Evolution

TWiM #285: How Plague Got Deadly

Podcast and Annotation Information

  • Annotation by Steven Cramer, Lauren Fritzinger, and Karen P. York, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #286 Podcast
  • Podcast transcript by Otter.ai and edited by Eden Anderson and Laurel Thompson: Access Podcast Transcripts
  • Papers Discussed:
    • Alqurainy N, Miguel-Romero L, Moura de Sousa J, Chen J, Rocha EPC, Fillol-Salom A, Penadés JR. 2023. A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature. Cell Host Microbe 31(1): 69-82. doi: 10.1016/j.chom.2022.12.001 
    • Kolodziejek AM, Bearden SW, Maes S, Montenieri JM, Gage KL, Hovde CJ, Minnich SA. 2023. Yersinia pestis Δail mutants are not susceptible to human complement bactericidal activity in the flea. Appl Environ Microb 89(2): e0124422–e0124422. doi: 10.1128/aem.01244-22

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 2:44 minutes

The Most Interesting Things (according to students)

A new type of satellite virus was described. While typically satellite viruses require the capsid proteins of a helper virus to produce complete infectious viral particles. This new bacteriophage virus encodes all the proteins needed except the tail. It makes a small capsid for its smaller genome, and acquires only the tail proteins from the helper virus.  

“Phage satellites are genetic elements that couple their life cycle to that of helper phages they parasitize, interfering with phage packaging through the production of small capsids, where only satellites are packaged. So far, in all analyzed systems, the satellite-sized capsids are composed of phage proteins. Here, we report that a family of phage-inducible chromosomal islands (PICIs), a type of satellites, encodes all the proteins required for both the production of small-sized capsids and the exclusive packaging of the PICIs into these capsids. Therefore, this new family, named capsid-forming PICIs (cf-PICIs) only requires phage tails to generate PICI particles. Remarkably, the representative of cf-PICIs are produced with no cost from their helper phages, suggesting that the relationship between these elements is not parasitic. Finally, our phylogenomic studies indicate that cf-PICIs are present in both gram-positive and gram-negative bacteria and have evolved at least three times independently to spread in nature.” (Alqurainy et al. 2023, with no changes)

1.2. Main paper; discussion starts at 26:21 minutes

The Most Interesting Things (according to students)

The causative agent of plague, Yersinia pestis, emerged from nonvirulent strains. This paper explored how the virulent strain emerged. It was known that serum complement (innate immunity) from all mammals except mice can kill Y. pestis. This paper showed that sera from humans was bactericidal when tested in vitro, but not bactericidal against Y. pestis when the bacterium was growing in fleas (in vivo). Thus, the flea host provided a protective niche where Y. pestis could grow and new strains could evolve. The podcast also described how Y. pestis biofilm formation of the flea causes fleas to seek more frequent blood meals that promotes the spread of Y. pestis to new mammalian hosts.

“Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescence microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain.” (Kolodziejek 2023, with no changes)

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

Snippet Main
Vision and Change Topics
  • Evolution (V&C_E)
  • Structure and Function (V&C_SF)
  • Information Flow & Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
ASM Fundamental Statements
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 11 (ASM_11): Obligate intracellular microbes require living host cells for replication.
  • Fundamental Statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.
  • 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 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recognize the structures of a bacteriophage virion.
  • Distinguish between a satellite virus and a helper virus.
 S L
  • Evaluate data to determine whether a satellite-helper phage interaction should be considered mutualistic or parasitic.
 S H
  • Describe how Yersinia pestis is transmitted from one host to another.
  • When provided examples, distinguish between in vivo and in vitro experiments.
  • Recall observations that support how biofilm formation of Yersinia pestis in the flea contributes to its transmission to more human hosts.
 M L
  • Predict the results of an experiment based on a hypothesis generated from this study’s results.
 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

  • Electron Microscopy (13:45–14:39): This is a high resolution microscopy method.  Here, it was used to image each type of virus.
  • Southern Blot Analysis (15:49–16:19): This is a molecular technique to analyze DNA sequences.  It typically involves using restriction enzymes to cut DNA, electrophoresis to separate the DNA fragments, transfer to a membrane, and then identifying specific DNAs by their complementarity to a probe. Here, it was used to determine the size of the genome.
  • Plaque Assay (16:20–16:34): This is an assay to identify the presence and quantity of bacteriophages by exposing a bacterial lawn to media containing bacteriophage.  Phage presence is noted as a clear spot in the bacterial lawn.  Here it was used to quantify the number of infectious particles (the PICIs).
  • Protein Purification & Analysis (16:34–16:59; 21:23–21:44): Typically protein purification relies on biochemical separation of cell lysates depending on the properties of the proteins being purified.  Here, the researchers purified helper or satellite virus and protein analysis was conducted to show which virus the capsid and tail are coming from.
  • DNA Sequencing (16:59–17:10): This is a technique to identify the base sequences of a DNA.  Here, it was used to further confirm which virus the capsid and tail are encoded in.
  • Mass Spectroscopy (17:10–17:18; 20:07–21:00): This is a technology that is used to identify and quantify molecules in a mixture.  Here, it was used to confirm that the satellites only need the protein for the tail from the helper.
  • Genomic Mining/Bioinformatics (21:44–22:22): This is a method to search for other sequences related to a target sequence using available genome resources (databases).  The researchers searched databases to look for other PICIs and what other genera have them.
  • Phylogenetic Analysis (22:22–22:37): This is a clustering method using either amino acid or DNA sequences to identify the similarity sequences, which infers an evolutionary relationship.  Here, the researchers used this clustering method and its visual output (a tree) to figure out whether each family of PICIs arose independently or not.

4.2. Main Paper

  • Immunostaining (53:21–54:44): This is a technique using fluorescence, dyes, or enzymes to identify the location or presence of a specific molecule.  Here, specific antibodies were used to identify specific proteins.
  • Fluorescent Single Cell Quantification (54:44–56:00): This is an application of fluorescent microscopy to quantify a molecule in single cells.  Here, fluorescent microscopy fluorescence was quantified for specific components in a cell.
  • Viable Count (56:00–57:35): This is one of many methods used to quantify the count of all cells that are alive.  This is typically done colony forming units or live/dead staining to show that intact cells are indeed alive.
  • Cloning (1:04:05–1:04:50): This is the process of creating a genetic replica of a specific cellular component to do further studies on it.

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

5.1. Snippet Paper

  • Natural Selection (7:55–8:49): Viruses have evolved through natural selection for preferential packaging
  • Phage Structure (10:41–12:21): Phages have icosahedral head, tail, tail fibers. Satellite virus can make its own small capsid but uses the tail produced by the helper virus.
  • Viral Satellites (4:09–7:01): Satellites are viruses that require a helper virus for replication
  • Phage Reproduction and Packaging (6:33–7:55; 8:50–10:37; 14:32–16:19; 21:00–26:21): A satellite virus makes a small capsid so its DNA can fit inside, but the helper virus’ DNA will not. This ensures preferential packaging of the satellite viral genome.

5.2. Main Paper

  • Biofilm Formation (38:09–40:09): Bacteria form layers of cells that blocks the esophagus of the flea. This causes the fleas to regurgitate bacteria in an attempt to clear the block, and to seek more frequent blood meals increase the spread Y. pestis to new hosts.
  • Bactericidal Complement (33:49–34:43): An innate immune response that can kill bacteria exposed to blood serum; most mammals, except mice, have sera that is bactericidal to Y. pestis.
  • Plague Cycles in Infectious Diseases (35:40–39:48): When the primary reservoir for plague is wiped out due to disease, the fleas (vector) will feed on other mammals, which could lead to humans being infected. There are different types of plague that are characterized by the way the infection is transmitted, which include septicemic, pneumonic, and bubonic.
  • Complement Activation (41:55–47:52): Complement is a part of the innate immune system. Activation of the complement cascade in blood serum results in a complex that can kill the invading microbe. The gene ail (encodes attachment invasion locus protein) is required to protect Y. pestis from serum complement.

6. Podcast Questions

Labeled phage: A is pointing to the top; B to the interior of the top nodule; C to the stem coming down to the "legs," and D to the "legs" of the phage.

  1. Match the labeled structures on the phage with their names.
    1. ________ Capsid
    2. ________Sheath
    3. ________Genome
    4. ________Tail fibers
  2. Which of the descriptions below distinguishes a satellite virus from a helper virus?
    1. The satellite virus replicates more slowly than the helper virus.
    2. The satellite virus requires a co-infected helper virus to reproduce.
    3. The satellite genome does not encode a capsid or tail protein.
    4. The satellite virus is significantly larger than a helper virus.
  3. The podcasters note that this satellite-phage interaction was mutualistic rather than parasitic by evaluating the number of each phage produced.  Which of the datasets (plaque forming units per milliliter) below indicates a parasitic rather than mutualistic interaction?  What is your evidence? [Experiment 1: Satellite phage = 245 PFU/mL and Helper phage = 230 PFU/mL; Experiment 2: Satellite phage = 245 PFU/mL and Helper phage = 96 PFU/mL]
    1. Experiment 1 is parasitic because both phage are produced equally.
    2. Experiment 1 is parasitic because the satellite phage is more abundant.
    3. Experiment 2 is parasitic because both phage are produced unequally.
    4. Experiment 2 is parasitic because the helper phage is less abundant.
  4. Bubonic plague caused by Yersinia pestis became one of the deadliest pathogens in recorded history. However, recent studies have shown that this bacteria is commonly found in ancient human remains. We are still learning how plague became a deadly pathogen. Which of the following describes how Yersinia pestis is transmitted to humans?
    1. Foodborne
    2. Contaminated water
    3. Flea blood meal
    4. Mouse bite
  5. The podcast described an unusual role of biofilm in the spread of the Yersinia pestis to new hosts. Which of the following is true?
    1. Biofilm formation in the flea facilitated spread to new mammalian host.
    2. Biofilm allowed Yersinia pestis to survive at higher temperatures.
    3. Biofilm formation in the mouse prevented transmission to the flea.
    4. Biofilm formation in the mouse facilitated mouse innate immune response.
  6. Kolodziejek et al. (2023) describe differences between the in vitro experimental results and the in vivo experiments. Which of the following is an example of an in vivo experiment described in the podcast main paper?
    1. Mouse and human blood serum incubated with Yersinia pestis at 28°C.
    2. Human blood serum incubated with Yersinia pestis containing a deletion of the ail gene.
    3. Plate cell count of Yersinia pestis after growth in fleas fed different bloodmeals.
    4. Immunofluorescence of proteins on Yersinia pestis to detect effects of complement proteins.
  7. Based on the main paper study, what would you predict for a Y. pestis strain that was unable to form a biofilm?
    1. It would be more deadly to fleas.
    2. It would be transmitted less often.
    3. It would be transmitted more often.
    4. It would be less deadly to fleas.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3) and one from the main paper (Figure 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features and experimental design aspects of microscopy images and Southern blot results.
  • Analyze electron microscopy images to draw conclusions about a new class satellite virus, “cf-PICI.”
  • Evaluate phage production results to make conclusions about the components encoded in a satellite phage.
  • Evaluate phage production results for phage with mutant components to make conclusions about the components necessary for phage production.
  • Predict the results of a Southern blot analysis given specific phage types.

Experimental Background (Alqurainy et al. Figure 3)

Phage satellites are phage-like genetic elements that require and are parasitic to helper phages.  They do this by altering phage packaging to generate small capsids that can only fit the smaller satellite DNA.  Additionally, these small capsids are composed of only phage-encoded proteins because these elements do not encode the infectious particle proteins.  These authors discovered a genetic element in an E. coli strain that appeared to be a fusion of a phage-inducible chromosomal island (PICI) satellite and a phage.   To characterize this element, they first sequenced and annotated genes within the element.  Unlike other satellites, this element appeared to encode putative genes for capsid formation and packaging.  It appeared to be missing only the genes encoding the protein tail. However, these in silico (computational) predictions needed to be tested in cells.  So, they used electron microscopy to test whether the putative genes in this element could in fact support capsid formation, as well as virus packaging and transfer.

They utilized different variants of helper virus and satellite phage DNA elements.  The helper phages were as follows: wild-type, called HK106; a terL packaging protein mutant called HK106 gp01*; or a capsid mutant called HK106 gp05*.  The satellite phage types were as follows: wild-type called EcClEDL933; a terL packaging protein mutant called EcClEDL933 1794*; or a capsid mutant calledEcClEDL933 1786*.  In this experiment, different helper phages (HK types) were mixed with E. coli containing the different satellite phage elements (Ec types), then induced to enter lysogenic stage so that phage particles would be produced. Electron microscopy was used to identify phage size and morphology (panels A1-6) and Southern blot (panel C) was used to confirm which genomes were present in the phage particles (Alqurainy et al 2023).

Several microscopy photos of lysogenic strains. 1 through 3 are denser groupings, whereas 4 has more sparse, separated cells, and 5 and 6 have smaller groupings.
Figure 3.

“EcCIEDL933 produces small capsids. (A) Electron microscopy analyses of different MC-induced HK106 lysogenic strains (WT, terL mutant [gp01*] or capsid mutant [gp05*]) in presence or absence of EcCIEDL933 (WT, terL mutant [1794*] or capsid mutant [1786∗]). Different fields are shown, containing HK106 phage particles (top images), EcCIEDL933 particles (bottom images), or both (bottom left; PICI particles highlighted with white arrows). EcCIEDL933 particles have smaller heads. Images were collected using a JEOL 1200 TEM microscope with 12K magnification. Scale bars represent 200 nm. (B) Lysogenic strains carrying the WT or the terL mutant (gp01*) HK106 prophage, in presence or absence of EcCIEDL933, were MC induced; the DNA was extracted from the purified infective particles and resolved on a 0.7% agarose gel, GelRed stained. The size of phage HK106 is 41,468 bp, while EcCIEDL933 has a size of 15,471 bp. (C) Southern blot of the purified DNA shown in (B) using either phage HK106- or EcCIEDL933-specific probes. First line contains the Southern blot molecular marker (DNA molecular weight marker VII; Roche). See also Table S4.”  (Alqurainy et al 2023, changes: section A panels were annotated with numbers 1–6)

7.1.2. Questions

  1. Which panel of the electron microscopy images is the control to show the size(s) of the wild-type helper phage?
    1. Panel A1
    2. Panel A2
    3. Panel A3
    4. Panel A4
    5. Panel A5
    6. Panel A6
  2. Which panel of the electron microscopy images is the control to show the size(s) of the wild-type satellite phage?
    1. Panel A1
    2. Panel A2
    3. Panel A3
    4. Panel A4
    5. Panel A5
    6. Panel A6
  3. When the satellite element/phage (Ec) lacks a functional terL protein (1794*) and the helper phage (HK) is wild-type (HK106), only satellite-type phage are produced (panel A2).  What does this indicate?
    1. Helper terL is sufficient for both satellite-type phage and helper-type phage particles to be produced.
    2. Satellite terL supports packaging and production of satellite phage only and not helper phage.
    3. Helper terL is not necessary for either satellite-type phage or helper-type phage particles to be produced.
    4. Helper terL is not necessary for helper-type phage particles, but it is necessary for satellite-type phage particles.
  4. Which panel of the electron micrographs indicates that the helper phage (HK) with a capsid mutation [gp05*] can still make phage tails?
    1. Panel A1
    2. Panel A2
    3. Panel A3
    4. Panel A4
    5. Panel A5
    6. Panel A6
  5. The authors show an agarose gel electrophoresis of DNA isolated from phage particles in panel B and a Southern blot analysis of the same DNA in panel C. Why was it necessary to perform the Southern blot analysis since agarose gel electrophoresis showed the two expected DNA sizes?
    1. Southern blot analysis is a more useful for observing large DNA size differences.
    2. Agarose gel electrophoresis confirms the size, but not the sequence of the DNAs.
    3. The agarose gel was shown as a step in the process of Southern blot analysis.
    4. Southern blot analysis shows the genes from each particle are being transcribed.
  6. What Southern blot analysis results would you expect to see if you isolated DNA from phage in panel A2 and used a mixture of phage HK106- EcCIEDL933-specific probes?
    1. 41,468 bp DNA fragment
    2. 15,471 bp DNA fragment
    3. Both 41,468 bp and 15,471 bp DNA fragments
    4. Neither DNA fragment

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features and experimental design aspects of the described experiments and data presented as line graphs.
  • Describe how a viability count is used to calculate/quantify survival.
  • Evaluate the data and make conclusions about bacterial survival following serum exposure.
  • Analyze the data and make conclusions about the circumstances in which the attachment invasion locus protein (Ail) is functional as complement resistance factor.
  • Defend the inclusion of a complementation strain and identify its value to the experiment.

Experimental Background (Kolodziejek et al., Figure 2)

Yersinia pestis, the causative agent of the bubonic plague, is transmitted to rodent and human hosts via a flea vector.  Generally, the mammalian host will activate the innate immune response to recognize and respond to the pathogen.  This response includes activation of the complement cascade that generates membrane-penetrating complexes that kill the pathogen. Y. pestis has a virulence factor, attachment invasion locus protein (Ail) that confers serum resistance at 37°C, which is mammalian body temperature.  Kolodziejek et al (2023) were interested in whether this protein was also able to confer serum/complement resistance at the flea body temperature of 28°C.  To examine this, they incubated wild-type (KIM6+), an ail mutant (Δail), or an ail mutant complemented with wild-type ail (Δail/ail+) with human serum (NHS), mouse serum (NMS), or rat serum (NRS), or heat-inactivated sera as the controls, for up to 160 minutes and assayed bacterial survival.  Survival was measured by plate counts (colony forming units or CFU) with the counts found for heat-inactivated sera assigned as 100% survival and all other values calculated relative to that.

 

Three line graphs plotting survival of NHS, NRS, and NMS over time. The first has all three lines relatively stable. The second has NMS stable, with the other two falling steeply at the hour mark. The third has all three relatively stable, and NRS growing.
Figure 2. “Rat or human but not mouse sera were bactericidal against the Y. pestis Δail mutant at 28°C. Y. pestis wild-type KIM6+, the Δail mutant, and Δail/ail+ were grown with aeration to mid exponential phase at 28°C in Lysogeny broth (LB). Cells were centrifuged, washed with PBS, and incubated in 50% normal human serum (NHS), normal rat serum (NRS), normal mouse serum (NMS), or the respective heat-inactivated sera (HIS) for 0, 60, and 150 min at 28°C. Bacteria were enumerated by plate counts and CFU after each HIS incubation were assigned as 100% survival. Results are means ± SEM (n = 4) from experiments performed twice in duplicate. Asterisk (*) indicates statistical differences between mouse compared to human or rat sera (repeated-measures ANOVA, P < 0.001).” (Kolodziejek et al 2023, no changes) 

 

7.2.2. Questions

  1. The authors report their data as % survival, which is a calculated value based on what?
    1. CFU for normal serum at each time point multiplied by CFU at time 0 for normal serum.
    2. CFU for normal serum at each time point divided by CFU for heat-inactivated serum at that time.
    3. CFU for normal serum at each time point subtracted from CFU at time 0 for normal serum.
    4. CFU for normal serum at each time point subtracted from an average CFU for heat-inactivated sera.
  2. Percent survival is quantified in the center panel for Y. pestis cells with a mutant ail gene Δail with exposures to different normal sera.  in the center panel.   What notation in this panel indicates the comparison being made is statistically significant?
    1. dot/line height
    2. whiskers
    3. asterisks
    4. nothing
  3. Which species’ serum/a contain/s effective antimicrobials against wild-type Y. pestis? How can you tell?
    1. Rat because wild-type bacteria are killed by rat serum.
    2. Mouse because wild-type bacteria are killed by mouse serum.
    3. Human because wild-type bacteria are killed by human serum.
    4. None because wild-type bacteria are not killed by any serum.
  4. Based on the data provided in the figure, Ail-deficient Y. pestis (center panel) are sensitive to the serum/a of which species? How can you tell? Pick all that apply.
    1. Rat because Δail bacteria are killed by rat serum.
    2. Mouse because Δail  bacteria are killed by mouse serum.
    3. Human because Δail bacteria are killed by human serum.
  5. What do the experiments with the Y. pestis strain denoted as having genotype Δail /ail+ (right panel) add to the conclusions? Pick all that apply.
    1. The bactericidal effect observed for Δail was due to the ail defect and not some other random mutation in the strain.
    2. The bactericidal effect observed for Δail was caused by host immune factors and not the ail defect in Y. pestis strain.
    3. The bactericidal effect observed for Δail was due to a change in growth media and not the ail defect in Y. pestis strain.
    4. The original bactericidal effect observed for Δail was due to plasmid loss and not due to the ail defect in Y. pestis strain.

8. Paper Information and Licensing

8.1. Snippet paper

  • Alqurainy N, Miguel-Romero L, Moura de Sousa J, Chen J, Rocha EPC, Fillol-Salom A, Penadés JR. 2023. A widespread family of phage-inducible chromosomal islands only steals bacteriophage tails to spread in nature. Cell Host Microbe 31(1): 69-82. doi: 10.1016/j.chom.2022.12.001
  • 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’s copyright information.

8.2. Main paper

  • Kolodziejek AM, Bearden SW, Maes S, Montenieri JM, Gage KL, Hovde CJ, Minnich SA. 2023. Yersinia pestis Δail mutants are not susceptible to human complement bactericidal activity in the flea. Appl Environ Microb 89(2): e0124422–e0124422. doi: 10.1128/aem.01244-22
  • 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’s copyright information.

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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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