Increased mistranslation protects E. coli from protein misfolding stress due to activation of a RpoS-dependent heat shock response

Abstract

The misincorporation of an incorrect amino acid into a polypeptide during protein synthesis is considered a detrimental phenomenon. A mistranslated protein is often misfolded and degraded or nonfunctional and results in an increased cost to quality control machinery. Despite these costs, errors during protein synthesis are common in bacteria. Here, we report that mistranslation in Escherichia coli increase the protein level of the heat shock sigma factor RpoH and protect cells against heat stress. Surprisingly, this increase in RpoH due to mistranslation is dependent on the presence of the general stress response sigma factor RpoS. This report provides evidence for a protective function of mistranslation and suggests a novel regulatory role of RpoS in the heat shock response.

Keywords: RpoH; RpoS; heat shock response; mistranslation.

Figure 1

The rpsD* strain is protected from heat killing and has an activated heat shock response. (A) MG1655 and rpsD* strains were grown to mid‐logarithmic phase at 30 °C, then treated at 50 °C for 90 min. Serial dilutions were made every 30 min and CFU counts were used to determine the rate of killing (N = 3). (B). MG1655 and rpsD* strains expressing promoter fusions to gfp on a low‐copy plasmid were grown to mid‐logarithmic phase. The fluorescence in each culture was determined using fluorescence spectroscopy (N = 3). *P‐value < 0.05. **P‐value < 0.01 by unpaired t‐test.

Figure 2

Increased mistranslation induces aggregate clearance. MG1655 and rpsD* strains expressing an IPTG‐inducible sfGFP‐ClpB fusion were grown to mid‐logarithmic phase in LB with 100 μm IPTG at 30 °C. Then, cells were treated at 42 °C (A, B) or with 100 μg·mL⁻¹ streptomycin (C, D) for 1 h. After treatment, cultures were treated with 100 μg·mL⁻¹ spectinomycin to stop protein synthesis. Images were taken immediately after and 2 h after spectinomycin treatment. (A, C) Representative images of cells after treatment. (B, D) The numbers of aggregates per cell were quantified manually using imageJ (N = 3). (E) MG1655 cells expressing sfGFP‐ClpB were grown in the presence or absence of the arginine analogue, Canavanine (Canav), to mid‐logarithmic phase in LB with 100 μm IPTG. After growth, cultures were treated at 42 °C for 1 h. After treatment, cultures were treated with 100 μg·mL⁻¹ spectinomycin to stop protein synthesis. Images were taken immediately after and 2 h after spectinomycin treatment. The numbers of aggregates per cell were quantified manually using imagej (N = 3). *P‐value < 0.05, **P‐value < 0.01 by unpaired t‐test compared to time point 0 h.

Figure 3

Time‐lapse microscopy reveals that increased mistranslation promotes aggregate clearance in single cells. MG1655 and rpsD* cells expressing the IPTG‐inducible sfGFP‐ClpB construct were grown in LB with 100 μm IPTG to mid‐logarithmic phase. Additionally, the rpsD* cells constitutively expressed mCherry on a plasmid. After growth, cultures were mixed and incubated at 42 °C for 30 min. Then, spectinomycin were added to the culture to stop protein synthesis. Cells were transferred to a 1.5% agarose pad. (A) Aggregate formation was visualized using fluorescence microscopy and recover was tracked for 3 h. (B) The number of cells containing aggregates in the MG1655 and rpsD* cells were quantified manually using imagej (N = 2 of > 60 individual cells). *P‐value < 0.05, **P‐value < 0.01 by unpaired t‐test.

Figure 4

RpoS is necessary for the mistranslation‐induced increase of RpoH. MG1655, rpsD*, rpsD* Δrpos strains were grown to mid‐logarithmic phase in LB at 30 °C. For comparison to RpoH induction by heat shock, MG1655 cells were incubated at 42 °C for 20 min. (A) A western blot of cell lysates using an α‐RpoH antibody to determine RpoH levels in each strain. RpoB protein levels were determined as a loading control. (B) Three independent repeats were quantified using volumetric analysis. **P‐value < 0.01 by unpaired t‐test.

Figure 5

Mistranslation‐induced heat protection is dependent on RpoS. (A) rpoS deletion strains of MG1655 and rpsD* were grown to mid‐logarithmic phase at 30 °C, then incubated at 50 °C. Heat killing was assayed via colony formation by serial dilution every 30 min. (B) The same experiment was performed with MG1655 with or without rpoS grown in the presence of canavanine (Canav). N ≥ 3 **P‐value < 0.01 by unpaired t‐test for each point compared to MG1655.