TWiM #242: Sourdough Starter Microbiome

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:45 minutes

“Humans have relied on sourdough starter microbial communities to make leavened bread for thousands of years, but only a small fraction of global sourdough biodiversity has been characterized. Working with a community-scientist network of bread bakers, we determined the microbial diversity of 500 sourdough starters from four continents. In sharp contrast with widespread assumptions, we found little evidence for biogeographic patterns in starter communities. Strong co-occurrence patterns observed in situ and recreated in vitro demonstrate that microbial interactions shape sourdough community structure. Variation in dough rise rates and aromas were largely explained by acetic acid bacteria, a mostly overlooked group of sourdough microbes. Our study reveals the extent of microbial diversity in an ancient, fermented food across diverse cultural and geographic backgrounds.” (Landis et al. 2021).

1.2. Main paper; discussion starts at 23:37 minutes

“Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle-like compartment in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryo-electron microscopy, we have determined the structure of the nanocompartment complex to 2.2 Å resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.” (Nichols et al. 2021).

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

3.  Potential Learning Objectives for the Podcast

¹ Papers: Snippet (S) or Main (M)

²Learning 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

• Ribosomal DNA Sequencing (rDNA) (10:25–11:34): Ribosomal DNA sequencing is a technique that can be used to identify a species and also to investigate its evolutionary history. Since bacteria often undergo horizontal gene transfer, using the seldom-changing rDNA is a key way to identify different species of bacteria present in a heterogeneous population.
• Microbial Culturing (15:47- 17:19): Culturing bacteria can allow for the examination of one or a few species. The researchers used this to demonstrate how many bacteria rely on each other for growth and development (which is especially true with primary and secondary fermenters). Combining the cultures with yeast mimicked that of the bread.

4.2. Main Paper

• Cyro-Electron Microscopy (Cryo-EM) (24:22–29:20): This is a technique for imaging 3D structures and works well for very small structures that typical microscopy would not be able to detect, such as prokaryote compartments. It involves cryogenic freezing of samples followed by staining and microscopy.

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

5.1. Snippet Paper

• Fermentation (18:31–19:33): Many fermentation byproducts are mentioned. Many bacteria have unique fermentation products and processes that they undertake to break down available energy sources. Furthermore, other bacteria take the products of an existing fermentation to break it down further and even work with other microbes to synergize and couple reactions.
• Horizontal Gene Transfer (10:25–11:34): Mechanisms that allow microorganisms from diverse backgrounds to acquire the same gene, which necessitates the use of rDNA sequencing to determine different species within a population.

5.2. Main Paper

• Cytoplasmic Contents (24:22–29:20): The authors focused on the carboxysomes and other internal structures and their similarity to phage heads. Typically, prokaryotes are not associated with internal structures but many do have them across evolutionary lines, mainly to create new environments to optimize reaction conditions.
• Oxidation Reduction Interactions (30:25–33:18): Oxidation-reduction reactions are the key reaction type that lets cells transform and use energy. Often, these reactions need specific conditions, which nano compartments can create. The researchers studied oxidation-reduction reactions involving sulfur removal from amino acids.

6. Podcast Questions

1. Which group of microorganisms were found in the sourdough cultures? Pick all that apply.
   1. Yeast
   2. Propionic acid bacteria
   3. Lactic acid bacteria
   4. Acetic acid bacteria
2. What characteristics of ribosomal RNA genes make them useful for species identification? Pick all that apply.
   1. These genes diverge quickly and distinctly.
   2. These genes do not undergo horizontal gene transfer.
   3. These genes change are common and change infrequently.
   4. These genes are not found in eukaryotic microbes.
3. What was one major function of acetic acid bacteria discussed in the context of sourdough starters?
   1. Producing gluten proteins that help dough rise
   2. Providing yeast with nutrients for growth
   3. Generating volatile compounds that influence flavor
   4. Protecting against pathogens in sourdough environments
4. Based on the podcasters’ discussion, if you identified a sourdough culture with a high abundance of Lactobacillus sanfranciscensis, which of the following would likely be true?
   1. Bread made from this culture will have a classic flavor.
   2. Bread made from this culture will have a high rise.
   3. The sourdough culture itself is an older culture.
   4. The sourdough culture is likely from Europe.
5. A nanocompartment is ________.
   1. a space between the inner and outer membranes in bacteria
   2. a cargo vesicle for delivery of nanomaterials in bioindustry
   3. a sulfur-containing particle with a spiky exterior to capture copper
   4. an organelle-like structure with a specialized function in bacteria
6. What did the electron microscopy and similarity studies reveal about the nanocompartment?
   1. The protein shell has features in common with capsid proteins of phages.
   2. The protein shell captures and sequesters all metal ions, including copper.
   3. The protein shell was unable to be degraded by extreme heat or acid.
   4. They appear to be structurally distinct from other encapsulins.
   5. Homologs of the shell protein are also found in mycobacteria pathogens.
7. If a new nanocompartment is found that functions in nitrogen metabolism rather than sulfur metabolism, what would likely be true about its structure based on the findings in the main paper’s discussion?
   1. It would contain identical structural proteins to sulfur nanocompartments.
   2. Its structure would likely vary to support unique enzymatic processes.
   3. It would rely on eukaryotic cellular machinery related to nitrogen metabolism.
   4. It would encapsulate enzymes involved in carbon metabolism as well.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 4) and one from the main paper (Figure 7).

7.1. First Figure Reading Exercise

Experimental Background (Landis et al., Figure 4)

Sourdough bread is produced by fermentation with a mixed culture of bacteria and yeast. These mixed cultures are commonly kept and grown by individual bakers, so they vary extensively. Landis et al. (2021) were interested in characterizing the microbial diversity and functional characteristics of sourdough cultures across the world. They assessed over 500 sourdough cultures for a variety of characteristics including identity of species present and two baking-relevant characteristics: 1) volatile organic compounds (VOC; which influence aroma and flavor) and 2) leavening (which influences dough rise and bread structure). The data for 40 of the cultures were visualized using a heatmap to show the distribution of acetic acid bacteria, aroma type, and rise time in the two large groups, which are a result of clustering the overall VOC profiles.

For this visualization, each column has data for a specific sourdough culture and each row is the level of a culture’s characteristic. The four horizontal bars at the top of the image have individual color keys (top left of image) and identify: 1) the acetic acid bacteria percentages in the culture (AAB%), 2) the dough rise rate, 3) a score of how much principal component 1 explains the variability in VOC (VOC NMDS1), and 4) the dominant sensory note (culture aroma). Each of the following rows indicate the relative level (color key at top right) of the specific volatile compound identified at the right of the image. The tree-like structure at the very top of the image shows how the data from the sourdough cultures clustered into two groups according to similarities in their VOC analyses. Let’s call them Group A (left) and Group B (right).

7.1.2. Questions

1. Using the color key for dough rise rate, what would be the color of the heatmap square for a sourdough culture with the fastest rise rate?
   1. blue
   2. green
   3. yellow
   4. white
2. Using the color key for mean acetic acid-producing bacteria percentage, what would be the color of the heatmap square for a sourdough culture with the lowest percentage of acetic acid bacteria?
   1. Black
   2. Dark gray
   3. Light gray
   4. Pale gray
3. Using the color key for VOC, what would be the color of the heatmap square for a sourdough culture that has extremely low 1-hexanol abundance compared to other cultures?
   1. Dark blue
   2. Pale blue
   3. Yellow
   4. Light red/orange
   5. Dark red/orange
4. What dominant sensory note is found for the culture with the highest dough rise rate?
   1. Acetic acid vinegar
   2. Yeasty
   3. Fermented sour
   4. Green apple
5. In which “tree cluster” (Group A or Group B), are acetic acid-producing bacteria most prevalent?
   1. Group A
   2. Group B
   3. They are equally prevalent in both clusters.
   4. They are not prevalently found in either cluster.
6. Match the dominant sensory note (aroma) with the appropriate cluster/group statement (A = exclusive to Group A; B = exclusive to Group B; C = found in both Groups)
   1. _______ Acetic acid vinegar
   2. _______ Yeasty
   3. _______ Fermented sour
   4. _______ Green apple
7. The sourdough cultures with the most similar VOC profiles to each other (Group B) are consistently lacking in which VOC type?
   1. Acetates
   2. Butanones
   3. Heptanes
   4. Alcohols
8. If a culture was shown to have a high AAB percentage, a small rise rate, and a yeasty aroma, which group would it likely be placed in?
   1. Group A
   2. Group B
   3. I can’t tell from these data.

7.2. Second Figure Reading Exercise

Experimental Background (Nichols et al., Figure 7)

A new and interesting feature of prokaryotic studies is the discovery of nanocompartments or encapsulins, organelle-like structures that have specialized functions. They generally are encoded by two genes, one encodes a shell protein and the other encodes its cargo protein. Nanocompartments provide highly specialized metabolism to the species in which they are found, such as iron storage. Nichols et al. (2021) identified a new type of nanocompartment in cyanobacterium Synechococcus elongatus (S. elongatus) that has the enzyme cysteine desulfurase (CyD) as its cargo. In their characterization they were interested in determining whether encapsulation affects enzyme activity, so they compared enzyme activity for encapsulated CyD, CyD alone, CyD lacking its amino terminal domain (ΔNTD-CyD), and an empty nanocompartment. The enzymatic activity for the cysteine desulfurase was quantified across a range of substrate concentrations using a couple fluorescent system and the final measure is reported here as nmol/minute.

7.2.2. Questions

1. What is the highest enzyme activity found in this experiment, and which enzyme version is this?
   1. 55 nmol per minute; ΔNTD-CyD
   2. 105 nmol per minute; CyD alone
   3. 145 nmol per minute; encapsulated CyD
   4. 210 nmol per minute; empty compartment
2. What does the difference in enzyme activity for the encapsulated CyD protein compared to enzyme activity for the non-encapsulated CyD protein suggest?
   1. Encapsulation hinders enzyme activity.
   2. Encapsulation enhances enzyme activity.
   3. Cysteine concentration has no impact on CyD.
   4. Empty nanocompartments increase enzyme activity.
3. What does the difference in enzyme activity for the non-encapsulated CyD protein compared to enzyme activity for the non-encapuslated CyD lacking the amino-terminal end of the protein (ΔNTD-CyD) suggest? The amino terminal portion of the protein _________.
   1. regulates encapsulation and desulfurase activity.
   2. Is not involved in suppressing desulfurase activity.
   3. has structural elements for enzymatic function.
   4. Is a loss of function mutant found in nature.
4. What conclusion can be drawn from the level of enzyme activity observed for the empty nanocompartment? Empty nanocompartments ___________
   1. have very low enzyme activity.
   2. have moderate enzyme activity..
   3. have significant enzyme activity.
   4. were not measured for enzyme activity.
5. What about these results might be of interest to industrial enzyme producers? These results suggest ______
   1. enzymes are more stable when encapsulated.
   2. enzymatic activity can be enhanced by encapsulation.
   3. encapsulation can be used to increase protein recovery.
   4. nanocompartments can be used as protein factories.

8. Paper Information and Licensing

8.1. Snippet paper

• Landis EA, Oliverio AM, McKenney EA, Nichols LM, Kfoury N, Biango-Daniels M, Shell LK, Madden AA, Shapiro L, Sakunala S, et al. 2021. The diversity and function of sourdough starter microbiomes. Elife. 10:e61644. doi: 10.7554/eLife.61644.
• 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://elifesciences.org/articles/61644#copyright

8.2. Main paper

• Nichols RJ, LaFrance B, Phillips NR, Radford DR, Oltrogge LM, Valentin-Alvarado LE, Bischoff AJ, Nogales E, Savage DF. 2021. Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism. Elife. 10:e59288. doi: 10.7554/eLife.59288.
• 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://elifesciences.org/articles/59288#copyright