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Transfer RNAs Mediate the Rapid Adaptation of Escherichia coli to Oxidative Stress

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Protein synthesis measurement under normal condition and oxidative stress using 15N metabolic labeling and mass spectrometry.

(A) Growth rate constant of E. coli BL21(DE3) cells grown under normal conditions in M9 minimal medium containing 14NH4Cl (14N) or 15NH4Cl (15N). (B) Experimental design for pulse labeling with 15N under normal and oxidative stress conditions. (C) Bacterial cells were transferred to M9 medium containing 15N, after which the 14N/15N ratio of the E. coli BL21(DE3) proteome was determined by mass spectrometry under normal conditions (upper plot) and oxidative stress induced by exposure to 0.5 mM H2O2 (lower plot). The green line indicates the best linear fit of the dataset. The Pearson r correlation coefficient and P-value of the fit are indicated in green text. (D) The same experiment represented in (C) was repeated using the wild-type E. coli BW25113 strain. (E) Examples showing changes in the 14N/15N ratio of 4 randomly selected proteins (AtpA, GpmA, Eda, and PurA) in BL21(DE3) cells over time. The fit was determined according to Eq 6 (Materials and Methods section). The slope of the linear fit is the synthesis rate constant (ksyn). The black dots indicate the actual data points following protein quantification under normal condition, while the red dots indicate the data points measured under oxidative stress. Linear fits were determined for each dataset, and the Pearson correlation coefficients (r) are indicated in the diagram. (F) The Pearson correlation coefficients (r) values of all proteins were fitted using Eq 6, under normal condition and oxidative stress. Data generated using the BL21(DE3) and wild-type BW25113 strains are shown. (G) P values (in -log10 scale) of linear fits of all proteins using Eq 6 under normal and oxidative stress conditions. The green line denotes the significance threshold (P = 0.05). The data generated using the BL21(DE3) and wild-type BW25113 strains are shown. (H) Histograms of protein half-lives under normal (blue) and oxidative stress (red) conditions. (I) Comparison of protein half-lives measured under normal and oxidative stress conditions. The grey line is at a 45° angle, which demarks the positions where the protein half-lives were unchanged. Half-lives longer than 500 min are shown as 500 min.

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doi: https://doi.org/10.1371/journal.pgen.1005302.g002