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Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

ABSTRACT: To cope with harsh circumstances, bacterial cells must initiate cellular stress response programs, which demands the de novo synthesis of many stress defense proteins. Reactive oxygen species (ROS) is a universal environmental stressor for both prokaryotic cells and eukaryotic cells. However, the physiological burden that limits the survival of bacterial cells during oxidative stress remains elusive. Here we quantitatively characterize the cell growth and translational elongation rate of Escherichia coli cells treated with different doses of hydrogen peroxide. Cell growth is immediately arrested by low to moderate levels of hydrogen peroxide, but completely recovers after a certain lag time. The lag time depends positively on the dose of hydrogen peroxide. During the lag time, translational elongation rate drops by as much as ?90% at initial stage and recovers to its normal state later, a phenomenon resulting from the dramatic alteration in cellular tRNA pools during oxidative stress. However, translational elongation is completely stalled at a certain threshold-level of hydrogen peroxide, at which cells ultimately fail to resume growth. Although the mRNA transcription of oxidative defense genes in oxyR regulon is dramatically induced upon hydrogen peroxide treatment, the extreme slow-down of translational elongation during high levels of hydrogen peroxide has severely compromised the timely synthesis of those oxidative defense proteins. Our study demonstrates that the tRNA-limited translational elongation is a key physiological bottleneck that the bacteria must overcome to counteract ROS, and the maintenance of translational elongation rate for timely synthesis of stress defense proteins is crucial for cells to smoothly get over the oxidative stress.

SUBMITTER: Zhu M

PROVIDER: S-EPMC6698664 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

Zhu Manlu M Dai Xiongfeng X

Nucleic acids research 20190801 14

To cope with harsh circumstances, bacterial cells must initiate cellular stress response programs, which demands the de novo synthesis of many stress defense proteins. Reactive oxygen species (ROS) is a universal environmental stressor for both prokaryotic cells and eukaryotic cells. However, the physiological burden that limits the survival of bacterial cells during oxidative stress remains elusive. Here we quantitatively characterize the cell growth and translational elongation rate of Escheri ...[more]

PMID: 31131413

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Project description:UNLABELLED:Elongation factor P (EF-P) is a universally conserved bacterial translation factor homologous to eukaryotic/archaeal initiation factor 5A. In Salmonella, deletion of the efp gene results in pleiotropic phenotypes, including increased susceptibility to numerous cellular stressors. Only a limited number of proteins are affected by the loss of EF-P, and it has recently been determined that EF-P plays a critical role in rescuing ribosomes stalled at PPP and PPG peptide sequences. Here we present an unbiased in vivo investigation of the specific targets of EF-P by employing stable isotope labeling of amino acids in cell culture (SILAC) to compare the proteomes of wild-type and efp mutant Salmonella. We found that metabolic and motility genes are prominent among the subset of proteins with decreased production in the ?efp mutant. Furthermore, particular tripeptide motifs are statistically overrepresented among the proteins downregulated in efp mutant strains. These include both PPP and PPG but also additional motifs, such as APP and YIRYIR, which were confirmed to induce EF-P dependence by a translational fusion assay. Notably, we found that many proteins containing polyproline motifs are not misregulated in an EF-P-deficient background, suggesting that the factors that govern EF-P-mediated regulation are complex. Finally, we analyzed the specific region of the PoxB protein that is modulated by EF-P and found that mutation of any residue within a specific GSCGPG sequence eliminates the requirement for EF-P. This work expands the known repertoire of EF-P target motifs and implicates factors beyond polyproline motifs that are required for EF-P-mediated regulation. IMPORTANCE:Bacterial cells regulate gene expression at several points during and after transcription. During protein synthesis, for example, factors can interact with the ribosome to influence the production of specific proteins. Bacterial elongation factor P (EF-P) is a protein that facilitates the synthesis of proteins that contain polyproline motifs by preventing the ribosome from stalling. Bacterial cells that lack EF-P are viable but are sensitive to a large number of stress conditions. In this study, a global analysis of protein synthesis revealed that EF-P regulates many more proteins in the cell than predicted based solely on the prevalence of polyproline motifs. Several new EF-P-regulated motifs were uncovered, thereby providing a more complete picture of how this critical factor influences the cell's response to stress at the level of protein synthesis.

Project description:In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE). We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG) at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert and Jakob presented in the accompanying paper (Leichert and Jakob 2004), which provide evidence that MetE is one of the proteins in E. coli most susceptible to oxidation. In eukaryotes, glutathionylation of key proteins involved in protein synthesis leads to inhibition of translation. Our studies suggest a simpler mechanism is employed by E. coli to achieve the same effect.

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Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

Publications

Maintenance of translational elongation rate underlies the survival of Escherichia coli during oxidative stress.

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