more  archaeal lipid in the araM– strain than the araM+ strain at the same IPTG

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in line graphs, data table, and experimental background
  • Analyze the data and make conclusions and predictions about bacterial gene functional groups and treatment conditions that affect nematode lifespan via the gut microbiome.
  • Justify whether particular genes identified should be studied further.
Experimental Background (Han et al. 2018, Figure 1)

The gut microbiome has been implicated in health and aging. For example, microbes populating the gut may be involved in production of health-related metabolites and inactivation of harmful exogenous compounds.  However, these studies are nearly all correlative studies because the mammalian gut microbiome is very complex and difficult to study.  One system that has proven useful for studying a simpler gut microbiome is the soil nematode Caenorhabditis elegans (C. elegans). This model organism has only one member in its gut microbiome, the bacterial model organism Escherichia coli (E. coli). Both model organisms also bring the power of genetics to this study of how the gut microbiome might impact aging and lifespan.  In this study, Han et al (2018) in determining which bacterial genes contributed to lifespan, so they fed strains of E. coli that were mutant for a single gene each to wild-type nematodes and quantified changes in nematode lifespan.  Bacterial genes identified as affecting lifespan were investigated and categorized by functional group, lifespan effect, whether the lifespan change was observed in one of both of the two wild-type E. coli strains used (strain BW25113 and strain MG1655), and whether the lifespan effect could be observed when feeding the mutant strain began in adulthood (panel A).  A representative lifespan experiment is quantified and shown for each functional group (panel B-F). The mutant gene in each strain is noted by a Δ preceding the gene name.  For example, Δhns in panel B indicates data for the strain that has a deletion in the bacterial hns gene.

  • 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. Nematodes were fed different kinds of E. coli and the lifespan measured (panel D).  What is different about the two E. coli strains?
    1. One strain produces a toxin that affects growth and the other does not.
    2. One has a wild-type secB gene and one has a deletion in the secB gene.
    3. One strain is resistant to all antibiotics, while the other is partially sensitive.
    4. One strain is genetically tolerant to glucose, while the other is not tolerant.
  2. Which bacterial gene, when deleted, extends the nematode lifespan to the greatest degree?
    1. hns
    2. aroG
    3. secB
    4. lpp
    5. pbl
  3. Each bacterial gene’s contribution to nematode lifespan was tested by deleting the gene in two different E. coli strains that were otherwise considered wild-type. Some of the genes show an effect only in one of the wild-type strain backgrounds, including psuK, yfiB, and uidC (panel A). Would these be important to continue studying or not? Why or why not?
    1. Yes; these genes are found in wild-type E. coli so they should be kept in the pool of possible mutants.
    2. No; if the effect is not observed in both E. coli strains then it is a false positive and should be ignored.
    3. Yes; these genes do contribute, but the other strain may have genetic differences that mask the effect.
    4. No; since the effect is not found in both strains, the gene is unlikely to be present in all E. coli strains.
  4. In which functional group do you find the most bacterial genes that affect nematode lifespan? Why might this group contribute the most?
    1. Transcription and translation; they change the environment stress-related gene expression.
    2. Metabolism and respiration; they alter the small molecules that are produced by the bacteria.
    3. Membrane and transport; they change the ability of the bacteria to move around in the gut.
    4. Proteases and chaperones; they alter the folding of the proteins into their correct structures.
  5. Each bacterial gene’s contribution to nematode lifespan was tested by feeding nematodes either their entire life or just as adults (panel A). To find these, compare the E. coli MG1655 column to the Adult Effect column.  If you were interested in studying treatments or supplements to be used in human adults,  which group of genes should you focus on? [pick all that apply]
    1. Genes where the lifespan effect was in lifetime, adult, and phage resistance, e.g., gmhA.
    2. Genes where the lifespan effect was found for lifetime, but not adult feeding, e.g., ycgL.
    3. Genes where the lifespan effect was found for both lifetime and adult feeding, e.g., ihfB
    4. Genes where the lifespan effect was found for adult, but not lifetime feeding, e.g., uidC.
  6. This study focused on gut microbiome bacterial strains that extend nematode lifespan when a bacterial gene was removed.  If you were to perform proteomic or metabolomic studies, which types of compounds/metabolites would you be most interested in following up on?
    1. Metabolites present in the wild-type strain that are lower or not in the mutant strain.
    2. Metabolites that are present in the mutant strain that are not in the wild-type strain.
    3. Metabolites that are at a lower level in the wild-type strain than in the mutant strain.
    4. Metabolites that are at a higher level in the mutant strain than the wild-type strain.

8. Paper Information and Licensing

8.1. Snippet paper

  • Caforio A, Siliakus MF, Exterkate M, Jain S, Jumde VR, Andringa RLH, Kengen SWM, Minnaard AJ, Driessen AJM, van der Oost J. 2018. Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane. Proc Natl Acad Sci U S A. 115(14):3704-3709. doi: 10.1073/pnas.1721604115
  • This article is licensed for Creative Commons use using CC BY 4.0 (https://creativecommons.org/licenses/by-nc-nd/4.0/), which allows re-use and adaptation with proper attribution and notation of any changes. See https://doi.org/10.1073/pnas.1721604115

8.2. Main paper

  • Han B, Sivaramakrishnan P, Lin CJ, Neve IAA, He J, Tay LWR, Sowa JN, Sizovs A, Du G, Wang J, Herman C, Wang MC. 2017. Microbial Genetic Composition Tunes Host Longevity. Cell. 169(7):1249-1262.e13. doi: 10.1016/j.cell.2017.05.036. doi: 10.1016/j.cell.2017.05.036
  • This article is not licensed for Creative Commons use; see https://doi.org/10.1016/j.cell.2017.05.036. Thus, the abstract and figures cannot be copied here.

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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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