Metabolic Pathways

TWiM #312: Cry Havoc! And Let Slip the Phages of Healing

Podcast and Annotation Information
  • Annotation by Alaina Devlin, Parker Flanagan, Megan Frank, Cara Leavitt, Spencer Ort, Caroline Scott, and Beth Potter
  • Podcast audio by TWiM: Listen to TWiM #312 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Rebecca Seipelt-Thiemann: Access Podcast Transcripts
  • Papers Discussed:
    • Al-Anany AM, Fatima R, Nair G, Mayol JT, Hynes AP. 2024. Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny. mBio 15(6):e0050424. https://doi.org/10.1128/mbio.00504-24
    • Yoo W, Shealy NG, Zieba JK, Torres TP, Baltagulov M, Thomas JD, Shelton CD, McGovern AG, Foegeding NJ, Olsan EE, Byndloss MX. 2024. Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis. Cell Host Microbe 32(6):887-899.e6. https://doi.org/10.1016/j.chom.2024.05.001

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:10 minutes

The Most Interesting Things (according to students)

This study highlights the fascinating ability of phage to gain an intimate knowledge of their environment/host. A previous paper to this study showed that ciprofloxacin, a quinolone antibiotic, could act synergistically with a temperate phage by forcing the temperate phage into a virulent lytic cycle. The authors show that the mechanism for the bias involves the activation of the SOS response and similar synergism occurs with other classes of antibiotics that involve an SOS response. Moreover, the authors were able to show temperate phage synergy with several classes of antibiotics that do not involve activation of the SOS-response, revealing another mechanism for the inhibition of lysogeny.

“A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some β-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.” (Al-Anany et al. 2024, no changes)

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

The Most Interesting Things (according to students)

The highlight of this paper was how the authors were able to break down a rather complex set of events of how aspartate increases in the inflamed gut and provides a fitness advantage to Salmonella enterica serovar Typhimurium through a series of logically-presented and eloquent experiments.

“Inflammation boosts the availability of electron acceptors in the intestinal lumen, creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection increases intestinal levels of the amino acid aspartate. S. Tm used aspartate-ammonia lyase (aspA)-dependent fumarate respiration for growth in the murine gut only during inflammation. AspA-dependent growth advantage was abolished in the gut of germ-free mice and restored in gnotobiotic mice colonized with members of the classes Bacteroidia and Clostridia. Reactive oxygen species (ROS) produced during the host response caused lysis of commensal microbes, resulting in the release of microbiota-derived aspartate that was used by S. Tm, in concert with nitrate-dependent anaerobic respiration, to outcompete commensal Enterobacteriaceae. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent S. Tm expansion during colitis.” (Yoo et al. 2024, no changes).

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

Snippet

Main

Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Impact of Microorganisms (V&C_IM)
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)

ASM Fundamental Statements
  • 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 18 (ASM_18): The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.
  • Fundamental Statement 7 (ASM_7): Microbes have evolved structures adapted for specific functions that are often associated with a fitness advantage in a particular environment
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 21 (ASM_21): Microbes and the environment interact with and affect each other.

3.  Potential Learning Objectives for the Podcast

The student will be able to:

Paper1

Order2
  • Describe the differences between lytic and temperate (lysogenic) phage.
  • Identify the evidence that checkerboard assays provided to this study.
  • Recall how RecA is involved in temperate phage-antibiotic synergy.

S

L
  • Evaluate hypothetical data related to the use of temperate phage in therapeutic practices.

S

H
  • Recall how the gut microbiome is beneficial to the host.
  • Identify key metabolic features of Salmonella (S. Tm) infecting the inflamed gut.
  • Identify the data that showed the importance of aspartate origin for Salmonella enterica serovar Typhimurium (S. Tm) gut pathogenesis

M

L
  • Propose general therapeutic interventions to curb S. Tm expansion in the gut.

M

H

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

2Learning 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

  • Checkerboard Assay (16:08): This is an assay used to show whether synergy occurs between various conditions or compounds.  Two compounds are serially diluted along the length and width of a 96 well plate.  This webpage has a nice visual of this set up: https://antiviral.creative-diagnostics.com/antimicrobial-synergy-testing-checkerboard-assay.html.  In this study, they are used to demonstrate synergy between temperate phage and various antibiotics. Results are displayed as heatmaps.

4.2. Main Paper

  • In vivo Mouse Models (29:22–29:42; 30:20–30:46; 32:05–32:27; 34:38–36:52): The term in vivo refers in an organism and the term mouse model refers to using mice as a model for another organism, typically humans.  Here, laboratory mice are used to study aspects of human disease. In this study, both germ-free (no microbes) and gnotobiotic (engineered to have specific microbiota) mice were used.
  • Salmonella enterica serovar Typhimurium Mutants (31:08–31:52; 32:36–33:07): Mutant strains of bacteria are those with variations in their DNA and can be isolated or engineered.  They are used to study the bacterial traits encoded in the genes or to identify the genes involved in conferring the trait.  Here, Salmonella enterica serovar Typhimurium, were engineered to have key genes removed (knocked out) which were then studied.
  • Genetically-engineered Bacteroides theta (37:05–38:42): This is a particular strain of bacteria.  It was used to colonize germ-free mice and show that lysis of commensal Bacteroides increased aspartate levels in the inflamed gut of mice.

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

5.1. Snippet Paper

  • Phage Therapy (4:06–5:04): These are bacterial infection treatments that rely on the use of viruses (bacteriophages) that specifically infect bacterial cells.
  • Temperate Versus Lytic Phage (5:07–7:40): Lytic phages are virulent and immediately kill the host while temperate (lysogenic) phages can enter a host in a non-virulent fashion, allowing them to ‘hide’ within the host.

5.2. Main Paper

  • Gut Microbiome (24:13–25:00): Microbes in the gut can provide essential nutrients and provide a formidable barrier to colonization by pathogenic bacteria.
  • Pathogenic Salmonella (25:02–26:07): Pathogenic Salmonella infections cause 1.3 million infections per year that involve diarrhea, cramps, and fever that can last for several days. There have been several recent examples of Salmonella outbreaks in different food products.
  • Anaerobic Respiration (27:37–28:40): This is a multistep process that extracts energy from carbon sources in the absence of oxygen.

6. Podcast Questions

  1. Which of the following is a unique characteristic associated with most temperate (lysogenic) phage?
    1. Phage DNA is injected into the bacterial cell
    2. Phage DNA integrates into the bacterial host genome
    3. Host machinery is utilized to make viral proteins
    4. Lysis of the bacteria sheds hundred of new phage progeny
  2. Checkerboard assays showed synergy between a temperate phage and several different classes of antibiotics because phage _______ .
    1. Enhanced the ability of the antibiotic to disrupt the host cell
    2. Lowered the minimum inhibitory concentration of the antibiotic
    3. Increased the phage replication when the antibiotic was present
    4. Increased the amount of DNA damage within the host cell
  3. Which of the following describes the role of RecA in temperate phage-antibiotic synergy (tPAS) involving SOS-inducing antibiotics?
    1. RecA increases bacterial host sensitivity to specific antibiotics
    2. RecA induces DNA damage which allows for phage induction
    3. RecA degrades the lambda repressor allowing lytic genes to turn on
    4. RecA stabilizes the cis-acting elements that promote the lysogenic cycle
  4. The podcasters note how the phage studies could be used clinically.  However, a next step would probably be to test this in mice first.  Let’s say you infected mice with E. coli and treated with different concentrations of drug B or a combination of drug B and the temperate phage KH97.  What would be your conclusion if the drug B MIC was the same as the drug B + phage MIC?
    1. Lysis was not effectively induced, so there was no synergy for drug B.  This would not support clinical use.
    2. Lysis was effectively induced, so there was synergy for drug B. This would be good support clinical use.
    3. Lysis was not effectively induced, so synergy was delayed for drug B. This would not support clinical use.
    4. Lysis was effectively induced, so there was synergy among the phages.  This would not support clinical use.
  5. Which of the following are functions of the gut microbiome described in the podcast? [pick all that apply]
    1. Production of vitamins, such as vitamin K
    2. Aids in degradation of dietary fiber
    3. Secretion of antibiotics into the gut
    4. Competition with pathogens for nutrients
  6. What is the terminal electron acceptor in anaerobic respiration for Salmonella (S. Tm) infecting the inflamed gut?
    1. Oxygen
    2. Glycerol
    3. Aspartate
    4. Fumarate
  7. In this study the researchers found that aspartate plays a key role in S. Tm gut pathogenesis, but there was no fitness advantage between S. Tm strains that lack or had a functional aspA gene when the researchers fed mice aspartate in the diet.  Instead, they found aspartate’s origin was elsewhere.  Where was the aspartate coming from and what evidence showed this?
    1. Lysis of normal gut microbiome species; they engineered a Bacteriodes species to lyse under specific conditions and then the AspA+ strains had a fitness advantage.
    2. Secretion by intestinal epithelial cells; they engineered several different cell lines, each to overexpress aspartate, and then the AspA+ strains had a fitness advantage.
    3. Production by host immune cells; they engineered and polarized tissue macrophages to release aspartate and then the AspA+ strains had a fitness advantage.
    4. Release from damaged host epithelial tissues; they engineered tissue injury models in mice of the same strain and then the AspA+ strains had a fitness advantage.
  8. Initially, we might have proposed to reduce aspartate as a therapeutic that to control Salmonella pathogenesis in the gut. However, these data suggest a different treatment might be more effective.  Based on the conclusions of this study, what would be a reasonable step forward for treatment?
    1. Find ways to increase the microbiome by using probiotics.
    2. Find ways to increase the microbiome by using prebiotics.
    3. Find ways to reduce microbiome death due to inflammation.
    4. Find ways to reduce pathogen load using engineered bacteria.

7. Figure Reading Exercises

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

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features, experiment design elements for checkboard assays, heatmaps, and bar charts.
  • Analyze checkerboard assay/heatmap data to make conclusions about minimum inhibitory concentrations.
  • Evaluate checkerboard assay/heatmap data to make calculate fold changes in MIC.
  • Analyze checkerboard assay/heatmap data to make conclusions about phage-antibiotic synergy (tPAS).

Experimental Background (Al-Anany et al., Figure 4A-F)

Using bacteriophages as therapeutics is increasing in popularity as antibiotic resistance has increased.  Al-Anany et al. (2024) explore this re-newed idea.  In prior experiments of this same study they showed that temperate phage-antibiotic synergy (tPAS) occurs with several classes of antibiotics that can induce a general bacterial stress response called the SOS-response. Here, the researchers investigate whether non-SOS-inducing antibiotics can also exhibit tPAS using an assay called a checkerboard assay (or synograph).  In this assay, different dilutions of phage and antibiotic are tested for their combined (synergistic) ability to show lysis. Temperate phage and four antibiotics that inhibit protein synthesis were tested with wild-type bacteria (panels A-D).  Please note that M.O.I. (multiplicity of infection) is the unit for phage concentration.  These data were then used to calculate parameters that represent bacterial growth in various ways, typically used in pharmacokinetic studies.  For example, in panels A-D, the authors present the area under the curve (AUC) -the growth curve- to evaluate the drugs effects over the entire growth cycle. In addition, the fold reduction in number of survivors for gentamycin alone (left side of panel E) and gentamycin + phage (right side of panel E) were calculated and compared.  The data are represented in relation to gentamycin’s known minimum inhibitory concentration (MIC), e.g., MIC, 1/2 MIC, 1/4 MIC, etc.  Next, to determine whether the effect they observed was synergistic, they calculated the expected effect by multiplying individual phage effect and individual antibiotic effect, and compared this to the observed reduction in survivors (panel F).  Other acronyms of note: HK97 = temperate phage model used in study, AUC = area under the curve, SD = standard deviation, P = p-value or probability.

Checkerboard assays show protein synthesis inhibitors result in temperate phage-antibiotic synergy.
Figure 4. “Protein synthesis inhibitors result in temperate phage-antibiotic synergy. Checkerboard assay of HK97 and (A) gentamicin, (B) kanamycin, (C) tetracycline, and (D) azithromycin. AUC relative to untreated bacterial control, averaged among three biological replicates, plotted as a heatmap. (E) Bars show the average number of survivors relative to untreated cultures in three biological replicates, each of three technical replicates. Each biological replicate is represented by its own shape: circle, square, or triangle. Limit of detection (10 CFU/mL) is represented at all points of MIC and 1/2 MIC tPAS data, as no counts were obtained, except “square” at 1/2 MIC. Error bars depict the SD, while ***P from 0.001 to 0.0001 from a one-way ANOVA and Tukey’s post hoc test. (F) Bars show the observed effect (green) versus the expected (gray) effect determined by multiplying the effect of the phage and antibiotic alone. Average from the three biological replicates for each observed tPAS data from Fig. 4E was compared to the calculated expected effect at the corresponding antibiotic concentration using a paired t test, *P ≤ 0.05. …. Area under the curve relative to untreated bacterial control, averaged among three biological replicates, plotted as a heatmap.” (Al-Anany et al. 2024)

7.1.2. Questions

  1. The minimum inhibitory concentration (MIC) is defined as ________ and the MIC for gentamycin alone (panel A) is _______ .
    1. The lowest concentration of an antibiotic that kills 99.9% of bacteria; 4100 ng/mL
    2. The lowest concentration of an antibiotic that shows toxicity to human cells; 128 ng/mL
    3. The lowest concentration of an antibiotic that inhibits bacterial growth; 1024 ng/mL
    4. The lowest concentration of an antibiotics that inhibits phage reproduction cycles; 5 ug/mL
  2. The data for the checkerboard assays are displayed as heatmaps (panels A-D, G, & H).  Which color indicates the greatest bacterial survival and what M.O.I./azithromycin concentration indicates this amount of survival?
    1. dark green; 0 M.O.I. and 0.5 ug/mL
    2. medium green; 20 M.O.I. and 0.39 ug/mL
    3. light green; 0.001 M.O.I. and 1.56 ug/mL
    4. white; 0.0001 M.O.I. and 3.13 ug/mL
  3. Which antibiotic(s) show phage-related decreases in MIC greater than 10 fold, regardless of M.O.I needed?
    1. Gentamicin (panel A)
    2. Kanamycin (panel B)
    3. Tetracycline (panel C)
    4. Azithromycin (panel D)
  4. All antibiotics showed some phage-related decreases in MIC, but one in particular required a much larger M.O.I.  Which antibiotic required more phage for the synergistic effect?
    1. Gentamicin (panel A)
    2. Kanamycin (panel B)
    3. Tetracycline (panel C)
    4. Azithromycin (panel D)
  5. The synergism of antibiotic and phage are particularly visible when the data are displayed as shown in panel F for gentamycin. At which concentration is there the most synergism between antibiotic and phage? What is your evidence?
    1. MIC
    2. 1/2 MIC
    3. 1/4 MIC
    4. 1/8 MIC
  6. If the researchers were to generate a graph for tetracycline data similar to that displayed in panel E for gentamycin, which of the following would most likely be correct regarding the tetracycline + phage visualization?
    1. There would be a similar trend in the fold reduction but a higher fold reduction would be expected for 1/16 MIC.
    2. There would be a similar decreasing trend but the fold reduction would not be as high as those seen for gentamicin.
    3. The graph would be different with all antibiotic concentrations having a similar fold reduction to that observed for MIC.
    4. The graphs would be completely different because tetracycline inhibits protein synthesis in a different way to gentamicin.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features and experimental design aspects of bacteria growth and competition data and graphs.
  • Analyze the data to make conclusions about growth and growth inhibition in different growing conditions.
  • Evaluate the data to make conclusions about competition and fitness due to metabolic environment, co-culture, and genetic differences
  • Evaluate the data to make conclusions about competition and fitness in vitro and in vivo.
Experimental Background (Yoo, et al., Figure 7)

Inflammation can play a critical role in infection by gut pathogens, but the mechanism is not understood.  Here, Yoo et al. (2024) investigate different gut lumen metabolites affect infection. In prior experiments they found that AspA (aspartate-ammonia lyase, which converts aspartate into fumarate) provided a fitness advantage to Salmonella enterica serovar Typhimurium (S. Tm) in the inflamed gut. Furthermore, they found that the aspartate necessary for this effect was provided by commensal Bacteroides that were lysed by reactive oxygen species produced in inflammation. Their prior experiments were performed using mice that lack a gut commensal bacterium known to have a crucial role in resisting Salmonella infection, therefore their next experiment was to investigate whether their conclusions about aspartate and aspA were also true when this bacterial species, an Enterobacteriaceae (Escherichia coli Mt1B1) was present.  The researchers first quantified the fitness by inoculating mice with a mixture of wild-type S. Tm and the ΔaspA mutant S. Tm. when the gut was or was not also colonized with wild-type E. coli, quantifying the growth for each strain, and calculating a ratio, which is a competitive index (panel A).  To confirm persistence in the gut, S. Tm strains were quantified from feces over 7 days and are expressed as colony forming units (CFU; panel B).  To determine whether the impact on fitness was due to metabolic changes in the gut environment, they tested in vitro anaerobic growth when the medium was supplemented with different metabolic components present in an inflamed gut (glycerol, nitrate, aspartate) for wild-type S. Tm (panel C), wild-type E. coli (panel D), and an ΔaspA mutant S. Tm (panel E).  Having established the growth they’d expect for bacteria grown in mono-culture, they next evaluated growth in an in vitro co-culture using the supplemented media conditions, and calculated the competitive index for wild-type S. Tm and wild-type E. coli  (panel F).  To establish the combined effects of including E. coli, the media supplements, and aspA, the competition experiments were performed in vitro (with media supplements; panel G) and in vivo (by mouse inoculation; panel H) for combinations of wild-type S. Tm or ΔaspA mutant S. Tm with E. coli.

bar graphs and dot plots suggesting that S. Tm may use a combination of anaerobic nitrate and aspartate-dependent fumarate respiration to outcompete commensal Enterobacteriaceae during intestinal inflammation.
Figure 7. “Aspartate-dependent fumarate respiration and nitrate-dependent respiration synergize to enable S. Tm to outcompete commensal Enterobacteriaceae. (A) Competitive index (CI) of S. Tm wild-type and isogenic ΔaspA mutant at day 7 post-infection in CBA/J mice colonized or not with E. coli Mt1B1. (B) Wild-type S. Tm and isogenic ΔaspA mutant burden in fecal samples at 1, 3, 5, and 7 days post-infection. from CBA/J mice colonized or not with E. coli Mt1B1. (C and D) Wild-type S. Tm (C) and wild-type E. coli (Mt1B1) (D) anaerobic growth in NCE media containing glycerol (10 mM) and supplemented with aspartate (30 mM) and/or nitrate (40 mM). Bacterial growth measured by OD600 at 18 h post-inoculation. (E) Wild-type S. Tm and isogenic ΔaspA mutant anaerobic growth in media conditions described in (C). Bacterial growth measured by OD600 at 18 h post-inoculation. (F) CI for wild-type S. Tm and wild-type E. coli co-cultured in conditions described in (C). (G) CI for wild-type S. Tm or isogenic ΔaspA mutant and wild-type E. coli co-cultured in NCE media containing glycerol (40 mM) and supplemented with aspartate (30 mM) and nitrate (40 mM). (H) CBA/J were colonized with E. coli Mt1B1 and were infected with 109 CFU of a 1:1 ratio of wild-type S. Tm and ΔaspA mutant for 10 days. CI of S. Tm strains in feces was determined at day 10 post-infection. Each dot represents data from one animal (biological replicate). Bars represent mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.” (Yoo, et al. 2024, no changes).

7.2.2. Questions

  1. What competitive index would you expect if the two strains you were testing grew equally well?
    1. 0
    2. 0.5
    3. 1
    4. 10
  2. The competitive indexes for WT and  ΔaspA mutant S. Tm when grown with and without E. coli are compared in panel A.  How much better does WT grow than the  ΔaspA mutant S. Tm when no Escherichia coli are present?
    1. greater than 100
    2. greater than 10
    3. less than 10
    4. less than 1
  3. Based on the competitive index data in panel A, is there a competitive advantage for S. Tm over ΔaspA mutant S. Tm  when grown with E. coli? What is your evidence?
    1. Yes, the highest dot in the panel is for no E. coli, so wild-type still has a fitness advantage, but only there are no E. coli.
    2. Maybe, the bar heights, which indicate means, and the whisker lengths, which indicate standard deviation are inconclusive.
    3. Yes, the bars are different heights with + E. coli being lower, so the advantage is for the ΔaspA mutant S. Tm.
    4. No, the bars are about the same height and no statistical measures/notations indicate there are statically different.
  4. When did a significant decrease in the amount of S. Tm ᐃaspA mutants compared to WT S. Tm happen in the inflamed gut of mice that contained commensal Enterobacteriaceae (panel B)?
    1. Day One
    2. Day Three
    3. Day Five
    4. Day Seven
  5. For each of the bacterial types grown in mono-culture (panels C, D, E), which supplemental medium was best for each bacterium? (1 = glycerol; 2 = glycerol + aspartate; 3 = glycerol + nitrate; 4 = glycerol + aspartate + nitrate)
    1. ________ wild-type S. Tm
    2. ________ ΔaspA mutant S. Tm
    3. ________ E. coli
  6. When wild-type S. Tm or E. coli were grown in co-culture, which medium favored with species (panel F)? (1 = wild-type S. Tm; 2 = ΔaspA mutant S. Tm; 3 = E. coli)
    1. ________ glycerol + aspartate
    2. ________ glycerol + nitrate
    3. ________ glycerol + aspartate + nitrate
  7. The last two panels compare competition/fitness of wild-type S. Tm and ΔaspA mutant S. Tm to E. coli in vitro and in vivo.  What can you conclude from these results? [pick all that apply]
    1. The wild-type S. Tm have a good fitness in vitro and in vivo.
    2. The ΔaspA mutant S. Tm have a good fitness in vitro and in vivo.
    3. The wild-type S. Tm have a good fitness in vitro only.
    4. The ΔaspA mutant S. Tm have a good fitness in vitro only.
    5. The wild-type S. Tm have a good fitness in vivo only.
    6. The ΔaspA mutant S. Tm have a good fitness in vivo only.

8. Paper Information and Licensing

8.1. Snippet paper

  • Al-Anany AM, Fatima R, Nair G, Mayol JT, Hynes AP. 2024. Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny. mBio 15(6):e0050424. https://doi.org/10.1128/mbio.00504-24
  • 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://doi.org/10.1128/mbio.00504-24

8.2. Main paper

  • Yoo W, Shealy NG, Zieba JK, Torres TP, Baltagulov M, Thomas JD, Shelton CD, McGovern AG, Foegeding NJ, Olsan EE, Byndloss MX. 2024. Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis. Cell Host Microbe 32(6):887-899.e6. https://doi.org/10.1016/j.chom.2024.05.001
  • 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 https://www.nature.com/articles/ismej201795#rightslink

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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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