Journal: Nucleic Acids Research

Article Title: OLD amputates the anticodon arm of tRNAs during P2–Lambda interference

doi: 10.1093/nar/gkaf874

Figure Lengend Snippet: A two-plasmid system for on-demand induction of P2–OLD toxicity in E. coli . ( A ) Spot dilution assay showing growth defects of E. coli MG1655 producing P2–OLD alongside Gam from Lambda (λ) or Gp5.9 from T7. Data are representative of n > 3 independent experiments. ( B ) Experimental workflow for activating P2–OLD through temperature-induced Gam expression used in panel (C – G). Culture sampling intervals: 15 min for measuring cell growth and viability, 20 min for microscopy, or 30 min for isolating genomic and plasmid DNA. ( C ) Growth curves of MG1655 cultures producing either wild-type (WT) P2–OLD or P2–OLD H332A E402A mutant upon induction of Gam expression. Data presented are from technical replicates and are representative of n = 2 independent experiments. ( D ) CFUs of MG1655 cultures producing WT P2–OLD or the P2–OLD H332A E402A mutant upon induction of Gam expression. Data presented are average of n = 2 independent experiments. ( E and F ) Integrity of genomic and plasmid DNA extracted from growing MG1655 cultures expressing WT or mutant P2–OLD at each indicated time point. 100 ng of DNA was loaded and resolved in 1% E-Gel EX with SYBR Gold II. Data are representative of n = 2 independent experiments. ( G ) Phase contrast and fluorescence micrographs showing nucleoid compaction upon P2–OLD activation. Relevant plasmids are indicated in parentheses. Phase contrast images are overlaid with mCherry fluorescence images (pseudo-colored red). Null indicates SK589 without plasmids. ( H ) Visualization of total RNA extracted from cultures expressing WT Old or Old mutant and Gam at indicated intervals. Data shown are representative of n = 2 independent experiments. ( I ) Visualization of total RNA extracted from cultures 2 h after treatment with Mitomycin C. RNA is run on an acid–Urea–PAGE gel and stained with methylene blue. Data shown are representative of n = 3 independent experiments.

Article Snippet: The mCherry filter cube (Nikon 96365) was used to visualize nucleoid area within the cells.

Techniques: Plasmid Preparation, Dilution Assay, Expressing, Sampling, Microscopy, Mutagenesis, Fluorescence, Activation Assay, Staining

Journal: Nucleic Acids Research

Article Title: OLD amputates the anticodon arm of tRNAs during P2–Lambda interference

doi: 10.1093/nar/gkaf874

Figure Lengend Snippet: Phage rescue tRNAs Thr delay translation inhibition caused by degradation of a E. coli tRNA ThrU isoacceptor. ( A ) Bar chart showing that the UGU anticodon of tRNA Thr6 is required for suppressing the P2–OLD toxicity. The spot dilution assay was carried out as described in the “Materials and methods” section. MG1655 E. coli expressing OLD and Gam were transformed with plasmids expressing tRNA Thr6 , tRNA Thr6(UAC) , or RNA Thr6(CGU) . ( B ) Schematic for inducing P2–OLD activation and micrograph showing that P2–OLD-induced nucleoid compaction is delayed and mitigated by rescue tRNA Thr6 , further detailed in the “Materials and methods” section. Experiments were performed with SK589 expressing OLD and Gam and a third plasmid (in parenthesis) for expressing the indicated phage tRNAs. Phase contrast images are overlaid with mCherry fluorescence images (red pseudo color). Null is indicated when the strain does not carry any plasmid. Images in the top two rows also appear in Fig. . ( C ) Northern blot analysis showing depletion of tRNA ThrU over time upon P2–OLD activation while tRNA ThrT and tRNA ThrW remain intact. Total RNAs extracted from E. coli MG1655 expressing P2–OLD or the P2–OLD H332A E402A mutant and Gam at the indicated time point. tRNA ThrV , which only differs by four bases from tRNA ThrT was omitted from this analysis.

Article Snippet: The mCherry filter cube (Nikon 96365) was used to visualize nucleoid area within the cells.

Techniques: Inhibition, Dilution Assay, Expressing, Transformation Assay, Activation Assay, Plasmid Preparation, Fluorescence, Northern Blot, Mutagenesis