Information Flow and Genetics

TWiM #304: A New Blue Cheese-Making Fungus

Podcast and Annotation Information
  • Annotation by Leia Baek, Savanneh Salmon, Ananya Venkatachala, and Michaela Gazdik Stofer
  • Podcast audio by TWiM: Listen to TWiM #304 Podcast
  • Podcast transcript by Grace Hagedorn and Laurel Thompson: Access Podcast Transcripts
  • Papers Discussed:
    • Crequer E, Ropars J, Jany J, Caron T, Coton M, Alodie Snirc, Jean‐Philippe Vernadet, Branca A, Giraud T, Coton E. 2023. A new cheese population in Penicillium roqueforti and adaptation of the five populations to their ecological niche. Evolutionary Applications. 16(8):1438–1457. doi: 10.1111/eva.13578
    • Heffernan JR, Wildenthal JA, Tran H, Katumba GL, McCoy WH, Henderson JP. 2024 Yersiniabactin is a quorum-sensing autoinducer and siderophore in uropathogenic Escherichia coli. mBio 15:e00277-23. doi: 10.1128/mbio.00277-23

1. Paper Abstracts

1.1. Snippet paper discussion starts at 2:04 minutes

The Most Interesting Things (according to students)

  • The description of how cheese is made, where you start off with pasteurized milk and then go through several processes including acidify, curd formation, cutting, draining, salting, and then aging the cheese where the flavor and texture depends on the time, temperature, and humidity.
  • Microbial diversity plays such a big role in the cheese-making process. There is complex interplay between various microorganisms including bacteria, yeasts, molds, and fungi, creating different  environments for metabolic processes to take place. This  contributes to the unique characteristics of each cheese variety.

“Domestication is an excellent case study for understanding adaptation and multiple fungal lineages have been domesticated for fermenting food products. Studying domestication in fungi has thus both fundamental and applied interest. Genomic studies have revealed the existence of four populations within the blue-cheese-making fungus Penicillium roqueforti. The two cheese populations show footprints of domestication, but the adaptation of the two non-cheese populations to their ecological niches (i.e., silage/spoiled food and lumber/spoiled food) has not been investigated yet. Here, we reveal the existence of a new P. roqueforti population, specific to French Termignon cheeses, produced using small-scale traditional practices, with spontaneous blue mould colonisation. This Termignon population is genetically differentiated from the four previously identified populations, providing a novel source of genetic diversity for cheese making. The Termignon population indeed displayed substantial genetic diversity, both mating types, horizontally transferred regions previously detected in the non-Roquefort population, and intermediate phenotypes between cheese and non-cheese populations. Phenotypically, the non-Roquefort cheese population was the most differentiated, with specific traits beneficial for cheese making, in particular higher tolerance to salt, to acidic pH and to lactic acid. Our results support the view that this clonal population, used for many cheese types in multiple countries, is a domesticated lineage on which humans exerted strong selection. The lumber/spoiled food and silage/spoiled food populations were not more tolerant to crop fungicides but showed faster growth in various carbon sources (e.g., dextrose, pectin, sucrose, xylose and/or lactose), which can be beneficial in their ecological niches. Such contrasted phenotypes between P. roqueforti populations, with beneficial traits for cheese-making in the cheese populations and enhanced ability to metabolise sugars in the lumber/spoiled food population, support the inference of domestication in cheese fungi and more generally of adaptation to anthropized environments.” (Crequer et al. 2023)

1.2. Main paper discussion starts at 22:28 minutes

The Most Interesting Things (according to students)

  • Iron is really important to bacteria and siderophore systems allow them to scavenge iron from the environment both for themselves and for other microbes in the population. Clinical strains of E. coli that cause UTIs had different siderophores than non-pathogenic strains. Their ability to recruit iron was important for pathogenesis.
  • Bacteria are able to measure the number of cells in a population through quorum sensing so they don’t waste resources and energy making products that are not needed yet. Siderophore production was controlled by quorum sensing so they were only produced when there were more cells in the population.

“Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.” (Heffernan et al. 2024)

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

Snippet Main
Vision and Change Topics
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics  (V&C_IFG)
  • Evolution  (V&C_E)
  • Information Flow and Genetics (V&C_IFG)
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 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 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analyses, molecular methods, and bioinformatic tools.
  • Fundamental Statement  13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the conclusions of the Penicillium roqueforti phylogenetic analysis.
  • Recall differences among Penicillium roqueforti strains, particularly in terms of genetic recombination and growth characteristics.
 S L
  • Use these data to make predictions about diversity of other food-related fungi.
 S H
  • Identify the function of the yersinisabactin siderophore.
  • Recall how quorum sensing impacts yersinisabactin siderophore production in E. coli.
  • Recall the potential benefits and costs of siderophore production for E.coli.
 M L
  • Hypothesize the benefits of using potential strategies for limiting siderophore function as a therapeutic intervention against urinary tract infections (UTIs).
 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

  • Whole Genome Sequencing and Phylogenetic Analysis (6:32–10:13): Whole genome sequencing is a method, usually next generation sequencing based, that determines the DNA sequence of a whole genome using small reads that are then used to piece together a whole genome (done by computational methods).  A way of comparing whole genomes to see how they are like and different to infer the taxonomy/relatedness of species is phylogenetic analysis, again using computation and statistical methods.  Here, nucleotide sequence differences between Penicillium genomes was used to estimate their evolutionary relatedness.
  • Microbial Growth Assays (10:23–12:57): These are assays to determine differences in growth rates.  Here, they were used to compare growth of the Penicillium roqueforti strains in a variety of environmental conditions such as varying pH, temperature, and carbon source.

4.2. Main Paper

  • Bacterial Growth Assays (41:30–42:30): These are assays to determine differences in growth rates.  Typically, they involve optical density measurements of liquid bacterial cultures.  The higher the OD, the more cells are present.  Here, the assays were used to compare growth differences of wild-type and mutant strains of E. coli in the presence and absence of iron and various siderophores.
  • Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (45:51–46:30): RT-PCR is an assay to measure the levels of specific mRNAs.  This is done by extracting RNA, converting it to DNA using reverse transcriptase, and then measuring the amount of DNA through PCR amplification. Here, it was used to quantify the level of RNAs encoded by siderophore genes.

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

5.1. Snippet Paper

  • Genomics (4:35–18:20): Phylogenic analysis of Penicillium roqueforti based on whole genome sequence analysis showed key differences in genome structure and the amount of horizontal gene transfer in wild vs commercial strains.
  • Effects of Heat, Salt, and pH on Microbes (10:23–12:57): Strains of Penicillium roqueforti showed different growth rates in different concentrations of salt and at different pH’s. These differences relate to if they are wildtype or commercial strains.
  • Microbial Adaptation (11:28–17:05): Penicillium roqueforti  strains adapt to various food sources and the microbial populations evolve based on available nutrients, showcasing the significance of their metabolic pathways.
  • Horizontal Gene Transfer (General) (12:58–14:30): Regions of the genomes of wild-type strains were obtained through horizontal gene transfer, which is seen in their phylogenetic relatedness.

5.2. Main Paper

  • Quorum Sensing (29:11–32:45; 42:47–51:39): Regulatory mechanism in which microbial cells release and detect signaling molecules, or autoinducers, which accumulate as population density increases. In this context, E. coli uses quorum sensing to regulate production of the siderophore yersiniabactin as cell density and iron demand increases. Siderophores are high-affinity iron-chelating molecules secreted by bacteria to acquire iron, which is often limited within host environments.
  • Phenotypic Variation (37:12–39:15): Different strains of E. coli encode different siderophore systems. These produce phenotypic variation between clinically relevant strains ascertain siderophore systems were identified more often in clinical urinary tract infection (UTI) strains.
  • Microbial Evolution and Mechanisms of Pathogenesis (39:24–43:11): E. coli strains with higher pathogenic potential take on the additional cost of synthesizing additional siderophores which may provide an evolutionary benefit to balance the energy cost.
  • Regulation of Gene Expression (45:51–46:30): The regulation of yersiniabactin production was examined in different environments. It’s transcription increased in correlation with E.coli population size and during growth in urine, mimicking a UTI infection.

6. Podcast Questions

  1. What does the presence of a “web-like” pattern in the phylogenetic tree of Termignon cheese strains indicate?
    1. A lack of genetic diversity within the strain.
    2. A high degree of genetic recombination and gene flow.
    3. Recent divergence from a common ancestor.
    4. The presence of harmful mutations in the strain.
  2. Based on the phylogenetic analysis, which of the following statements is/are true about the relationship between different Penicillium roqueforti lineages? [pick all that apply]
    1. Strains isolated from lumber and spoiled food are most closely related to commercial cheese strains.
    2. The Termignon cheese strains exhibit greater genetic diversity compared to commercial strains.
    3. All Penicillium roqueforti strains belong to a single, closely related lineage regardless of niche area.
    4. Silage-derived strains are genetically distinct from all other lineages, but particularly from cheese strains.
  3. How did the growth characteristics of Penicillium roqueforti strains isolated from silage and lumber differ from those isolated from cheese?
    1. Silage/lumber strains grew better on plant sugars.
    2. Silage/lumber strains were more tolerant of acidic conditions.
    3. Silage/lumber strains exhibited faster growth rates overall.
    4. Silage/lumber strains were more susceptible to salt.
  4. Based on these studies, would you expect to find if you compared the genetic diversity between and among commercially-available sourdough cultures and naturally-fermented sourdough cultures? What is your reasoning?
    1. The commercial strains will be more different to each other and natural strains will be more similar to each other and to commercial strains.
    2. The commercial strains will be diverse and natural strains will be diverse; each group will show similar variation within their group.
    3. The commercial strains will be more different to each other and natural strains will be more similar to commercial strains than to each other.
    4. The commercial strains will be more similar to each other and natural strains will be more different to each other and to commercial strains.
  5. What is the primary function of the siderophore yersinisabactin in E.coli during a urinary tract infection?
    1. It assists in the synthesis of siderophores to promote cellular growth in nutrient-rich environments.
    2. Serves as a quorum sensing molecule that regulates biofilm formation only in low-density populations.
    3. It enables iron uptake in an iron-limited environment, supporting bacterial survival and growth
    4. It facilitates nitrogen fixation to allow E.coli to grow under anaerobic, low nutrient conditions
  6. How does quorum sensing play a role in yersiniabactin production in E. coli?
    1. Quorum sensing allows individual microbes to produce yersiniabactin to scavenge iron and other rare metals for that individual cell.
    2. Quorum sensing allows coordinated gene expression of yersiniabactin, increasing it’s production based on increases in cell density.
    3. Quorum sensing allows the direct transfer of the yersiniabactin gene from one cell to another, helping the overall population to survive.
    4. Quorum sensing allows yersiniabactin to bind to iron more effectively by triggering localized environmental changes at low cell density.
  7. Why might E. coli strains producing yersiniabactin avoid producing other siderophores like enterobactin under certain conditions?
    1. Yersiniabactin may provide a dual function of iron uptake and chelating damaging metals such as copper.
    2. Yersiniabactin is more effective in high-oxygen environments, eliminating the need for other siderophores.
    3. Enterobactin production is inhibited by human immune defenses, making yersiniabactin the preferred option.
    4. Other siderophores require more energy to produce, so production has a very high fitness cost during infection.
  8. How could targeting siderophore function potentially benefit the treatment of urinary tract infections (UTIs) caused by antibiotic-resistant E. coli strains?
    1. Siderophore inhibitors could provide an alternative therapeutic approach when antibiotics are ineffective.
    2. Targeting siderophores would reverse the antibiotic resistance, making the antibiotic a viable option again.
    3. Siderophore inhibitors would prevent horizontal gene transfer of antibiotic resistance genes to sensitive strains.
    4. Siderophore inhibitors would prevent E. coli from colonizing the bladder by inhibiting biofilm formation genes.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1a) and one from the main paper (Figure 9).

7.2. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify the purpose of a phylogenetic analysis, as represented by a phylogenetic tree.
  • Evaluate the data to make conclusions about the most and least genetically diverse groups.
  • Analyze the data to make conclusions about the relationships between and among the five groups of Penicillium roqueforti isolates.
  • Hypothesize why different groups of P. roqueforti show different levels of genetic diversity.
Experimental Background (Crequer et al. Figure 1a)

Microbes are used for many human purposed including agriculture, production of medically-relevant proteins, and food production.  Among these applications, fungi are particularly important in making bread, cheese, and beer. Fungal species that are used in commercial cheese production are considered domesticated strains because they have been repeatedly selected to produce consistent cheese characteristics. Domestication often leads to adaptive divergence where different strains can be highly different from each other. In this study of fungal adaptation, Crequer et al. (2023) were interested in the evolutionary relationships of domesticated and naturally occurring fungi so they used genome sequencing to analyze and compare the genomes of 51 P. roqueforti isolates from five populations: Roquefort cheese strains, non-Roquefort cheese strains, Termignon cheese strains, silage food spoiler strains, and lumber spoiled food strains. Termignon cheese strains are specific to French Termignon cheeses that are produced using traditional practices by a small number of cheese makers in the French Alps. The strains used in these traditional practices come from spontaneous colonization during the cheese making process from spores in the environment. These are not cultivated isolates like those used in commercial cheese production. The data are represented as a phylogenetic tree, which is a diagram that represents evolutionary relatedness predicted by sequence similarity and differences.  An unusual feature in this particular tree is webbing between the branches, which denotes horizontal gene transfer.

Phylogenetic tree of P. roqueforti species
Figure 1. “Representation of the genomic data and the differentiation between the five populations of Penicillium roqueforti: the three cheese populations (Roquefort, non-Roquefort and Termignon) and the two non-cheese populations (silage/food spoiler and lumber/spoiled food). (a) Reticulated network of P. roqueforti strains based on 190,387 single nucleotide polymorphisms, showing the five distinct populations with pictures of their respective environments of collection. The ID of the strains used for phenotyping are in bold…” (Text and image modified from Crequer et al, 2023 to include only panel A).

7.1.2. Questions

  1. What can be identified from analysis of a phylogenetic tree?
    1. The average lifespans of each listed species in relation to one another.
    2. The evolutionary relatedness of the species/isolates analyzed.
    3. Unique phenotypic characteristics associated with each group of fungi.
    4. The number of linked and coordinately expressed traits each family has.
  2. Which of the following groups was the least genetically diverse?
    1. Lumber spoiled food strains
    2. Silage spoiled food strains
    3. Termignon cheese strains
    4. Non-Roquefort strains
  3. Which of the following groups was highly genetically diverse and had high levels of recent genetic recombination and/or horizontal gene transfer?
    1. Lumber spoiled food strains
    2. Silage spoiled food strains
    3. Termignon cheese strains
    4. Non-Roquefort cheese strains
  4. Which of group is most closely related to the Termignon group?
    1. Lumber spoiled food strains
    2. Non-Roquefort cheese strains
    3. Roquefort cheese strains
    4. Silage spoiled food strains
  5. What is a likely explanation for why the Termignon strains are more genetically diverse than the other cheese making strains?
    1. These strains likely have lower rates of mutation than the other strains used by commercial cheese producers.
    2. These strains likely have lower rates of sexual reproduction and recombination compared to the strains used by commercial cheese producers.
    3. These strains do not undergo selective breeding and thus are exposed to a wider range of environmental pressures and diverse environments.
    4. These strains likely have higher growth rates than the other strains used by commercial cheese producers.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in the model schematic, including processes and molecules.
  • Analyze the model processes and processes to match key elements with their functions.
  • Hypothesize how disruptions in the Ybt system would affect cells and iron uptake.
Experimental Background (Heffernan et al., Figure 9)

Urinary tract infections (UTI) are common infections with more than 400 million diagnosed each year. The most common pathogen for UTI is  E. coli which uses acquired siderophore mechanisms to scavenge iron in this nutrient poor environment.  E. coli have a constitutively expressed enterobactin (Ent) siderophore system and clinical UTI isolates commonly also have a genetically distinct yersiniabactin (Ybt) siderophore system. The researchers wanted to determine if these two systems were redundant or distinctive, so they compared the growth and gene expression characteristics of wild-type and mutant strains to create a model of how the Ybt siderophore system worked.  They found the Ybt system is a distinct siderophore system from Ent, and that the Ybt has an integrated quorum sensor system, which is shown in the figure.  Recall that quorum sensing is the process in which bacteria can communicate with each other in a population density dependent manner, typically to regulate gene expression.

Within this diagram, the gray rod shapes represent E. coli cells alone (left side) and in a population (right side). Yersiniabactin (Ybt), siderophore, is represented as blue diamonds. YbtA, the transcription activator protein that turns on transcription of the Ybt biosynthesis genes (ybtSEirp1irp2ybtU; noted as text), is represented by an orange circle with a “cutout.”  Iron is not represented here, but would be present at low levels external to the cell, and could be bound to the Ybt siderophore, including the Ybt that enters the cell. Note that the conformational change of YbtA-Ybt promotes high levels of Ybt biosynthesis gene activation (bold arrows; right panel).

Diagram of E. coli secreting Ybt
Figure 9. “Model of Ybt-mediated QS autoregulation. At low cell density and limited iron availability (left), Ybt biosynthesis is insufficient to match the iron uptake activity of the Ent system. Extracellular Ybt accumulates slowly and autoinduction is minimal. With increasing cell density (right), extracellular Ybt accumulates sufficiently to bind YbtA and stimulate increased Ybt biosynthesis by each cell. This autoinductive cycle rapidly increases extracellular Ybt as cell density increases.” (Heffernan et al 2018, no changes)

7.2.2. Questions

  1. What symbol represents the iron-binding siderophore molecule in this schematic, and what is the name of that molecule?
    1. blue diamond; Ybt
    2. empty orange circle; YbtA
    3. filled orange circle; YbtA+Ybt
    4. gray rod shape; E. coli
  2. What feature in the schematic identifies Ybt as the quorum sensor molecule?
    1. It activates communication within bacterial community increasing cell-cell competition.
    2. It inhibits the production of Ybt by inactivating YbtA, which regulates Ybt biosynthesis.
    3. The concentration of Ybt increases when there are more cells in the local population.
    4. It inhibits the binding of Ybt to iron, reducing iron acquisition in high-density populations.
  3. What feature in the schematic identifies Ybt as an autoinducer, that is a molecule that upregulates its own expression?
    1. Ybt binds to the activator protein, allowing it to activate transcription.
    2. Ybt has a positive feedback loop, causing an increase in its own expression.
    3. E. coli takes in YbtA released from other bacterial species to trigger gene expression.
    4. Ybt is secreted into the environment by E. coli at any cell concentration.
  4. Based on this model, how would a bacterial population that has a mutation that prevents YbtA from binding Ybt be affected?
    1. The bacteria would lose the enhanced ability to sequester iron at a high cell numbers.
    2. The bacteria would produce increased levels of Ybt even at low cell numbers.
    3. The bacteria would produce more YbtA protein to compensate for a loss of function.
    4. The bacteria would produce an increase in Ybt to compensate for the mutation.
  5. You now have a bacterial population that has a mixture of cells, some have a mutation that prevents YbtA from binding Ybt and some are wild-type for all Ybt-related genes. What effect would this have?
    1. The wild-type bacteria that produce increased levels of Ybt would have enhanced iron uptake, but the mutant bacteria would not.
    2. The wild-type bacteria would produce more YbtA protein to compensate for the loss of YbtA activation in the mutant cells.
    3. All bacteria would lose the enhanced ability to sequester iron at a high cell numbers because the mutation is dominant.
    4. The wild-type bacteria would produce a lot of Ybt, so iron uptake would be enhanced at high cell numbers for both cell types.

8. Paper Information and Licensing

8.1. Snippet paper

  • Crequer E, Ropars J, Jany J, Caron T, Coton M, Alodie Snirc, Jean‐Philippe Vernadet, Branca A, Giraud T, Coton E. 2023. A new cheese population in Penicillium roqueforti and adaptation of the five populations to their ecological niche. Evolutionary Applications. 16(8):1438–1457. https://doi.org/10.1111/eva.13578.
  • 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 https://onlinelibrary.wiley.com/doi/10.1111/eva.13578.  

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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