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.

Modulation of Escherichia coli Translation by the Specific Inactivation of tRNAGly Under Oxidative Stress

“…In agreement with our observations, reports of experiments performed in other E. coli strains also indicate that the relevance of elongation in determining the overall speed of protein production increases at stronger oxidative stress conditions, in this case induced by higher concentrations of H 2 O 2 (Zhu and Dai, 2019). In contrast to what we have observed, but in agreement with previous reports (Zhong et al, 2015), the authors of these experiments observe a decrease in the concentration of all tRNAs.…”

“…Out of 10 tested tRNAs, only tRNA Gly showed a statistically significant decrease in the levels of active tRNA after stress by exposure to either paraquat or H 2 O 2 ( Figure 1A). Previous reports have shown a general decrease in total tRNA levels under oxidative stress in minimal media (Zhong et al, 2015;Zhu and Dai, 2019). In contrast, we observed that tRNA levels remained fairly constant (Figure 1B), suggesting the observed decrease in the levels of active tRNA Gly was a specific response to oxidative stress.…”

“…The response to oxidative stress has been extensively studied, in particular because generation of an oxidative attack by macrophages and polymorphonuclear leukocytes is one of the main defense strategies of the human body against invading bacteria once they have crossed the primary physical barriers (Slauch, 2011;Nguyen et al, 2017). In order to adapt to conditions that induce oxidative stress, bacteria may: (I) reduce motility, increase exopolysacharide production, and induce biofilm formation, thereby reducing accessibility to molecules that produce oxidative stress (Gambino and Cappitelli, 2016); (II) inhibit replication, preventing DNA mutagenesis at sites of base oxidation (Imlay, 2013) and possibly also reducing the toxicity of replication near the site of repair for oxidized bases (Charbon et al, 2014); (III) reduce the rate of global translation (Katz and Orellana, 2012;Zhong et al, 2015;Zhu and Dai, 2019); (IV) selectively induce the production of several proteins involved in the reduction, repair, or degradation of oxidant molecules or oxidized biological targets such as thiol groups and Fe/S clusters in proteins (Imlay, 2013); and (V) decrease the production of reduced nicotinamide adenine dinucleotide (NADH) in order to increase production of reduced nicotinamide adenine dinucleotide phosphate (NADPH), required to reduce oxidant molecules and oxidized targets (Rui et al, 2010;Shen et al, 2013). Finally, oxidative stress also induces some members from a bacterial community to enter a partially quiescent state known as "persistence" (Wu et al, 2012), where several primary metabolic pathways are repressed and stress responses are induced (Lewis, 2010;Cohen et al, 2013).…”

“…It has been shown that changes in error rate of aminoacyl-tRNA synthetases are altered by oxidation of editing domains that may increase or decrease their activities under oxidative stress (Ling and Söll, 2010;Wu et al, 2014;Steiner et al, 2019). Others have found that tRNA may be oxidized (Liu et al, 2012) and, in at least some E. coli strains, oxidative stress induces a general and undiscriminated degradation of tRNAs that strongly reduce translation elongation, eventually provoking cell death (Zhong et al, 2015;Zhu and Dai, 2019). Others have found that, in E. coli and other bacteria, oxidation of translation elongation factors may also inhibit elongation (Kojima et al, 2009;Nagano et al, 2015;Yutthanasirikul et al, 2016).…”

O2-Tuned Protein Synthesis Machinery in Escherichia coli-Based Cell-Free System

“…To gain more insight into the effects of the oxygen concentration on the protein synthesis, the oxygen byproducts that might affect the protein synthesis were further analyzed. The level of ROS (reactive oxygen species, O 2− , H 2 O 2 , and OH) was investigated in the oxygen-tuned cell-free system, which was considered as the metabolic intermediate of oxygen and affecting many physiological processes (Schieber and Chandel, 2014;Ezraty et al, 2017;Reichmann et al, 2018;Zhu and Dai, 2019).…”