TWiM #295: Uncultured and Unmutable

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 3:38 minutes

“Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases.” (van den Berg et al. 2023)

1.2. Main paper; discussion starts at 26:35 minutes

“Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.” (Shukla et al. 2023).

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 (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

• SEA-PHAGE Database (8:02–11:32): SEA-PHAGES (Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science) is an undergraduate community phage project to identify bacteriophages from the environment.  The database for this project can be used  by anyone.  Here, the researchers used it to identify tRNAs in viral genomes and tRNA nucleases in genomes of Mycobacterium smegmatis.

4.2. Main Paper

• Isolation Chip (iChip) (30:32–32:19; 36:31–38:35): This is method for culturing single environmental cells in miniature chambers.  Here, the researchers used it to cultivate bacteria from soil derived samples.
• Minimum Inhibitory Concentration Assay (38:24–39:13): This is a method for screening antimicrobials for the least concentration that is effective.  This determines the MIC.
• Whole Genome Sequencing (40:17): This is a method for determining the base sequence for an entire genome.  Here, the researchers used it to screen for  certain genetic sequences such as operons.
• High Performance Liquid Chromatography (HPLC) (41:05–41:37): HPLC is a chemical method to separate, quantify, and identify components of a mixture.  Here, it was used to separate fermentative byproducts of Eleftheria terrae to identify antimicrobials.
• Live/Dead Staining (48:55–49:42): This is a microscopy method for characterizing cell membrane integrity using stains.  The researchers used it to determine which cells were alive.
• Murine Model of Infection (51:21–52:58): Mouse models are often used to mimic infection in humans.  Here, mice were infected with Staphylococcus aureus to test the effectiveness of the antibiotic clovibactin.
• Resistance Assay (53:35–54:32): This is a method for determining which genes are involved in antimicrobial resistance using genomic analysis of sensitive parent strains and resistant strains.
• Radiolabeling of Metabolic Precursors (55:11–56:16): This is a method where precursor molecules are labeled and followed while metabolism occurs. Here it was used to identify clovibactin targets in biosynthetic pathways.

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

5.1. Snippet Paper

• tRNA (4:34 — 6:55; 7:37–8:01):  In protein synthesis, transfer RNA (tRNA) complements anti-codons with codons on mRNA.  The podcasters discuss theories about why phages might code for their own set of tRNAs, such as that this characteristic can increase the phages’ resistance to host anticodon nucleases
• Anticodon Nucleases (6:50–7:35):  Anticodon nucleases target sites in tRNA for cleavage
• Mycobacterium smegmatis (8:46–9:09; 11:13–11:22): This is a gram-positive bacteria that can be infected by phages.
• Phage Therapy (9:56–10:18): Bacterial phages can be used to target specific bacteria in certain infections.
• Enterobacteriaceae (14:36–15:00): These are a family of gram negative bacteria present in the intestinal tract.

5.2. Main Paper

• Streptomycin (28:01–28:13, 35:59–36:08):  This is an antibiotic used against mycobacterium that can cause tuberculosis
• Natural Products Scaffold (29:30–30:32):  This is used to predict how certain molecules might impact growth of cells
• Great Plate Anomaly (30:57–31:18):  There is a known difficulty of replicating conditions necessary to extract bacterial colonies in the lab
• Teixobactin (34:22–35:11): The mechanism of this antibiotic involves inhibiting the synthesis of the cell wall
• Lassomycin (35:22–35:59):  This antibiotic targets an inhibitor of a universal export protein complex in mycobacteria
• Kalimantacin (39:53–40:59): This antibiotic was isolated from E. terrae
• Clovibactin (41:27–42:10; 42:57–44:15): A depsipeptide antibiotic isolated from E. terrae using HPLC. Clovibactin has antimicrobial effects against gram positive bacteria such as MRSA.
• Biosynthetic Gene Clusters (42:12–42:43):  This is the idea that operons encode multiple genes that work together to produce complex molecules in specific metabolic pathways
• Bactericidal (44:28–45:20): This is the ability of an antimicrobial, such as clovibactin, to induce killing of cells
• Lipid II (50:23–51:21): Lipid II plays a role in cell wall synthesis
• Peptidoglycan (55:5–56:45): Targets of clovibactin include molecules important for peptidoglycan synthesis.
• Pyrophosphate (57:45–1:01:50): This is a moiety on many precursors that gram positive bacteria use in cell wall synthesis; it is a target of clovibactin.

6. Podcast Questions

1. What are bacteriophages?
   1. Bacteria that are resistant to antibiotics.
   2. Viruses that infect fungal or human cells.
   3. Viruses that can infect bacteria only.
   4. Bacteria that are resistant to virus infection.
2. What is the target of the bacterial nucleases described in the podcast?
   1. phage-encoded mRNA
   2. phage-encoded tRNA
   3. bacterial-encoded tRNA
   4. bacterial-encoded rRNA
3. Which of the following would provide a selective advantage for phage that infect bacteria with anticodon nucleases?
   1. Bacteria could evolve anticodon nucleases that degrade the viral genomic DNA.
   2. Viruses could produce small anticodon RNA decoys to overwhelm the nucleases.
   3. Bacterial could evolve a higher G-C content to counteract the viral DNA replication.
   4. Viral tRNAs with mutations in their anticodons would be able to escape degradation.
4. The newly discovered antibiotic clovibactin works by _______ .
   1. Inhibiting the enzyme used in cell wall synthesis.
   2. Lysing the cell membrane to make cells leak.
   3. Binding and sequestering cell wall precursors.
   4. Inhibiting the replication of bacterial DNA.
5. What is the main advantage of the iChip method described in the podcast?
   1. Allows isolation of “difficult to culture” soil bacteria.
   2. Increases the sample size for colony counts agar plates.
   3. Decreases airborne contamination found in laboratories.
   4. Decreases the cost of culturing anaerobic halophiles.
6. Knowing the mechanism of action for clovibactin, what effectiveness level would you predict against gram negative bacteria?  What is your reasoning?
   1. High effectiveness; all bacteria replicate their DNA.
   2. Moderate effectiveness; some bacteria host phages.
   3. Low effectiveness; cell walls vary by bacterial type.
   4. We can’t tell from these data.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 1) and one from the main paper (Figure 3a-e).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Experimental Background (van den Berg et al., Figure 1A-D)

Bacteriophages are viruses that infect bacteria, have extremely reduced genome sizes, and maintain few viral genes.  Despite this reduced genome size and gene number, many phages encode their own transfer RNAs (tRNAs) rather than depending on their host’s tRNAs.  This has puzzled researchers for many years, so these researchers approached this puzzle by first identifying the location of tRNA genes within the genomic structure of a C1 mycobacteriophage (panel A).  Having done that, they next investigated the abundance of the many types of tRNAs in the C1 mycobacteriophage phage cluster (panel B).  To determine how the phage and host tRNAs differed, and whether there might be a clue to why phage retained their own tRNAs, they next identified differences in phage tRNA anticodons compared to bacterial tRNA anticodons (panel C).   Additionally, the researchers highlight cleavage sites for known bacterial anticodon nucleases. The authors present a model of infection that visualizes how host and phage tRNAs may be affected differently during infection (panel D).

 

7.1.2. Questions

1.  The C1 mycobacteriophage genome regions that encode tRNAs are diagrammed in panel A.  How many of the tRNAs are targeted by anticodon nucleases and how many are not?
   1. 9; 26
   2. 15; 10
   3. 18; 17
   4. 0; 35
2. Gene clusters are formed from multiple genes located close together that have related functions and/or evolved by gene duplication.  What conclusions can you draw from the  diagram of C1 mycobacteriophage genome regions where the phage tRNAs are located (panel A)?
   1. There is no clustering of phage tRNAs, so it is unlikely they evolved by gene duplication.
   2. Most clusters have both targeted and untargeted tRNAs, so they likely evolved independently.
   3. The tRNAs targeted by anticodon nucleases are clustered, so they resulted from gene duplication.
   4. All phage tRNAs can be targeted by anticodon nucleases, so they evolved by gene duplication.
3. The frequency of phage-encoded tRNAs per codon is shown in panel B.  What conclusions can you draw from these data?
   1. Most phage encode all of their own tRNAs so they do not rely at all on host tRNAs.
   2. Most bacteria that are infected with phage utilize many of the phage tRNAs.
   3. Most phage encode tRNAs that are distinct and unique from bacterial tRNAs.
   4. Most phage use the host tRNAs for Ser and Ile or do not use those amino acids.
4. In the study, the researchers find that Mycobacterium phages encode around 30-33 tRNAs on average. Anticodon sequences for ten of the tRNAs that are targeted by anticodon nucleases are shown in panel C.   What notation is used for bases that are mutant in the phage tRNA when compared to the bacterial-encoded tRNA?
   1. red triangles
   2. red circles
   3. blue circles
   4. blue highlight
5. What relationship do you see between the “mutant” bases of phage tRNA and the bases of the anticodon nuclease cleavage and/or recognition sites? What might this indicate?
   1. The mutations are found in regions know to be flexible; therefore, the mutations do not affect translation.
   2. The mutations are found only in the stem portion of the stem-loop; the mutations have no substantial effect.
   3. The mutations are often found in the cleavage or recognition site; the mutations reduce tRNA degradation.
   4. The mutations are often located in the anticodon bases of the loop; the mutations affect phage translation.
6. According to this figure, and in particular panel D, what is the advantage for phages to encode their own tRNAs?
   1. There is no advantage to either since host anticodon nucleases do not target phage tRNA.
   2. To speed up phage translation and thus also speed up the replication of the phage life cycle.
   3. The host uses different codons, so the phage needs to supply their own phage-specific tRNAs
   4. All tRNAs are potential nuclease targets; mutations help phage to avoid nuclease recognition.

7.2. Second Figure Reading Exercise

Experimental Background (Shukla et al., Figure 3A-E)

Resistance to current antimicrobials is a major concern because it is responsible for many deaths worldwide.  One path to resolving this problem is the discovery of new antibiotics. Shukla et al. (2023) used a new technology called iChip to culture soil microbes and discovered one that produced a novel antimicrobial they named clovibactin.   To first investigate the mechanism of action for their new antibiotic, the researchers traced radioactively labeled metabolic precursors in S. aureus treated with the new drug clovibactin or with antibiotics that inhibit specific metabolic processes, including DNA synthesis (ciproflaxin), RNA synthesis (rifampicin), peptidoglycan synthesis (vancomycin), and protein synthesis (erythromycin) (panel A).  To confirm their metabolic tracking results, they next used a reporter assay where lacZ expression was controlled by a promoter of a gene that is known to be involved in each metabolic process (panel B).  In this reporter system, the B-galactosidase (lacZ) product would be produced in the engineered bacteria if the antibiotic affects transcription of genes in that pathway.  B-galactosidase can cleave X-gal and produce a blue precipitate. Based on their results, the next step the researchers took was to compare the physical appearance of B. subtilis bacteria treated with clovibactin and other antibiotics (panel C).

Panel D was generated from P_lial-lux induction. P_lial is a promoter that responds to antibiotics, particularly those that interfere with the membrane-anchored steps of cell wall biosynthesis and cellular distress. The lux gene encodes for luciferase, an enzyme that catalyzes the reaction of luciferin the presence of oxygen and light; it produces bioluminescence.  Panel E utilized NMR spectroscopy to measure the absorbance levels of bioluminescent products in the cytoplasm. Panel F utilized biosynthesis assays to measure purified enzymes and substrates in cells exposed to clovibactin. Panel G used microtiter plates to track growth of S. aureus after reintroducing purified lipid intermediates to challenge the effects of clovibactin in the cells.

 

7.2.2. Questions

1.  The scientists hypothesized that clovibactin has a mechanism of action involving DNA synthesis is tested in panel A (first two bars).  The dependent variable for this experiment is ______ and the independent variable for this experiment is _______.
   1. erythromycin treatment; DNA replication
   2. clovibactin treatment; ciproflaxin treatment
   3. specific antibiotic treatment; % incorporation
   4. clovibactin treatment; DNA and RNA synthesis
2. The hypothesis that clovibactin has a mechanism of action involving protein synthesis is tested in panel A (last two bars).  The positive control for this experiment is ______ and the negative control for this experiment is _______.
   1. erythromycin treatment; no negative control
   2. clovibactin treatment; ciproflaxin treatment
   3. no positive control; erythromycin treatment
   4. water treatment; antibiotic treatment
3. Which Staphylococcus aureus metabolic process(es) are primarily affected by clovibactin (panel A)? What is your evidence? [Pick all that apply]
   1. DNA synthesis; we see a high level of DNA synthesis precursors incorporated
   2. RNA synthesis: we see a high level of RNA synthesis precursors incorporated
   3. Peptidoglycan synthesis; we see low levels of the PG precursors incorporated
   4. Protein synthesis: we see a high level of protein synthesis precursors incorporated
4. According to the figure legend, what is the indicator of a positive result in the bioreporter assays using B. subtilis (panel B)?
   1. Decrease in cell size
   2. Change in cell shape
   3. Increase in cell division
   4. a blue halo around cells
5. Which Bacillus subtilis metabolic process(es) are primarily affected by clovibactin (panel B)? What is your evidence? [Pick all that apply]
   1. Cell wall synthesis; we see a blue halo when bacteria are exposed to clovibactin
   2. DNA synthesis; we see a distinct tan halo when bacteria are exposed to clovibactin
   3. RNA synthesis: we see a large zone of inhibition when bacteria are exposed to clovibactin
   4. Protein synthesis: we see dark patch of growth when bacteria are exposed to clovibactin
6. Bacillus subtilis is a rod-shaped bacterium that was treated with different antibiotics and microscopy performed to identify structure effects caused by antibiotic exposure (panel C). Categorize each statement as true or false.
   1. Clindamycin treatment caused no deformities of the cell shape.
   2. Clovibactin treatment caused the rod-shaped bacterium to become coccus shaped.
   3. Hypeptin treatment caused no deformities of the cell shape.
   4. Clovibactin treatment caused no deformities of the cell shape.
   5. Teixobactin treatment caused deformities of the cell shape.
7. If clovibactin had a mechanism of action that involved degrading ribosomes, how would the results shown in panel A have been different?
   1. Instead of clovibactin having reduced peptidoglycan precursor incorporation, we’d have seen protein precursor incorporation reduced.
   2. Instead of erythromycin having reduced protein precursor incorporation, we’d have seen protein precursor incorporation increased.
   3. Instead of having reduced incorporation of precursors for the controls, the other antibiotics would show variable precursor incorporation.
   4. Instead of reducing the incorporation of peptidoglycan, clovibactin treatment would increase peptidoglycan and reduce all the others.

8. Paper Information and Licensing

8.1. Snippet paper

• van den Berg DF, van der Steen BA, Costa AR, Brouns SJJ. 2023. Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations. Elife. 12:e85183. doi: 10.7554/eLife.85183
• This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.  Please see the article’s copyright information.

8.2. Main paper

• Shukla R, Peoples AJ, Ludwig KC, Maity S, Derks MGN, De Benedetti S, Krueger AM, Vermeulen BJA, Harbig T, Lavore F, Kumar R, Honorato RV, Grein F, Nieselt K, Liu Y, Bonvin AMJJ, Baldus M, Kubitscheck U, Breukink E, Achorn C, Nitti A, Schwalen CJ, Spoering AL, Ling LL, Hughes D, Lelli M, Roos WH, Lewis K, Schneider T, Weingarth M. 2023. An antibiotic from an uncultured bacterium binds to an immutable target. Cell. 2023 Sep 14;186(19):4059-4073.e27. doi: 10.1016/j.cell.2023.07.038
• This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.