Microbial Ecology

TWiM #308: Living in a Community World

Podcast and Annotation Information
  • Annotation by Zainab Aljabri, Nadira Haji, Osman Alam, Melanie Petrillo, Sophia Rizzo, Kayla Villaraza, Jeremy Ritzert, Rebecca Seipelt-Thiemann, and Mel Melendrez-Vallard.
  • Podcast audio by TWiM: Listen to TWiM #308 Podcast
  • Podcast transcript by Otter.ai and edited by Harshita Sharma and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Zepeda-Rivera M, Minot SS, Bouzek H, Wu H, Blanco-Míguez A, Manghi P, Jones DS, LaCourse KD, Wu Y, McMahon EF, Park SN, Lim YK, Kempchinsky AG, Willis AD, Cotton SL, Yost SC, Sicinska E, Kook JK, Dewhirst FE, Segata N, Bullman S, Johnston CD. 2024. A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche. Nature. 628(8007):424-432. doi: 10.1038/s41586-024-07182-w
    • Lozano GL, Bravo JI, Garavito Diago MF, Park HB, Hurley A, Peterson SB, Stabb EV, Crawford JM, Broderick NA, Handelsman J. 2019. Introducing THOR, a Model Microbiome for Genetic Dissection of Community Behavior. mBio. 10(2). doi: 10.1128/mBio.02846-18

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:45 minutes

The Most Interesting Things (according to students)

  • Bacteria play a large part in cancer in addition to genetics and environmental factors.
  • Fusobacterium is normally present in the human oral cavity and rarely found in the lower GI tract of healthy individuals, but has been found to be enriched in the colons of Individuals with colorectal cancer, I found this interesting because it has me wondering how this can happen and why. Another interesting thing was that there is an odor to Fusobacterium that is apparently so strong and intense that you can recognize it from far away, this shows just how profound the smell is.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals, is enriched in human colorectal cancer (CRC) tumours. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche. (Zepeda-Rivera et al. 2024, no changes)

1.2. Main paper; discussion starts at 44:00 minutes

The Most Interesting Things (according to students)

  • Different bacteria are able to form colonies with each other and interact in a way that allows for microbial community success.
  • B. cereus uses a strategy which involves modifying alkaloid antibiotics that P. koreensis produces that inhibits F. Johnsoniae from growth suppression. It provides protection and stability to outside threats only when B. cereus enters the stationary phase. Additionally,  THOR exhibits emergent properties. Only when three species interact, emergent properties are seen such as improved biofilm formation and colony expansion.

The quest to manipulate microbiomes has intensified, but many microbial communities have proven to be recalcitrant to sustained change. Developing model communities amenable to genetic dissection will underpin successful strategies for shaping microbiomes by advancing an understanding of community interactions. We developed a model community with representatives from three dominant rhizosphere taxa, the Firmicutes, Proteobacteria, and Bacteroidetes. We chose Bacillus cereus as a model rhizosphere firmicute and characterized 20 other candidates, including “hitchhikers” that co-isolated with B. cereus from the rhizosphere. Pairwise analysis produced a hierarchical interstrain-competition network. We chose two hitchhikers, Pseudomonas koreensis from the top tier of the competition network and Flavobacterium johnsoniae from the bottom of the network, to represent the Proteobacteria and Bacteroidetes, respectively. The model community has several emergent properties, induction of dendritic expansion of B. cereus colonies by either of the other members, and production of more robust biofilms by the three members together than individually. Moreover, P. koreensis produces a novel family of alkaloid antibiotics that inhibit growth of F. johnsoniae, and production is inhibited by B. cereus. We designate this community THOR, because the members are the hitchhikers of the rhizosphere. The genetic, genomic, and biochemical tools available for dissection of THOR provide the means to achieve a new level of understanding of microbial community behavior. (Lozano et al., 2019, no changes)

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

Snippet Main
Vision and Change Topics
  • Impact of Microorganism (V&C_IM)
  • Microbial Ecology (V&C_ME)
  • Microbial ecology (V&C_ME)
  • Impact of Microorganism  (V&C_IM)
  • Structure and Function (V&C_SF)
ASM Fundamental Statements
  • Fundamental Statement 20 (ASM_20): Microbes in the environment interact with and affect each other.
  • Fundamental Statement 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society and ecosystems.
  • 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 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with minority having detrimental impact on their host
  • 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
  • Define clade.
  • Recall what happens to accessory genome size as the number of Fusobacterium genome sequences increases.
 S L
  • Predict tumor formation in a model using multiple Fusobacteria.
 S H
  • Define biofilm.
  • Identify the species in the THOR biofilm.
 M L
  • Predict the results of a hypothetical experiment based on bacterial community interaction.
 M H

1 Papers: Snippet (S) or Main (M)

2 Learning Objectives: Lower Order(L) 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

  • Metagenomics (7:24): Metagenomics is one method using next generation sequencing of a mixture of DNAs.  Here, it was used to sequence DNAs from a tumor environment and then bacterial genomes assembled from the sequences.
  • Comparative Genomic Analysis (13:09; 18:41): This is a method of comparing genome sequences.  Here, the researchers leveraged that availability of the large genome databases and data availability to conduct comparative studies to identify the clade associated with colorectal cancer.
  • Culturing (15:30): These are methods used to grow bacteria.  Here the culture requirements were used to identify niche enriched genes and subspecies.
  • Epigenetic Methylome Analysis (18:50): Methylated DNA is a marker of repressed gene expression, which can be identified and quantified by examining genomes using this method.  Here, the researchers characterized the methylated sites in the genome.
  • Anvi’o Analysis (20:32): This is a tool to examine -omics data.  Here the researchers used it to analyze 135 genomes.  It is beneficial for this use because it optimizes viewing the pangenome of microbes.
  • Principal Component Analysis (PCA) (26:41): This is a clustering methods used in statistics.  Here, the researchers used it to show the Fusobacterium clade 2 strains (FNA clade 2) is associated with colorectal cancer compared to FNA clade 1 strains.
  • Mouse Experiments (30:27): Mice are often used as models for studying biological processes.  Here, the researchers used a mouse model of colitis.
  • Stool Metagenomics (33:20): This is an analysis using sequenced genomes from stool using next generation sequence data from publicly available datasets.  It was used to compare the Fusobacterium in stool of humans with and without colorectal cancer.

4.2. Main Paper

  • Insertion Sequencing (In-Seq) (47:32–48:40): A method used to determine the location of transposon mutants in a microbial community.
  • In-frame Deletion (49:56): A frameshift deletion is a mutation that causes a deletion in a DNA sequence that could shift the way a sequence is read.

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

5.1. Snippet Paper

  • Microbiome (6:42): Description of Fusobacterium nucleatum normally present in the human oral cavity and GI tract of normal healthy individuals and is enriched in those with colorectal cancer.
  • Clade versus Strain (9:55–12:30): Definition of a group of organisms that evolve from a recent common ancestor versus strains which are bacteria evolved from the same species but diverging. A clade can encompass multiple strains.
  • Pathogenicity (14:27): Generates the pathology of colorectal cancer.
  • Commensalism (15:55): Fusobacterium is an oral patho-biont (it’s a commensal until it is not). Example given–H. pylori.
  • Virulence factors (26:16): How Fusobacterium has virulence factors that enable it to cause colorectal cancer, such as bone resorption.
  • Pangenome (21:00): Identify all genes in species being analyzed/compared and discerns between genes conserved among most members (core genome) and those that are ‘extra’ or accessory genes (genome)

5.2. Main Paper

  • Cell Wall Structure (45:21): Bacillus cereus is a gram positive model rhizosphere firmicute. It has 50 cell layers thick of peptidoglycan.
  • Biofilms (48:05; 51:28–53:40): Biofilms form and  is a survival strategy for bacteria to live in areas where there is competition for resources. Discussed as “zorbs”.
  • Type IX Secretory System (51:42): Flavobacteria use this as part of their gliding motility.
  • Colony Expansion (54:15  -55:15): Spread an organism then stab in a ‘challenger’, sometimes the challenger will expand ‘like crazy’.
  • Sociomicrobiology (55:42): Interaction among members of THOR–fitness, expansion of colonies, attachment.

6. Podcast Questions

  1. A clade is ______.
    1. A group of organisms that share a common habitat and interact with one another.
    2. All descendants of a common ancestor, regardless of evolutionary history.
    3. A group of species with similar physical characteristics regardless of origin.
    4. A classification based on the superficial similarities between organisms.
  2. What happens to accessory genome size as the number of Fusobacterium genome sequences increase?
    1. They increase.
    2. They decrease.
    3. They remain the same.
    4. They deteriorate.
  3. The data from the mouse model support that Fusobacterium infection increases adenoma formation.  Clade 1-infected mice showed: five mice had one adenoma, and two mice had two adenomas, and one mouse had three adenomas.  Clade 2-infected mice showed: five mice has three adenomas, one mouse had six adenomas, and one mouse had eight adenomas. The control treated mice had no adenomas.  What would you predict for mice infected a combination of both clade 1 and clade 2 Fusobacteria?
    1. You would expect fewer adenomas than either clade 1 or clade 2.
    2. You would expect as many adenomas as the clade 2-infected mice.
    3. You would expect more adenomas than the clade 2-infected mice.
    4. You would expect more adenomas than clade 1, but not more than clade 2.
  4. What is a biofilm?
    1. The lab environment built to mimic a natural environment.
    2. A complex co-existing, stable, structured bacterial community.
    3. A group of nonrelated species coexisting in an environment.
    4. An environmental structure where a single species lives.
  5. Which species are present in the THOR community? [pick all that apply]
    1. Bacillus cereus
    2. Flavobacterium johnsoniae 
    3. Rhizobium leguminosarum
    4. Pseudomonas koreensis
    5. Niallia circulans
  6. A concept called emergent properties was discussed in the podcast, but not named explicitly.  It is when new characteristics or abilities are observed when multiple species are able to interact.  You co-culture two species and find a new and unique polysaccharide is produced.  To test whether this new characteristic you found in the co-culture requires physical contact between two microbes or was due to an excreted metabolite you grow each species in a liquid media, remove the bacteria cells, and grow each in “the other’s used media.”  What would you expect to see if a metabolite was responsible for the new characteristic?
    1. Neither species will grow or produce the polysaccharide.
    2. Neither of the species will produce the polysaccharide.
    3. Each species will produce a new and different polysaccharide.
    4. One or both of the species will produce the polysaccharide.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1ABD) and one from the main paper (Figure 6EF).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features of schematics, bar charts, and box plots.
  • Analyze microbial metagenomic data to make conclusions about the abundance/enrichment of species from particular niches.
  • Analyze microbial metagenomic data to identify the gene clusters of interest for further study.
Experimental Background (Zepeda-Rivera et al., Figure 1ABD)

Fusobacterium nucleatum (Fn) are bacterial strains that are mainly found in the human oral cavity in healthy individuals.  They are also prevalent in colorectal cancer (CRC) tumor sites, suggesting that features either specific to the environment (oral vs tumor) or specific to the strain (genomic composition) might contribute to CRC.  To investigate this, Zepeda-Rivers et al. (2024) isolated oral cavity Fusobacteria species from healthy individuals and tumor-associated Fusobacteria species from CRC patients (panel a).  These researchers also investigated the number of different bacterial strains found in each location.  They were looking for differences in abundance per originating location (niche), so the researchers quantified the genomes/species present for each of the six Fusobacterim species (panel b).  Following this, they looked at how the genomes of these strains of each of six Fusobacterim species are different, so they completed an analysis to identify and quantify regions of the genome (gene clusters) that were enriched in each niche (gut vs oral), that is the abundance of CRC gene clusters and Oral gene clusters for each environment/niche (panel d).

A schematic of two patients, one presumed healthy and one with CRC. A bar chart of Fusobacterium in genomes, and a box plot.
Figure 1. “Fn niche features. a, A schematic of Fusobacterium strain collection (n = 146) and the sequencing strategy for unique strains. SMRT, single-molecule real-time sequencing. b, A column graph depicting the proportion of Fusobacterium genomes, subset by species, within the CRC (orange) and oral (blue) niches. The inset shows all non-Fn species of Fusobacterium (Fnec, F. necrophorum; Fu, F. ulcerans; Fp, F. pseudoperiodonticum; Fc, F. canifelinum; Fv, F. varium). c, The composition of the Fn pangenome subset by niche. Anvi’o21 gene cluster (GC) prevalence was used to define core (≥95%), accessory (≥5% and <95%) and rare (<5%) features conserved in both CRC-associated and oral-associated strains (collection core, ≥95% in all strains within the collection; collection cloud, ≥5% and <95% in all strains within the collection; collection rare, <5% in all strains within the collection). Disparate features are those that do not fall into any of the other noted bins. d, The proportion of niche-enriched gene clusters across CRC-associated and oral-associated Fn genomes. The plot box shows the 25th percentile, median and 75th percentile. The plot whiskers indicate the minima and maxima. e, KofamKOALA KEGG orthologue analysis 27 of niche-enriched gene clusters.” (Zepeda_Rivera et al., cropped to include panels a-d of image and figure legend).

7.1.2. Questions

  1. How many strains were isolated from CRC patients and what color is used to designate data for these strains?
    1. 28; orange
    2. 59; blue
    3. 65; orange
    4. 81; blue
  2. Which species of Fusobacterium is the most abundant of those isolated from CRC patients’ intestines?
    1. F. varium
    2. F. ulcerans
    3. F. necrophorum
    4. F. nucleatum
  3. Which species of Fusobacterium is the most abundant of those isolated from healthy individuals’ oral cavities?
    1. F. necrophorum
    2. F. nucleatum
    3. F. varium
    4. F. ulcerans
  4. The proportion of niche-enriched gene clusters was quantified in the different strains and the data are compared using box plots (panel d). Match the box plot feature with its description. For help on boxplots, you can go here: Atlassian Box Plot Guide

1 = mean; 2 = median; 3 = outlier; 4 = interquartile range; 5 = individual measures; 6= full data range excluding outliers

    1. ________ Box
    2. ________ Horizontal line in box
    3. ________ Whiskers
    4. ________ Symbols, such as circles
  1. Compare the CRC-enriched gene clusters present in strains originating in the CRC patients and in the healthy oral cavity. What does this comparison suggest?
    1. They are much more prevalent in CRC strains than oral cavity strains suggesting they are a good place to look for genes affecting tumor formation.
    2. They are equally distributed in CRC strains and oral cavity strains suggesting they are unlikely to be involved in genes affecting tumor formation.
    3. They are more abundant in oral cavity strains than CRC strains suggesting they may help protect the host from genes affecting tumor formation.
    4. They are present in both CRC and oral cavity strains suggesting they serve a general role not related to genes affecting tumor formation.
  2. Compare the oral-enriched gene clusters present in strains originating in the CRC patients and in the healthy oral cavity. What does this comparison suggest?
    1. They are equally distributed in CRC strains and oral cavity strains suggesting they are unlikely to be involved in genes affecting tumor formation.
    2. They are more abundant in CRC strains than oral cavity strains suggesting they may help protect the host from genes affecting tumor formation.
    3. They are much less prevalent in CRC strains than oral cavity strains suggesting they are also good place to look for genes affecting tumor formation
    4. They are present in both CRC and oral cavity strains suggesting they serve a general role not related to genes affecting tumor formation.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  •  Identify key features of line graphs and bar charts.
  • Read line graphs to identify conditions that promote biofilm formation.
  • Analyze biofilm data to determine how different bacterial species influence biofilm formation and maintenance.
  • Evaluate time-course biofilm data to determine when and under which conditions bacteria form the most exopolysaccharide.
Experimental Background (Lozano et al., Figure 6EF)

A biofilm is a community of microorganisms bound by an exopolysaccharide matrix that the bacteria living in this community produce collectively. Biofilm communities are important in agricultural, environmental, and clinical settings because microbial communities are involved in many biological processes including nitrogen fixation, gut microbiome interactions with hosts,  and the development of microbial antibiotic resistance.  Biofilm environments provide opportunities for microbes to produce compounds or activate biological pathways in co-culture that would not normally happen when the participating microbes are cultured separately.  In addition, to producing emergent properties, this cooperative lifestyle allows them to adapt over time. In this study, Lozano et al. investigate the validity of a biofilm model with three members, one from each of the three major phyla in plant root and human gut microbiomes. The species are: Bacillus cereus, Flavobacterium johnsoniae, and Pseudomonas koreensis.  Their focus for this study is P. koreensis, so they first wanted to examine the impact of each of the other two species on the growth of P. koreensis.  To do this, they grew P. koreensis alone, co-cultured P. koreensis with each of the other two species, and polyc-cultured all three together.  They quantified biofilm growth by optical density measures combined with staining procedures (crystal violet; panel E).  To determine the impact of monoculture, co-culture, and poly-culture they calculated the proportional biofilm growth of each culture condition compared to monoculture (panel F).  Because biofilms take time to develop, they sampled the monoculture, co-culture (2 participants) and polyculture (3 participants) for 12, 18, 24, and 36 hours (X axis of Figure 6, Panel F).   They stained the resulting biofilms with crystal violet, dissolved the crystal violet, and measured the optical density of the resulting solution (higher numbers indicate more stain, therefore more biofilm that was formed).  Since the research questions was “how does co-culture influence biofilm formation, the research team reported the proportion of biofilm in co-culture compared to biofilm made when P. koreensis  was a monoculture (Y axis of Figure 6, Panel F).

Two charts, one line chart and one bar chart, showing biofilm over time.
Figure 6. “Biofilm formation by rhizosphere isolates. Biofilm was quantified by measuring the optical density at 595 nm (OD595) after staining with crystal violet. (E) Biofilm formation by P. koreensis CI12 growing alone, in coculture with either F. johnsoniae CI04 or B. cereus UW85, and in triple culture at 12, 18, 24, and 36 h. (F) P. koreensis CI12 biofilm production when grown with two other isolates normalized against P. koreensis growth in pure culture. *, P < 0.01. Colored bars under the x axis indicate phylogenetic groups as in Fig. 1. Gray dotted line, limit of detection.”(Lozano et al 2019, cropped image and legend to include only panels E and F)

7.2.2. Questions

  1. What color dots and bars represent the data for the poly-culture (all three species grown together)?
    1. blue
    2. red
    3. orange
    4. purple
  2. When grown alone, at what point did P. koreensis reach maximum biofilm formation (panel E)?
    1. 12 hours
    2. 18 hours
    3. 24 hours
    4. 36 hours
  3. What effect did adding B. cereus to P. koreensis have on biofilm production (panel E)?
    1. It reduced biofilm formation and increased dissociation rate.
    2. It had no effect on biofilm formation or disassociation rate.
    3. It increased biofilm formation and reduced the rate of dissociation.
    4. It inhibited biofilm formation and increased the rate of dissociation.
  4. What effect did adding F. johnsoniae to P. koreensis have on biofilm production (panel E)?
    1. It increased biofilm formation and reduced the rate of dissociation.
    2. It inhibited biofilm formation and increased the rate of dissociation.
    3. It had no effect on biofilm formation or on biofilm dissociation rate.
    4. It reduced biofilm formation and increased dissociation rate.
  5. What was the outcome when P. koreensis was co-cultured with both B. cereus and F. johnsoniae (panel E)?
    1. It inhibited biofilm formation and increased the rate of dissociation.
    2. It had no effect on biofilm formation or on biofilm dissociation rate.
    3. It reduced biofilm formation and increased dissociation rate.
    4. It increased biofilm formation and reduced the rate of dissociation.
  6. Since biofilm formation (production and dissociation) differs over time, the authors normalized the data by calculating a relative proportion of biofilm growth.  They did this by dividing the co-culture biofilm by the P. koreensis biofilm growth.   What do these data suggest is the time point where the bacteria are most helpful to each other in biofilm formation, and which culture?
    1. 24 hours; culture of all three species
    2. 24 hours; culture with F. johnsoniae
    3. 36 hours; culture of all three species
    4. 36 hours; culture with F. johnsoniae
  7. Based these data, were the authors successful in generating a biofilm model they could use for further biofilm studies?
    1. Yes, because co-culture benefitted biofilm production and maintenance.
    2. No, because there were no combinations that increased biofilm production.
    3. No, co-culture reduces biofilm production due to competition between species.
    4. Yes, co-culture has an effect on biofilm production, but only past 36 hrs of incubation.

8. Paper Information and Licensing

8.1. Snippet paper

  • Zepeda-Rivera M, Minot SS, Bouzek H, Wu H, Blanco-Míguez A, Manghi P, Jones DS, LaCourse KD, Wu Y, McMahon EF, Park SN, Lim YK, Kempchinsky AG, Willis AD, Cotton SL, Yost SC, Sicinska E, Kook JK, Dewhirst FE, Segata N, Bullman S, Johnston CD. 2024. A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche. Nature. 628(8007):424-432. doi: 10.1038/s41586-024-07182-w
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journal’s web page.

8.2. Main paper

  • Lozano GL, Bravo JI, Garavito Diago MF, Park HB, Hurley A, Peterson SB, Stabb EV, Crawford JM, Broderick NA, Handelsman J. 2019. Introducing THOR, a Model Microbiome for Genetic Dissection of Community Behavior. mBio. 10(2). doi: 10.1128/mBio.02846-18.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journal’s web page.

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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