Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: Serine miscoding is toxic in E. coli . Chimeric tRNA Ser variants were generated to decode at either the Ser (anticodon, GGA), Ala (UGC), or Thr (GGU) codon (top). Mutant tRNAs were expressed in wild-type E. coli , and growth was monitored (bottom). Both tRNA mutants caused growth defects in MG1655. All growth experiments were performed in triplicate, and error bars indicate the standard deviation of the replicates.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Generated, Mutagenesis, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: AlaRS fidelity is required for optimal growth in E. coli . (A) AaRS mutants and the AlaRS C666A complement strain (pLK- alaS ) were grown in LB, and their growth was monitored. In rich medium, AlaRS fidelity is required for optimal growth. (B) AlaRS C666A was grown in M9 minimal medium supplemented with exogenous amino acids to determine the toxicity of noncognate stress. (C) Serine mistranslation was observed in the AlaRS C666A mutant using a β-lactamase S68A mistranslation reporter. (D) The severity of the AlaRS C666A growth defect was enhanced when grown at 42°C. All growth experiments were performed in triplicate, and error bars indicate the standard deviation of the replicates. WT, wild type.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Mutagenesis, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: AlaRS-mediated mistranslation disrupts proteome homeostasis. Total proteome analysis was performed on wild-type and AlaRS C666A E. coli . Depicted is a volcano plot of the 833 significantly enriched or underrepresented proteins when AlaRS fidelity is impaired.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques:

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: Swimming motility is impaired in the absence of AlaRS fidelity. (A) Representative images (top) and quantification (bottom) of swimming motility data. In the absence of AlaRS proofreading, E. coli has a swimming defect, and this can be rescued by complementation. (B) The observed swimming defect is not exclusively due to small RNA inhibition, as an hfq mutant was unable to rescue the defect. All data were plotted relative to a wild-type control for each experiment. Bar graphs shown the averages of the data collected from three experiments, and error bars represent the standard deviation of those experiments.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Inhibition, Mutagenesis, Control, Standard Deviation

Journal: mBio

Article Title: Alanyl-tRNA Synthetase Quality Control Prevents Global Dysregulation of the Escherichia coli Proteome

doi: 10.1128/mBio.02921-19

Figure Lengend Snippet: The E. coli membrane is affected when AlaRS fidelity is impaired. (A) The AlaRS C666A mutant (blue) is sensitive to an array of antibiotics, as observed using a disk diffusion assay compared to wild-type (black) and ThrRS C182A (red) E. coli . All experiments were performed in triplicate, with error bars indicating the standard deviation. The asterisks indicate statistical significance as determined using one-way analysis of variance (ANOVA) with Tukey post hoc comparison ( P < 0.05). (B) TEM analysis indicated altered nucleoid morphology in the absence of AlaRS fidelity.

Article Snippet: E. coli AlaRS (96 kDa) was probed with an anti- E. coli AlaRS antibody (1:1,000 dilution; ProSci custom antibody) and anti-rabbit horseradish peroxidase (anti-HRP; 1:5,000 dilution; GE Healthcare).

Techniques: Membrane, Mutagenesis, Diffusion-based Assay, Standard Deviation, Comparison