Structure and Function

TWiM #277: To Stop or Not to Stop

Podcast and Annotation Information
  • Annotation by Audrey Coleman, Grace Gutzman, Rebecca Seipelt-Thiemann, and Regina McGrane.
  • Podcast audio by TWiM: Listen to TWiM #277 Podcast
  • Podcast transcript by Otter.ai and edited by Nick Bellavia, Gwendowlyn Knapp, Marvin Romo, Harshita Sharma: Access Podcast Transcripts
  • Papers Discussed:
    • Borges AL, Lou YC, Sachdeva R, Al-Shayeb B, Penev PI, Jaffe AL, Lei S, Santini JM, Banfield JF. 2022. Widespread stop-codon recoding in bacteriophages may regulate translation of lytic genes. Nat Microbiol. 7(6):918-927. doi: 10.1038/s41564-022-01128-6.
    • Kvich L, Crone S, Christensen MH, Lima R, Alhede M, Alhede M, Staerk D, Bjarnsholt T. 2022. Investigation of the Mechanism and Chemistry Underlying Staphylococcus aureus’ Ability to Inhibit Pseudomonas aeruginosa Growth In Vitro. J Bacteriol. 204(11):e0017422. doi: 10.1128/jb.00174-22.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:29 minutes

The Most Interesting Things (according to students) 

  • Recoding of stop codons could potentially be a signal to switch between the lysogenic and lytic replication cycle. This was deduced because there is no recoding found in integrase genes, but there are in late genes used for the lytic cycle.
  • There are alternatives for the genetic code in different organisms which are not completely universal. Many bacteriophages recode TAG and TGA, which are normally stop codons, to add an amino acid instead of terminating the sequence.

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.1.

1.2. Main paper; discussion starts at 24:55 minutes

The Most Interesting Things (according to students)

  • Pseudomonas aeruginosa is a challenging infection specifically for patients with cystic fibrosis. The bacterium secretes a great amount of extracellular polymeric substances, which consolidates sputum in the lung leading to difficulty breathing.
  • If inoculated first, S. aureus is able to inhibit the growth of P. aeruginosa only in the presence of glucose and an acidic environment. However, when cocultured it is actually P. aeruginosa  that is able to outnumber the S. aureus.

“Pseudomonas aeruginosa inhibits or eradicates Staphylococcus aureus in most in vitro settings. Nonetheless, P. aeruginosa and S. aureus are commonly isolated from chronically infected, non healing wounds and lungs of people with cystic fibrosis (CF). Therefore, we hypothesized that S. aureus could protect itself from P. aeruginosa through glucose-derived metabolites, such as small organic acids, preventing it from  being eradicated. This in vitro study demonstrated that S. aureus populations, in the presence of glucose, secrete one or more substances that efficiently eradicate P. aeruginosa in a concentration-dependent manner. These substances had a molecular mass lower than three kDa, were hydrophilic, heat- and proteinase-resistant, and demonstrated a pH-dependent effect. Nuclear magnetic resonance analysis identified acetoin, acetic acid, and oligopeptides or cyclic peptides in glucose-grown S. aureus supernatants. All the tested wild-type and clinical S. aureus strain inhibited P. aeruginosa growth. Thus, we proposed a model in which a cocktail of these compounds, produced by established S. aureus populations in glucose presence, facilitated these two species’ coexistence in chronic infections.” (Kvich et al 2022)

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics  (V&C_IFG)
  • Structure and Function (V&C_SF)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 10 (ASM_10): Microbial reproductive cycles consist of sequential processes.
  • Fundamental Statement 18 (ASM_18).The regulation of gene expression is influenced by external and internal molecular cues and signals.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority  having a detrimental impact on their host.
  • Fundamental Statement 6 (ASM_6): The distinct structures and processes in microbes can be targets for interspecies competition, antimicrobial treatments, and host immunity.
  • 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 28 (ASM_28): A minority of microbes are pathogens that can cause diseases and harm host organisms, society and ecosystems.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Identify the data used to discover recoding was occurring.
  • Identify the types of codons and genes whose genetic code was recoded most often.
  • Recall why recoding was not found in particular genes.
 S L
  • Hypothesize potential bacterial defenses for phage recoding.
 S H
  • Recall the two bacterial species discussed.
  • Identify the name of the compound and which bacteria are inhibited in this study.
  • Identify the conclusion of the study regarding S. aureus and P. aeruginosa culture interactions.
 M L
  • Analyze hypothetical data to evaluate growth and growth inhibition in co-culture experiments.
 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

  • Bioinformatics (7:25–17:51): The researchers did not do any “wet” lab work, rather all of their analysis was done using computer methods to analyze biological data.  Here they compared phage genome sequences to the phage protein sequence to identify where expected codons did not match the amino acids in the protein.  This is called recoding.

4.2. Main Paper

  • In vivo Studies (24:55): In vivo studies occur in living organisms and, in this case, the paper focused on lung infections with Staphylococcus aureus and Pseudomonas aeruginosa in cystic fibrosis patients. Contrary to in vitro studies, they found in vivo that S. aureus does not outcompete and inhibit P. aeruginosa, and rather there are coinfections with both populations present. The goal of the paper was to identify why that difference in phenotype occurs.
  • Shake Flasks (25:00): This is a method of culturing and observing the competition between Staphylococcus aureus and Pseudomonas aeruginosa in vitro. In these shaking cultures P. aeruginosa was shown to outcompete and inhibit S. aureus.
  • In vitro Studies (26:29): In vitro experiments refer to those conducted outside of a living organism, and the paper used in vitro techniques to establish the mechanisms between interactions of Staphylococcus aureus and Pseudomonas aeruginosa bacterial colonies. In vitro it was found that S. aureus was able to outcompete and inhibit the growth of P. aeruginosa colonies.
  • Cell Culture Supernatant (33:13–34:19): This is the cell-free liquid remaining after centrifugation, which were used to observe growth of Staphylococcus aureus and Pseudomonas aeruginosa with various conditions.
  • Streak Plating (40:29): To test the inhibitive nature, bacterial streak plating was used. Staphylococcus aureus was streaked straight across a plate with Pseudomonas aeruginosa streaked perpendicularly. Depending on conditions (glucose presence and order of colonization) different phenotypes were observed.
  • Luria/Lysogeny Broth Complex Media (41:03–41:23): This is a nutrient-rich media, whose exact composition is not known, due to undefined ingredients (like extracts and digests), which was used to promote growth of Staphylococcus aureus and Pseudomonas aeruginosa
  • High Performance Liquid Chromatography (42:23): This is a method of separating components of liquids by elution point.  Here it was used to start the process of identifying the unknown product of the Embden-Meyerhof (EM) pathway that was able to inhibit the growth of Pseudomonas aeruginosa.

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

5.1. Snippet Paper

  • Genetic Code (3:30): The genetic code describes which amino acids added to a polypeptide based on the codon during translation of mRNA into proteins. The podcast touches on how we are taught that this is ubiquitous in all organisms; however, this is a false claim, and the snippet focuses on how differences in genetic codes between organisms is a target for phages who are able to influence the functionality of stop codons.
  • Genetic Recoding (3:59–6:24): Different organisms are able to recode their genetic code to modify which amino acids are paired to each codon, and this was used by some bacteriophages to modify TGA and TAG codons to code for different amino acids. The paper’s focus was looking into why this is evolutionarily advantageous.
  • Central Dogma (4:53): The flow of genetic information. They briefly mention that microbes do not always follow the central dogma.
  • Translation (5:41–6:25): Translation of mRNA into an amino acid sequence, which occurs as an amino acid bound tRNA aligns with its corresponding codon and a peptide bond is formed to add the amino acid to a growing polypeptide.
  • Bacteriophage (6:40): Viruses which infect bacteria. The study focuses on phage found in the gut microbiome in both humans and other animals (baboon, pig, cattle, horses, giant tortoises).
  • Phylogenetic Tree (9:06–9:35): A diagram reflecting how species are related. As bacteriophage evolve over time, the phylogenetic tree becomes branched to depict genetic changes.
  • Release Factors (9:56): These trigger the termination of translation by causing the release of the ribosome from the amino acid chain.
  • Late Genes (14:58): Viral genomes are organized into early and late (sometimes intermediate as well) genes, which are sequentially turned on and off depending on the stage of the viral lifecycle to prevent excess depletion of resources. In the snippet, the researchers found that this stop codon was only occurring in the late genes which are used to signal lytic processes.
  • Lytic Viral Reproduction (15:07–15:43; 16:20–17:25; 19:25–20:07): Bacteriophage infect the host cell and use the cell’s machinery to make more copies of itself. At the end of the cycle, the host cell is lysed releasing the bacteriophage copies.
  • Prophage (18:33): Prophages occur when a bacteriophage integrates their genome into the host genome, which is part of the lysogenic life cycle.
  • Integrase Genes (18:33): The genes that encode for integrase proteins which integrate the phage genome into the host genome.
  • Lysogenic Viral Reproduction (18:33–18:51;19:25–20:07): Bacteriophage integrates into the host cell’s DNA, which allows bacteriophage to replicate without killing the host cell.
  • Epistatics (21:38–22:50): Two or more genes interact to influence expression.
  • Epigenetics (21:38–22:50): Change in gene expression caused by environmental factors, not the DNA sequence itself.

5.2. Main Paper

  • Gram Negative Cell Envelope (26:46–27:12; 30:49–31:09; 40:06–40:15; 43:28–44:11): A structure composed of an inner membrane, thin peptidoglycan, and an outer membrane, which is characteristic of some bacteria such as Pseudomonas aeruginosa.
  • Biofilms (26:59): Aggregates of bacteria that form and secrete polysaccharides that prevent the penetration of antibiotics. In this paper they discussed how Staphylococcus aureus and Pseudomonas aeruginosa form biofilms and are especially harmful in lung infections in patients with cystic fibrosis.
  • Virulence Factors (27:52–28:09): These are molecules or substances utilized by Staphylococcus aureus and Pseudomonas aeruginosa that are used to cause disease in hosts or to inhibit/kill other microbes in order to decrease competition.
  • Gram Positive Cell Envelope (30:49  -31:09; 43:28–44:11): A structure composed of a cytoplasmic membrane and thick peptidoglycan layer, which is characteristic of some bacteria such as Staphylococcus aureus.
  • Embden Meyerhof Pathway (31:53): This is one of the glycolysis pathways which breakdowns glucose. In this paper they discuss that in the presence of glucose, there is a byproduct of its metabolism that acidifies the medium S. aureus is growing in. This byproduct of the EM pathway along with the acidification event are enough to inhibit the growth of P. aeruginosa.
  • pH (32:15–34:19; 35:51–37:31; 38:38–39:18): a measure of the free proton concentration in a substance to determine alkalinity or acidity.
  • Bactericidal (38:12–38:37; 44:49–45:01): A substance, such as acetoin, which has the ability to kill bacteria.
  • Opportunistic Pathogens (49:09): Pseudomonas aeruginosa is an opportunistic pathogen, which means that it will over colonize and establish itself in the host if given an opportunity that is not the normal conditions.

6. Podcast Questions

  1. Regarding the snippet paper, which data comparison was used to identify recoded codons?
    1. Phage protein sequences were compared to translated phage genome sequences.
    2. Phage genome sequences were compared to each other in phylogenetic trees.
    3. Phage codon tables were compared to eukaryotic and prokaryotic codon tables.
    4. Phage protein sequences were compared to each other in phylogenetic trees.
  2. Which codons were being recoded in the bacteriophage described in the snippet paper to code for different amino acids?
    1. Serine codons (AGU, AGC)
    2. Lysine codons (AAA, AAG)
    3. Stop codons (UAG and UGA)
    4. Start codon (AUG)
  3. Regarding the snippet paper, in which genes did recoding happen most frequently and why do the podcasters think was the reason?
    1. Regulator genes that are encoded in the host eukaryotic or prokaryotic genomes because they are necessary to control lysogeny.
    2. Late genes in the lytic bacteriophage cycle because bacteriophage RNAs and proteins must first be made to enable recoding.
    3. Genes involved in bacteriophage genome integration because they are a step in the lysogenic bacteriophage reproductive cycle.
    4. Early genes in the lytic bacteriophage cycle because the suppressor tRNAs involved in recoding are made during DNA replication.
  4. The podcasters speculate as to why the host has been unable to have evolve a defense against the recoding process. What is their reasoning?
    1. The recoding process has already undergone selection, so the host cannot recover.
    2. The recoding process is only found in certain bacteriophages among the eight clades.
    3. The recoding process doesn’t affect the host proteins, so the host has no selection.
    4. The recoding process occurs late in infection, so the host already is near to death.
  5. The podcasters speculate as to why the host has been unable to have evolve a defense against the recoding process. What types of defense strategies could the bacteria theoretically use to combat this bacteriophage’s strategy? [pick all apply]
    1. Degrading specific phage tRNAs.
    2. Preventing bacteriophage entry.
    3. Degrading bacterial release factors.
    4. Inhibiting ribosomal assembly.
  6. Identify the two bacterial species discussed in the main paper as major biofilm formers.
    1. Streptococcus pneumoniae and  Escherichia coli 
    2. Pseudomonas aeruginosa and Escherichia coli 
    3. Staphylococcus aureus and Pseudomonas aeruginosa 
    4. Streptococcus pneumoniae and Staphylococcus aureus
  7. Which molecule inhibits the growth of which species in a glucose rich medium?
    1. A product of acetoin produced by S. aureus inhibits the growth of P. aeruginosa 
    2. A product of acetoin produced by P. aeruginosa inhibits the growth of S. aureus.
    3. A product of the S. aureus Embden Meyerhof pathway inhibits P. aeruginosa growth.
    4. A product of the P. aeruginosa Embden Meyerhof pathway inhibits S. aureus growth.
  8. You find another pair of species whose growth may have a similar pattern to that observed for  S. aureus and P. aeruginosa,  so you perform co-culture experiments with colony forming growth measures (CFU) as your output.  What can you conclude based on these results?
Species Species A colony forming units (CFU/mL) Species B colony forming units (CFU/mL)
Species A and B co-cultured 503 511
Species A cultured, then species B added 499 502
Species B cultured, then species A added 255 816
Species A alone 1003 n/a
Species B alone n/a 986
    1. A mature culture of species A may produce a compound that inhibits the growth of a new culture of species B.
    2. A mature culture of species B may produce a compound that inhibits the growth of a new culture of species A.
    3. Species A immediately produces a very potent compound that inhibits the growth of all cultures of species B.
    4. Species B immediately produces a very potent compound that inhibits the growth of all cultures of species A.
  1. In a glucose rich medium, which statement correctly identifies the finding of the main paper?
    1. If S. aureus and P. aeruginosa were introduced, then S. aureus would use the Embden Meyerhof pathway and acidify the medium and produce a product of acetoin which inhibits the growth of  P. aeruginosa 
    2. If  S. aureus and  P. aeruginosa  were co-introduced, then P. aeruginosa  would use the Embden Meyerhof pathway and acidify the medium and produce a product of acetoin which inhibits the growth of S. aureus
    3. If S. aureus was introduced alone first, S. aureus would use the Embden Meyerhof pathway and acidify the medium and produce a product of acetoin which inhibits the growth of P. aeruginosa.
    4. If P. aeruginosa  was introduced alone first, P. aeruginosa would use the Embden Meyerhof pathway and acidify the medium and produce a product of acetoin which inhibits the growth of S. aureus.

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 2AC and 4).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify independent and dependent variables.
  • Explain the relationship of OD600 to population density/growth.
  • Analyze the data to make conclusions about which media types and conditions affect bacterial growth.
  • Defend why the researchers used both “TSB” and “Sa sup” at the same pH.
  • Predict growth results for different hypothetical interactions between P. aeruginosa and S. aureus.
Experimental Background (Kvich et al., Figure 2AC)

Pseudomonas aeruginosa and Staphylococcus aureus often co-exist in chronic clinical infections such as lung infections of cystic fibrosis (CF) patients. Yet, when studied in in vitro assays, P. aeruginosa typically overpopulates S. aureus.  To better understand how the bacterial species can co-exist in certain environments, but not others, Kvich et al. (2022) first wanted to identify S. aureus metabolites that affected P. aeruginosa growth.  To investigate this, they grew P. aeruginosa in a variety of media that had already been been used to grow S. aureus.  This can be called “used” or “spent” media; these researchers called this S. aureus supernatant (Sa sup.).  To make these media, the researchers first grew S. aureus, then cleared the medium of S. aureus, and finally, used the medium to grow P. aeruginosa.

Because glucose and pH levels of media can impact growth, they also adjusted and varied glucose and pH levels in the media. For the first experiment, the S. aureus culture media had different starting glucose concentrations (panel A).  In the second experiment, the researchers used plain, new media (Tryptic Soy Broth; TSB) or “used” media (Sa sup.) adjusted to two different pH levels (panel C). Pseudomonas aeruginosa growth was measured by spectroscopy (optical density of 600 nm; OD(600) in each experiment.

Histograms showing growth inhibition in P. aeruginosa after different treatments.
Figure 2. “The growth-inhibiting substance was glucose concentration-dependent and pH-dependent. The relative growth (measured as absorbance at an optical density of 600 nm, OD600) of Pseudomonas aeruginosa in Staphylococcus aureus supernatants from overnight cultures with different glucose concentrations (A). The relative growth (OD600) of P. aeruginosa when adjusting the pH of the same S. aureus supernatant up and down (C). Error bars represent standard deviation (SD). Statistical difference was calculated using a one-way ANOVA with Dunnett correction (n = 3) (A) or Kruskal-Wallis with Dunn’s correction (n = 3) (C). *, adjusted P  < 0.05; **, adjusted P  < 0.01; ****, adjusted P < 0.0001. TSB, tryptic soy broth.” (Kvich et al. 2022, cropped to Panels A and C only).

 

7.1.2. Questions

  1. What is an OD measurement and how is that related to the population density in a liquid bacterial culture?
    1. Ocular Distance is a measure of how far apart bacterial cells are from each other; therefore, a higher OD indicates a higher population density.
    2. Ocular Distance is a measure of how far apart bacterial cell are from each other; therefore, a higher OD indicates a lower population density.
    3. Optical Density is a measure of how much light is scattered by bacterial cells in a culture;  therefore, a higher OD readout indicates a lower population density.
    4. Optical Density is a measure of how much light is scattered by bacterial cells in a culture; therefore, a higher OD readout indicates a higher population density.
  2. What is the independent variable for the experiment in Panel A?
    1. Glucose concentration in Staphylococcus aureus culture
    2. Growth of Staphylococcus aureus measured using OD
    3. pH of medium for the Staphylococcus aureus culture
    4. Growth of Pseudomonas aeruginosa measured using OD
  3. What is the dependent variable for the experiment in Panel C?
    1. Glucose concentration in Staphylococcus aureus culture
    2. Growth of Staphylococcus aureus measured using OD
    3. pH of medium for the Staphylococcus aureus culture
    4. Growth of Pseudomonas aeruginosa measured using OD
  4. In Panel A, the data that support growth inhibition is when you compare _____ to ______ .
    1. 0% glucose to 1% glucose
    2. 0% glucose to 0.125% glucose
    3. 0.5% glucose to 1% glucose
    4. 0.25% glucose to 0.5% glucose
  5. Bacterial growth still occurred in which types of media (panel C)? (Pick all that apply)
    1. TSB pH 7
    2. TSB pH 4.8
    3. Sa sup. pH 4.8
    4. Sa sup. pH 7
    5. Sa sup. pH adj. back to 4.8
  6. What was the researchers’ purpose in including P. aeruginosa growth in Tryptic Soy Broth (TSB) medium (Panel C)?
    1. TSB is a control to show the normal growth level expected for P. aeruginosa at each pH.
    2. TSB  is a control to show the normal growth level expected for S. aureus at each pH.
    3. TSB  is a control to show P. aeruginosa growth inhibition as measured by OD at each pH.
    4. TSB  is a control to show how P. aeruginosa cells would scatter light differently at each pH.
  7. We see in Panel C that there is a growth difference when P. aeruginosa is grown in “Sa sup.” at different pH levels, but not when P. aeruginosa is grown in “TSB” at different pH levels.  What do these results indicate?
    1. The substance produced by Staphylococcus aureus that promotes Pseudomonas aeruginosa growth is pH dependent.
    2. Growth of Pseudomonas aeruginosa is completely inhibited by Staphylococcus aureus regardless of pH level.
    3. The substance produced by Staphylococcus aureus that inhibits Pseudomonas aeruginosa growth is pH dependent.
    4. Growth of Staphylococcus aureus is completely inhibited by Pseudomonas aeruginosa regardless of pH level.
  8. If the researchers had performed an experiment with different maltose levels (rather than glucose levels) and reported their results as in Panel A, what would you have expected to see if there were no growth inhibition?
    1. The OD would decrease with increasing maltose concentrations.
    2. The OD would increase in proportion with increasing maltose concentrations.
    3. The OD would be low for 0% maltose, but increase after 0.125% maltose.
    4. You would see approximately the same OD for all maltose concentrations.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key aspects of experimental design including, types of variables, as well as inoculation timing and location.
  • Distinguish the physical characteristics of each bacterial species.
  • Analyze the data to make conclusions about which bacterial species is inhibited in a cross streak plate assay.
  • Predict the outcome of a hypothetical cross streak plate assay when given hypothetical species and their interaction.
Experimental Background (Kvich et al., Figure 4)

Pseudomonas aeruginosa and Staphylococcus aureus often co-exist in chronic clinical infections such as lung infections of cystic fibrosis (CF) patients. Yet, when studied in in vitro assays, P. aeruginosa typically overpopulates S. aureus.  In this study, Kvich et al. (2022) found that S. aureus (Sa) utilizes glucose metabolism to produce a metabolite that affects P. aeruginosa (Pa) growth.  To further characterize how these bacterial species can co-exist under specific glucose-dependent environmental conditions, they investigated the timing of inoculation using a plate streak assay with and without glucose in the medium.  In this experiment (see the right diagram of each panel), one species (Pa or Sa) is streaked onto solid agar medium (top to bottom) in the absence or presence of 1% glucose. Then the other species is streaked across the same solid agar medium (left to right) on the same day (Panel A) or a day later (Panels B and C).

Streak plate assay results, with three sets of photos showing how bacteria was streaked and changes with glucose.
Figure 4. “Staphylococcus aureus inhibits Pseudomonas aeruginosa in a streak-assay. Cross-streak assays with S. aureus and P. aeruginosa on LB agar plates +/− 1% glucose. Drawings to the right of the image show how bacteria were streaked. The white arrow indicates zones of P. aeruginosa inhibition, and the black arrows indicate zones of S. aureus inhibition (A to C). S. aureus and P. aeruginosa were inoculated together (Sa+Pa) or at 1-day intervals (Sa, Pa later).” (Kvich et al. 2022, no changes)

 

7.2.2. Questions

  1. How did inoculation of S. aureus and P. aeruginosa differ between Panels A and B?
    1. In Panel A both were inoculated at the same time, but in Panel B P. aeruginosa was inoculated first and S. aureus one day later.
    2. In Panel A S. aureus was inoculated first and P. aeruginosa one day later, and in Panel B P. aeruginosa was inoculated first and S. aureus one day later.
    3. In Panel A both were inoculated at the same time, but in Panel B S. aureus was inoculated first and P. aeruginosa one day later.
    4. There were no inoculation timing differences between Panels A and B.  Instead the difference is in whether there was glucose in the medium.
  2. Which statement best describes the difference in appearance of the Pseudomonas aeruginosa (Pa) and Staphylococcus aureus (Sa) appearance in the cross streak plate assay?
    1. Pa streaks were yellow and thicker than the white and thin Sa streaks.
    2. Pa streaks were white and thicker than the white and thick Sa streaks.
    3. Pa streaks were yellow and thinner than the yellow and thick Sa streaks.
    4. Pa streaks were white and thicker than the yellow and thin Sa streaks.
  3. What are the dependent and independent variables for the cross streak assay shown in Panel A?
    1. Inoculation timing and glucose
    2. Glucose level and bacterial growth
    3. Inoculation timing and bacterial species
    4. Bacterial growth and glucose level
  4. Which species’ growth inhibition is dependent on glucose and which data in any of the panels support this?
    1. Pseudomonas aeruginosa, the growth inhibition shown by the white arrow in Panel B
    2. Pseudomonas aeruginosa, the growth inhibition shown by the black arrows in Panel C
    3. Staphylococcus aureus, the growth inhibition shown by the black arrows in Panel C
    4. Staphylococcus aureus, the growth inhibition shown by the black arrows in Panel A
  5. What does the difference in growth observed for the plates in Panel B indicate?
    1. In order for S. aureus to inhibit P. aeruginosa, it must be grown without glucose.
    2. In order for P. aeruginosa to inhibit S. aureus, it requires glucose metabolism.
    3. In order for S. aureus to inhibit P. aeruginosa, it requires glucose metabolism.
    4. For both S. aureus and P. aeruginosa to be grown, they both require glucose.
  6. Which statement best describes the conclusions of all of the experiments shown in this figure?
    1. Pseudomonas aeruginosa always inhibits growth of S. aureus no matter when it is inoculated or the presence of glucose.
    2. Which species is inhibited only depends on which is one is inoculated first and has nothing to do with glucose.
    3. S. aureus inhibits growth of P. aeruginosa when it is inoculated first and is dependent on glucose for inhibition.
    4. No conclusions can be made, since the plate streak results are not quantitative and cannot be used in statistical tests.

cross streak plate layout7. If species A and species B were used in a plate streak assay as shown in the drawing (also like panel A) and species A (top to bottom streak) inhibited the growth of species B (left to right streak), but only in the presence of compound X, what would you expect to see?

    1. Growth inhibition at the intersection and all the way across the right side for both the plate containing and the plate missing compound X.
    2. Growth inhibition at the intersection on the plate without compound X and uninhibited growth on the plate with compound X.
    3. Growth inhibition at the intersection and all the way across the right side of the “B streak,” but only on the plate containing compound X.
    4. Growth inhibition at the intersection and species A growth on the right side of the “B streak,” but only on the compound X plate.

8. Paper Information and Licensing

8.1. Snippet paper

8.2. Main paper

  • Kvich L, Crone S, Christensen MH, Lima R, Alhede M, Alhede M, Staerk D, Bjarnsholt T. 2022. Investigation of the Mechanism and Chemistry Underlying Staphylococcus aureus’ Ability to Inhibit Pseudomonas aeruginosa Growth In Vitro. J Bacteriol. 204(11):e0017422. doi: 10.1128/jb.00174-22.
  • 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 https://journals.asm.org/doi/10.1128/jb.00174-22

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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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